ANTIBODIES AND ANTIBODY FRAGMENTS AGAINST HNA _V 1.7 CHANNEL AND THEIR USE

IN PAIN AND CANCER INDICATIONS

TECHNOLOGICAL FIELD

The present disclosure concerns antibodies and compositions comprising antibodies for antagonizing the human Na _v 1.7 (HNa _v 1.7) channel for various indications including treatment and/or alleviation of symptoms of pain and cancer.

BACKGROUND

Ion channels are proteins that form pores in cell membranes and regulate the flux of ions between the intracellular and extracellular spaces. They are involved in the control of many fundamental physiological processes in various tissues and alterations in their functions give rise to a wide range of conditions, including neurological disorders, diabetes, cancer, hypertension and arrhythmia. There are ~60 different inherited ion channel diseases, known as “channelopathies”, that have been identified across cardiovascular, neuronal, neuromuscular, musculoskeletal, metabolic, and respiratory systems; and this number is destined to grow as knowledge about ion channel functions increases.

Sodium channels (Na _v ) are involved in the control of many fundamental physiological processes in various tissues of human body. In excitable cells, they are essential for the initiation and propagation of electrical signals.

Na _v has also been demonstrated to play a role in cancer. Ion channels are at the basis of the electrical variations that accompany the transitions along the different phases of cell cycle. Blocking the channels responsible for these electrical variations can inhibit the progression of cell cycle and consequently impair cell proliferation. Ion channels modify the osmotic gradients across the cell membrane and therefore induce changes in cellular volume and shape. Such changes, coordinated with polymerization/depolymerization of actin filaments underlie cell motility and, consequently, the metastatic potential of tumor cells. The degree of malignancy of various tumors also correlates with the de-novo expression of some specific ion channels. Na _v are totally absent in non excitable cells, however, in various cancers (e.g. breast, prostate, lung) de-novo functional expression of Na _v channels has been found and linked to malignant phenotypes (Fraser et al. 2005; Diss et al., 2001 ; Roger et al., 2007).

The Na _v family has nine members (nine types of pore-forming a subunits), which in mammals are numerically named from Na _v 1.1 to 1.9. These isoforms share a common overall structural motif and each Na _v type has a different function and expression profile. Na _v 1.1 , Na _v 1.2, Na _v 1.3, and Na _v 1.6 are mainly expressed in the central nervous system (CNS). Na _v 1.7, Na _v 1.8, and Na _v 1.9 are present in the peripheral nervous system (PNS), where they are known to accumulate in the region of peripheral nerve injury and consequently may be important in chronic and neuropathic pain. Na _v 1.4 is the muscle sodium channel and Na _v 1.5 is the predominant cardiac myocyte channel.

The Na _v 1.7 channel is expressed in nociceptive neurons (Dorsal Root Ganglion, DRG) of the PNS. When nociceptive stimuli (i.e., injury or inflammation) are initiated at periphery receptors, they are transduced at peripheral termini by Na _v s dependent action potentials. Na _v 1.7 channels are also present in the central axonal projections of DRG neurons and their presynaptic terminals within the dorsal horn of the spinal cord where they may facilitate impulse invasion or evoked release of neurotransmitters. Homology modelling based on crystal structures of ion channels suggests an atomic-level structural basis for the altered gating of mutant Na _v 1.7 that causes pain. Genetic, structural and functional studies have shown that Na _v 1.7 regulates sensory neuron excitability which is a major contributor to several sensory modalities, and have established the contribution of Na _v 1.7 to human pain disorders. In particular, genetic studies show that mutations in SCN9A gene which encode for Na _v 1.7 produce familial pain disorders. These mutations can be described as gain of function mutations (increase activity of Na _v 1.7) which cause paroxysmal extreme pain disorder (PEPD) or loss of function mutations (reduction of Na _v 1.7 activity) which are linked to complete insensitivity to pain (CIP). Importantly, people totally lacking Na _v 1.7 have minimal cognitive, cardiac, motor and sensory deficits, supporting Na _v 1.7 as a valid and indeed attractive target for development of drug against pain.

De novo functional expression of Na _v 1.7 has been documented in prostate, non small cell lung cancer and leukemia. The expression of Na _v 1.7 in these cancers is reported to promote cell invasion. It has been shown that in vitro application of tetrad otoxin (TTX), a potent Navs blocker, in prostate cancer cells inhibits cancer invasion, proliferation and migration (Laniado at al., 1997; Grimes at al., 1995). Likewise, in vivo injection of TTX directly into primary tumours, to avoid systemic toxicity of TTX, in a Copenhagen rat model of prostate cancer, reduced by >40% the number of lung metastasis (Yildirim at al., 2012). Consequently, blocking the Na _v 1.7 is a valid target to develop a drug against cancer malignancy.

The first generations of molecules developed against Na _v s were analgesics. These drugs were clinically effective, but, due to their non-selective Na _v activity, their analgesic potential was limited by numerous side effects (dizziness, sedation, convulsions, and cardio-toxicity). For example: lidocaine (as well as other Na _v blockers) is known to relieve pain but can only be used as topical jelly or ointment; other administration routes can be fatal. In the last decades a large number of programs have been put in place by pharmaceutical companies to develop a second generation of Na _v inhibitors which target specific subunits with a high degree of specificity in order to develop more effective and better tolerated compounds for pain management. However, the development of a novel therapeutic selectively targeting Na _v 1.7 represents a challenge due to the high degree of amino acid sequence homology among the different Na _v subtypes. In addition, Na _v s are complex structures which have different dynamic states (closed, open, or inactivated). Nevertheless, large bodies of evidence concerning the sequences containing the lower homology between the subtypes of human Na _v channels, regions containing mutations causing CIP and interaction regions for pore blockers (i.e., small molecules and natural toxins) are now available to help the development of new inhibitors.

Biologies such as monoclonal antibodies (mAbs) have the great advantage of being extremely selective, however, the majority of mAbs developed against ion channels so far are binders lacking functional efficacy. The difficulty to produce functional mAbs is because these targets have multiple transmembrane regions and small extracellular loops, are difficult to purify (unstable antigens) and the functional epitope is generally inside the pore. However, the tarantula toxin ProTx-ll a 30 amino acid peptide which is a highly selective Na _v 1.7 inhibitor does not bind the Na _v 1.7 pore region, demonstrating that targeting molecular regions outside the pore could be a successful strategy in identifying subtype-selective compounds.

Data seem to suggest that Na _v 1 7-mediated antinociception is obtained via both peripheral and central mechanisms. However, since the majority of therapeutics, biologies and small molecule, do not penetrate the blood-brain barrier (BBB) sufficiently to induce pharmacologically meaningful effects on central nervous system (CNS) targets, a successful therapeutic could be developed by increasing brain penetration.

There is need to be provided with Na _v 1.7 inhibitors that not only bind to the Na _v 1.7 but also lowers or inhibits its biological activity in mediating pain and halt or reduce cancer progression. In some embodiments, there is a need to be provided with Na _v 1.7 inhibitors which are capable of pene

BRIEF SUMMARY

In a first aspect, the present disclosure provides an immunogen for making a specific antibody to a peptide epitope, the immunogen comprising a single-domain antibody having at least one complementary determining region (CDR), wherein the at least one CDR has been modified to include the peptide epitope. In an embodiment, the at least one CDR has been modified by deleting at least one amino acid residue. In another embodiment, the single-domain antibody comprises a first CDR, a second CDR and a third CDR. In a further embodiment, the third CDR has been modified to include the peptide epitope. In an embodiment, the antibody heavy chain is derived from a camelid antibody heavy chain. In a further embodiment, the camelid antibody heavy chain is derived from a FC5 single-domain antibody. In yet a further embodiment, the immunogen has the amino acid formula (I):

NH ₂ - SS-FR1 -CDR1 -FR2-CDR2-FR3-CDR3-FR4 - COOH wherein SS is an optional signal sequence; FR1 has the amino acid sequence of SEQ ID NO: 60; CDR1 is the first CDR; FR2 has the amino acid sequence of SEQ ID NO: 61 ; CDR2 is the second CDR; FR3 has the amino acid sequence of SEQ ID NO: 62; CDR3 is the third CDR; FR4 has the amino acid sequence of SEQ ID NO: 63; and is an amide bond. In an embodiment, CDR1 has the amino acid sequence of SEQ ID NO:64 or has been modified to include the peptide epitope. In yet another embodiment, CDR2 has the amino acid sequence of SEQ ID NO: 65 or has been modified to include the peptide epitope. In still a further embodiment, CDR3 has the amino acid sequence of SEQ ID NO: 66 or has been modified to include the peptide epitope. In an embodiment, the immunogen has SS which can comprise or consist essentially of, for example, amino acid sequence of SEQ ID NO: 2 or 67. In another embodiment, the immunogen lacks SS and can comprise or consist essentially of the amino acid sequence of SEQ ID NO: 3 or 68.

According to a second aspect, the present disclosure also provides an immunogen for making an antibody specific to a Nav1.7 polypeptide. The immunogen having the amino acid sequence of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 67 or 68, be an immunogenic variant of the amino acid sequence of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 67 or 68 or be an immunogenic fragment of amino acid sequence of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 67 or 68.

According to a third aspect, the present disclosure provides the immunogen as described herein for selecting an antibody specific for the peptide epitope as well as method using same to select an antibody specific for the peptide epitope. In some embodiments, the method comprises selecting the antibody having an affinity to the immunogen as described herein. In an embodiment, the immunogen is intended for administration or is administered to an animal and a cell producing the antibody specific for the peptide epitope is intended to be selected or is selected from an organ of the animal having received the immunogen (the spleen, the lymph node or the bone marrow for example). In another embodiment, the selected cell is intended to be fused or is fused to a cancer cell to make an hybridoma. Alternatively or in combination, the immunogen is intended for contacting or is contacted with a library of cells producing antibodies and a cell producing the antibody specific for the peptide epitope is intended to be selected or is selected from the library. In such embodiment, the library of cells producing antibodies can be intended to be obtained or can be obtained from immunizing an animal with the immunogen. In a further embodiment, the affinity and/or specificity of the selected antibody is intended to be or is characterized with an immunological assay and/or surface plasmon resonance.

According to a fourth aspect, the present disclosure provides an antibody (which can be a monoclonal antibody) specifically recognizing a Na _v 1.7 polypeptide. The antibody of the present disclosure is specific for an epitope located on a loop 3 of a domain I (Dl) of the Na _v 1.7 polypeptide. The antibody is also capable of antagonizing the biological activity of the Na _v 1.7 polypeptide. In an embodiment, the Na _v 1.7 polypeptide is a human Na _v 1.7 polypeptide and as such, the antibody specifically recognizes the human Na _v 1.7 polypeptide. In another embodiment, the epitope is located on a first peptide comprising or consisting essentially of the following amino acid sequence:

KHKCFRNSLENNETLESIMNTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPEGYTCV KIGR

NPDYGY (SEQ ID NO: 1).

In a further embodiment, the antibody is a monoclonal antibody which can have, for example, a dissociation constant (K _D ) between 1 and 20 nM with the first peptide. In yet a further embodiments, the antibody is a single domain antibody, which can have, for example, a dissociation constant (K _D ) of about 1 nM to about 1000 mM. In still another embodiment, the epitope is located on a second peptide comprising or consisting essentially of the following amino acid sequence:

TLESEEDFRKYFYYLEGSKDALLCGFSTDS (SEQ ID NO: 5).

In still another embodiment, the epitope is located on a second peptide comprising or consisting essentially of the following amino acid sequence:

ALLCGFSTDSGQCPEGYTCVKIGRNPDYGY (SEQ ID NO: 6).

In such embodiments, the antibody can be a monoclonal antibody, a camelid antibody or is in a multivalent display format.

In a further embodiment, the antibody can have at least one complementary determining region (CDR) comprising or consisting essentially of an amino acid sequence of: GYTFTNYW (SEQ ID NO: 7), a variant thereof or a fragment thereof; INPSNGRA (SEQ ID NO: 8), a variant thereof or a fragment thereof; ARSPYGYYDY (SEQ ID NO: 9), a variant thereof or a fragment thereof; QSLLHSNGNTY (SEQ ID NO: 13), a variant thereof or a fragment thereof; KVS (SEQ ID NO: 14), a variant thereof or a fragment thereof; and/or SQITHVPLT (SEQ ID NO: 15), a variant thereof or a fragment thereof. In another embodiment, the antibody can have a first CDR having an amino acid sequence of GYTFTNYW (SEQ ID NO: 7), the variant thereof or the fragment thereof; a second CDR having an amino acid sequence of INPSNGRA (SEQ ID NO: 8), the variant thereof or the fragment thereof; and a third CDR having an amino acid sequence of ARSPYGYYDY (SEQ ID NO: 9) , the variant thereof or the fragment thereof. In still a further embodiment, the antibody of can have a heavy chain of amino acid sequence of SEQ ID NO: 12, a variant thereof or a fragment thereof. In another embodiment, the antibody can have a fourth CDR having an amino acid sequence of QSLLHSNGNTY (SEQ ID NO: 13), a variant thereof or a fragment thereof; a fifth CDR having an amino acid sequence of KVS (SEQ ID NO: 14), a variant thereof or a fragment thereof; and a sixth CDR having an amino acid sequence of SQITHVPLT (SEQ ID NO: 15), a variant thereof or a fragment thereof. In a further embodiment, the antibody can have a light chain of amino acid sequence of SEQ ID NO: 18, a variant thereof or a fragment thereof. In still another embodiment, the antibody can have the following amino acid sequences: GYTFTNYW (SEQ ID NO: 7), a variant thereof or a fragment thereof; INPSNGRA (SEQ ID NO: 8), a variant thereof or a fragment thereof; ARSPYGYYDY (SEQ ID NO: 9), a variant thereof or a fragment thereof; QSLLHSNGNTY (SEQ ID NO: 13), a variant thereof or a fragment thereof; KVS (SEQ ID NO: 14), a variant thereof or a fragment thereof; and SQITHVPLT (SEQ ID NO: 15), a variant thereof or a fragment thereof. In a further embodiment, the antibody can have a heavy chain of amino acid sequence of SEQ ID NO: 12, a variant thereof or a fragment thereof.

In still another embodiment, the antibody can have at least one a complementary determining region (CDR) comprising or consisting essentially of an amino acid sequence of: GFSLSRYN (SEQ ID NO: 75), a variant thereof or a fragment thereof; IWGGGST (SEQ ID NO: 76), a variant thereof or a fragment thereof; ARNGANWDWFAY (SEQ ID NO: 77), a variant thereof or a fragment thereof; QSLLYSSNQKNY (SEQ ID NO: 80), a variant thereof or a fragment thereof; WAS (SEQ ID NO: 81), a variant thereof or a fragment thereof; and/or QQYYSYPFT (SEQ ID NO: 82), a variant thereof or a fragment thereof. In another embodiment, the antibody can have a first CDR having an amino acid sequence of GFSLSRYN (SEQ ID NO: 75), the variant thereof or the fragment thereof; a second CDR having an amino acid sequence of IWGGGST (SEQ ID NO: 76), the variant thereof or the fragment thereof; and a third CDR having an amino acid sequence of ARNGANWDWFAY (SEQ ID NO: 77), the variant thereof or the fragment thereof. For example, the antibody can have a heavy chain of amino acid sequence of SEQ ID NO: 74 or 1 13, a variant thereof or a fragment thereof. In yet another embodiment, the antibody can have a fourth CDR having an amino acid sequence of QSLLYSSNQKNY (SEQ ID NO: 80), a variant thereof or a fragment thereof; a fifth CDR having an amino acid sequence of WAS (SEQ ID NO: 81), a variant thereof or a fragment thereof; and a sixth CDR having an amino acid sequence of QQYYSYPFT (SEQ ID NO: 82), a variant thereof or a fragment thereof. For example, the antibody can have a light chain of amino acid sequence of SEQ ID NO: 79 or 1 15, a variant thereof or a fragment thereof. In still another embodiment, the antibody can have the following amino acid sequences: GFSLSRYN (SEQ ID NO: 75), a variant thereof or a fragment thereof; IWGGGST (SEQ ID NO: 76), a variant thereof or a fragment thereof; ARNGANWDWFAY (SEQ ID NO: 77), a variant thereof or a fragment thereof; QSLLYSSNQKNY (SEQ ID NO: 80), a variant thereof or a fragment thereof; WAS (SEQ ID NO: 81), a variant thereof or a fragment thereof; and QQYYSYPFT (SEQ ID NO: 82), a variant thereof or a fragment thereof. In still another embodiment, the antibody can have a heavy chain of amino acid sequence of SEQ ID NO: 1 13, a variant thereof or a fragment thereof and a a light chain of amino acid sequence of SEQ ID NO: 1 15, a variant thereof or a fragment thereof. In yet another embodiment, the antibody can have at least one a complementary determining region (CDR) comprising or consisting essentially of an amino acid sequence of: GYTFTTYW (SEQ ID NO: 85), a variant thereof or a fragment thereof; INPSNGRA (SEQ ID NO: 86), a variant thereof or a fragment thereof; LRSLGYFDY (SEQ ID NO: 87), a variant thereof or a fragment thereof; QSLVHSNGNTY (SEQ ID NO: 90), a variant thereof or a fragment thereof; KVS (SEQ ID NO: 91), a variant thereof or a fragment thereof; and/or SQSTHVPYT (SEQ ID NO: 92), a variant thereof or a fragment thereof. In some embodiments, the antibody can have a first CDR having an amino acid sequence of GYTFTTYW (SEQ ID NO: 85), the variant thereof or the fragment thereof; a second CDR having an amino acid sequence of INPSNGRA (SEQ ID NO: 86), the variant thereof or the fragment thereof; and a third CDR having an amino acid sequence of LRSLGYFDY (SEQ ID NO: 87), the variant thereof or the fragment thereof. For example, the antibody can have a heavy chain of amino acid sequence of SEQ ID NO: 84 or 1 17, a variant thereof or a fragment thereof. In a further embodiment, the antibody can have a fourth CDR having an amino acid sequence of QSLVHSNGNTY (SEQ ID NO: 90), a variant thereof or a fragment thereof; a fifth CDR having an amino acid sequence of KVS (SEQ ID NO: 91), a variant thereof or a fragment thereof; and a sixth CDR having an amino acid sequence of SQSTHVPYT (SEQ ID NO: 92), a variant thereof or a fragment thereof. For example, the antibody can have a light chain of amino acid sequence of SEQ ID NO: 89 or 1 19, a variant thereof or a fragment thereof. In another embodiment, the antibody can have the following amino acid sequences: GYTFTTYW (SEQ ID NO: 85), a variant thereof or a fragment thereof; INPSNGRA (SEQ ID NO: 86), a variant thereof or a fragment thereof; LRSLGYFDY (SEQ ID NO: 87), a variant thereof or a fragment thereof; QSLVHSNGNTY (SEQ ID NO: 90), a variant thereof or a fragment thereof; KVS (SEQ ID NO: 91), a variant thereof or a fragment thereof; and SQSTHVPYT (SEQ ID NO: 92), a variant thereof or a fragment thereof. In still a further embodiment, the antibody can have a heavy chain of amino acid sequence of SEQ ID NO: 1 17, a variant thereof or a fragment thereof and a light chain of amino acid sequence of SEQ ID NO: 1 19, a variant thereof or a fragment thereof.

In still another embodiment, the antibody can have at least one a complementary determining region (CDR) comprising or consisting essentially of an amino acid sequence of: GFTFRSYA (SEQ ID NO: 95), a variant thereof or a fragment thereof; ISSGGST (SEQ ID NO: 96), a variant thereof or a fragment thereof; ARGYDGYYERIWYYAMDY (SEQ ID NO: 97), a variant thereof or a fragment thereof; QNVGTI (SEQ ID NO: 100), a variant thereof or a fragment thereof; SAS (SEQ ID NO: 101), a variant thereof or a fragment thereof; and/or QQYNTYPLT (SEQ ID NO: 102), a variant thereof or a fragment thereof. In some embodiments, the antibody can have a first CDR having an amino acid sequence of GFTFRSYA (SEQ ID NO: 95), the variant thereof or the fragment thereof; a second CDR having an amino acid sequence of ISSGGST (SEQ ID NO: 96), the variant thereof or the fragment thereof; and a third CDR having an amino acid sequence of ARGYDGYYERIWYYAMDY (SEQ ID NO: 97), the variant thereof or the fragment thereof. For example, the antibody can have a heavy chain of amino acid sequence of SEQ ID NO: 94 or 121 , a variant thereof or a fragment thereof. In additional embodiments, the antibody can have: a fourth CDR having an amino acid sequence of QNVGTI (SEQ ID NO: 100), a variant thereof or a fragment thereof; a fifth CDR having an amino acid sequence of SAS (SEQ ID NO: 101), a variant thereof or a fragment thereof; and a sixth CDR having an amino acid sequence of QQYNTYPLT (SEQ ID NO: 102), a variant thereof or a fragment thereof. For example, the antibody can have a light chain of amino acid sequence of SEQ ID NO: 99 or 123, a variant thereof or a fragment thereof. In still another embodiment, the antibody can have the following amino acid sequences: GFTFRSYA (SEQ ID NO: 95), a variant thereof or a fragment thereof; ISSGGST (SEQ ID NO: 96), a variant thereof or a fragment thereof; ARGYDGYYERIWYYAMDY (SEQ ID NO: 97), a variant thereof or a fragment thereof; QNVGTI (SEQ ID NO: 100), a variant thereof or a fragment thereof; SAS (SEQ ID NO: 101), a variant thereof or a fragment thereof; and QQYNTYPLT (SEQ ID NO: 102), a variant thereof or a fragment thereof. In a further embodiment, the antibody can have a heavy chain of amino acid sequence of SEQ ID NO: 121 , a variant thereof or a fragment thereof and a light chain of amino acid sequence of SEQ ID NO: 123, a variant thereof or a fragment thereof.

In another embodiment, the antibody can have at least one complementary determining region (CDR) comprising or consisting of an amino acid sequence of GFAFSSAP (SEQ ID NO: 39), a variant thereof or a fragment thereof; IESDQDHTI (SEQ ID NO: 40), a variant thereof or a fragment thereof; and/or QKRGEKKT (SEQ ID NO: 41), a variant thereof or a fragment thereof. In still another embodiment, the antibody can have a first CDR having an amino acid sequence of SEQ ID NO: 39, the variant thereof or the fragment thereof; a second CDR having an amino acid sequence of SEQ ID NO: 40, the variant thereof or the fragment thereof; and a third CDR having an amino acid sequence of SEQ ID NO: 41 , the variant thereof or the fragment thereof. In another embodiment, the antibody can have a heavy chain of amino acid sequence of SEQ ID NO: 38, a variant thereof or a fragment thereof. In another embodiment, the antibody can have the following amino acid sequences: GFAFSSAP (SEQ ID NO: 39), a variant thereof or a fragment thereof; IESDQDHTI (SEQ ID NO: 40), a variant thereof or a fragment thereof; and QKRGEKKT (SEQ ID NO: 41), a variant thereof or a fragment thereof. In another embodiment, the antibody can have a heavy chain of SEQ ID NO: 38.

In yet another embodiment, the biological activity of the Na _v 1.7 polypeptide can be measured as the amplitude of current associated with the Na _v 1.7 polypeptide and the antibody can be capable of substantially reducing the amplitude of current associated with the Na _v 1.7 polypeptide. In a further embodiment, the biological activity of the Na _v 1.7 polypeptide can be measured as a normalized current associated with the Na _v 1.7 polypeptide and the antibody can be capable of substantially maintaining or reducing the normalized current associated with the Na _v 1.7 polypeptide. In still a further embodiment, the biological activity of the Na _v 1.7 polypeptide can be measured in an hyperalgesia animal model and the antibody can be capable of substantially increasing the latency of paw withdrawal and/or increasing the percentage of the maximal possible effect in the hyperalgesia animal model.

According to a fifth aspect, the present disclosure provides a chimeric protein comprising the antibody described herein and a moiety, preferably an antibody, capable of transmigrating the blood-brain barrier, crossing the spinal cord and/or entering the central nervous system.

According to a sixth aspect, the present disclosure provides a pharmaceutical composition comprising the antibody described herein or the chimeric protein described herein and a pharmaceutically acceptable carrier. In an embodiment, the pharmaceutical composition is formulated or intended for intravenous administration.

According to a seveth aspect, the present disclosure provides the antibody described herein, the chimeric protein described herein or the pharmaceutical composition described herein for alleviating the symptoms of pain and/or managing pain in a subject in need thereof. The present disclosure also provides the use of the antibody described herein, the chimeric protein described herein or the pharmaceutical composition described herein for alleviating the symptoms of pain and/or managing pain in a subject in need thereof. The present disclosure further provides the use of the antibody described herein, the chimeric protein described herein or the pharmaceutical composition described herein for the manufacture of a medicament for alleviating the symptoms of pain and/or managing pain in a subject in need thereof. In an embodiment, the pain is a neuropathic pain. In another embodiment, the pain is a chronic pain. In still another embodiment, the subject is a human subject. In yet another embodiment, the subject is an animal subject.

According to a eigth aspect, the present disclosure provides a method of alleviating the symptoms of pain and/or managing pain in a subject in need thereof, the method comprising administering an effective amount of the antibody described herein, the chimeric protein described herein or the pharmaceutical composition described herein to the subject under conditions so as to alleviate the symptoms of pain and/or manage pain. In an embodiment, the pain is a neuropathic pain. In another embodiment, the pain is a chronic pain. In still another embodiment, the subject is a human subject. In yet another embodiment, the subject is an animal subject.

According to a ninth aspect, the present disclosure provides an antibody defined herein, a chimeric protein defined herein or a pharmaceutical composition defined herein for treating or alleviating the symptoms of an hyperproliferative disease in a subject in need thereof. The present disclosure also provides using and antibody defined herein, a chimeric protein defined herein or a pharmaceutical composition defined herein for treating or alleviating the symptoms of an hyperproliferative disease in a subject in need thereof. The present disclosure further provides using an antibody defined herein, a chimeric protein defined herein or a pharmaceutical composition defined herein for the manufacture of a medicament for treating or alleviating the symptoms of an hyperproliferative disease in a subject in need thereof. In an embodiment, the hyperproliferative disease can be cancer. In some additional embodiments, the subject can have a cancerous tumor. In specific embodiments, the cancerous tumor comprises at least one cancerous cell expressing a Na _v 1.7 polypeptide. In an embodiment, the the subject is a human subject. In another embodiment, the subject is an animal subject.

According to a tenth aspect, the present disclosure provides a method of treating or alleviating the symptoms of an hyperproliferative disease in a subject in need thereof. Broadly, the method comprising administering an effective amount of antibody defined herein, a chimeric protein defined herein or a pharmaceutical composition defined herein to the subject under conditions so as to treat or alleviate the symptoms of the hyperproliferative disease. In an embodiment, the hyperproliferative disease can be cancer. In some additional embodiments, the subject can have a cancerous tumor. In specific embodiments, the cancerous tumor comprises at least one cancerous cell expressing a Na _v 1.7 polypeptide. In an embodiment, the the subject is a human subject. In another embodiment, the subject is an animal subject.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the nature of the invention, reference will now be made to the accompanying drawings, showing by way of illustration, a preferred embodiment thereof, and in which:

Figure 1 provides a schematic representation of the Na _v 1.7 channel.

Figures 2A to C provide a 3D view of V _h H FC5 lacking 6 amino-acid residues from the inner region of its CDR3. (Figure 2A) shows the molecular model of V _h H FC5 lacking 6 amino-acid residues from the inner region of its CDR3. Retained segments of CDR3 are highlighted in black. The molecular model of FC5 V _h H was constructed as described below. (Figure 2B) provides a modeled three-dimensional (3D) structure of DIE3 loop including two disulfide bonds (labeled). (Figure 2C) provides the 3D-location of DIE3 loop (black) relative to cell membrane (planes) shown on the cryo-EM structure (PDB ID 5XSY) of the homologous electric eel Na _v 1.4 channel a subunit complexed with b-1 subunit (bI-lgL).

Figure 3 provides the results of a Western blot showing the expression of hNa _v 1.7 in transfected cells. Results are shown for control cells transfected with a control plasmid (HEK293 Neo) and cells transfected with the plasmid for Na _v 1.7 (hNa _v 1.7-HEK293 or SNC9) for both the Na _v 17 polypeptide (top panel) and GADPH control polypeptide (lower panel).

Figures 4A to 4C show that HEK293 cells stably transfected with the SNC9A plasmid (hNa _v 1.7- HEK293 cells) have significantly larger Na ⁺ currents (n=8) compared to non-transfected HEK293 cells (Control HEK293 cells; n=8). (Figure 4A) shows the current-voltage relationship of the peak current amplitude (left panel) and current density (right panel) in hNa _v 1.7-HEK293 cells (open circles) and non-transfected HEK293 cells (filled circles). (Figure 4B) shows current traces (lower trace) generated by voltage steps (from -85 to +60 mV; 5mV increments; upper trace). (Figure 4C) shows the activation (filled circles) and inactivation (diamonds) curves for Na+ currents recorded from hNa _v 1.7-HEK293 cells. Half-maximal activation occurred at -28.82 ± 4.33 mV (n=8). Half-maximal inactivation occurred at -63.47 ± 3.98 mV (n=8). These values correspond to literature values for hNa _v 1.7 channels expressed in HEK293 cells. Statistics shown as mean ± SEM.

Figure 5 provides the results of an ELISA of purified mAbs on FC5DIE3IR recombinant protein and evaluation of Bmax and apparent affinity for antibody 3A8 (·), 1 G5 (■), 1 H5 (x), 1 B6 (▼) and 2G1 1 (¨). Results are provided as the OD at 405 nm in function of the antibody concentration (provided on a logarithmic scale in nM).

Figures 6A to F show the effect of the anti-hNa _v 1.7 mAbs 3A8 and 1G5 on the amplitude of the Na _v 1.7 currents recorded at resting-closed state using patch-clamp whole-cell technique in HEK293 cells overexpressing the hNa _v 1.7 channels. (Figure 6A) Examples of hNa _v 1.7 current recordings in hNa _v 1.7-HEK293 cells in control and in response to the application of 3A8 (left; voltage step to -15 mV) and 1 G5 (right; voltage step to -20 mV) respectively. 3A8 at concentrations of 1 mM and 2 pM reduced the hNa _v 1.7 currents in a dose dependent manner, whereas 1 G5 (1 pM) had no effect. (Figure 6B) Time course of the effect of 3A8 (1 pM; open circles) and 1 G5 (1 pM; filled circles) on the hNa _v 1.7 currents. 1 G5 did not reduce hNa _v 1.7 currents, whereas 3A8 reduced the current of approx. 13%. The onset time of the mAb effect was estimated by curve-fitting the 3A8 induced current decay with an exponential function (solid line; T=176 sec). (Figure 6C) Normalized current-voltage (l-V) relationships for hNa _v 1.7 currents in control (open circles) and after the application of 3A8 (1 pM filled circles; 2 pM, open squares). Note the dose dependent decrease in the current amplitudes and the shift of the current onset to more hyperpolarized voltages. (Figure 6D) Activation and steady state fast inactivation curves for hNa _v 1.7 in control (open circles; curve fitting, solid lines) and after application of 3A8 (1 pM, filled circles; curve fitting, dotted lines). Curve fitting of the experimental data to Boltzmann sigmoidal curves, allowed the estimation of half effect voltages. (Figure 6E) Summary of the effects of different mAbs on hNa _v 1.7 currents. At the concentration of 1 pM, 3A8 was able to significantly reduce the Na _v currents (8.2% ± 1.7; n= 7), while 1 G5 did not change the amplitude of the currents (0.77% ± 1.34,_n=12). 3A8 at 2 mM reduced the Na _v currents of 16.0% ± 2.1 (n=5). (Figure 6F) Dose-response curve of the effect of 3A8 on hNa _v 1.7 currents. Results are provided as the percentage of Na _v 1.7 inhibition as a function of the concentration of the antibody (in mM, logarithmic scale). The IC ₅ o for 3A8 was measured to be 0.9 ± 0.2 mM.

Figures 7A to F show the effect of the anti-hNa _v 1.7 mAb 1 B6 on the amplitude of the Na _v 1.7 currents recorded at resting-closed state using patch-clamp whole-cell technique in HEK293 cells overexpressing the hNa _v 1.7 channels. (Figure 7A) Examples of hNa _v 1.7 current recordings in hNa _v 1.7-HEK293 cells in control and in response to the application of 1 B6 (voltage step to -15 mV). 1 B6 at concentrations of 2 mM reduced the hNa _v 1.7 currents. (Figure 7B) Time course of the effect of 1 B6 (2 mM) on the hNa _v 1.7 currents. The onset time of the mAb 1 B6 effect was estimated by curve-fitting the 1 B6 induced current decay with an exponential function (solid line; T=89 sec). (Figure 7C) Normalized current-voltage (l-V) relationships for hNa _v 1.7 currents in control (filled squares) and after the application of 1 B6 (1 mM filled circles; 2 mM, filled triangles). (Figure 7D) Activation and steady state fast inactivation curves for hNa _v 1.7 in control (filled squares; curve fitting, solid lines) and after application of 1 B6 (1 mM, filled circles; curve fitting, dotted lines). Curve fitting of the experimental data to Boltzmann sigmoidal curves, allowed the estimation of half effect voltages. (Figure 7E) Summary of the effects of different mAbs on hNa _v 1.7 currents. At the concentration of 1 mM, 1 B6 was able to significantly reduce the Na _v currents (27.7% ± 2.9; n= 20), while 1 G5 did not change the amplitude of the currents (0.77% ± 1.34, n=12). 1 B6 at 2 mM reduced the Na _v currents of 40.3%± 5.7 (n=12). (Figure 7F) Dose-response curve of the effect of 1 B6 on hNa _v 1.7 currents. Results are provided as the percentage of Na _v 1.7 inhibition as a function of the concentration of the antibody (in mM, logarithmic scale). The IC ₅ o for 1 B6 was measured to be 1.02 ± 0.19 mM.

Figures 8A to F show the effect of the anti-hNa _v 1.7 mAb 2G1 1 on the amplitude of the Na _v 1.7 currents recorded at resting-closed state using patch-clamp whole-cell technique in HEK293 cells overexpressing the hNa _v 1.7 channels. (Figure 8A) Examples of hNa _v 1.7 current recordings in hNa _v 1.7-HEK293 cells in control and in response to the application of 2G1 1 (voltage step to -15 mV). 2G1 1 at concentrations of 2 pM reduced the hNa _v 1.7 currents. (Figure 8B) Time course of the effect of 2G1 1 (2 mM) on the hNa _v 1.7 currents. The onset time of the mAb 2G1 1 effect was estimated by curve-fitting the 2G1 1 induced current decay with an exponential function (solid line; T=57 sec). (Figure 8C) Normalized current-voltage (l-V) relationships for hNa _v 1.7 currents in control (filled squares) and after the application of 2G11 (1 mM filled circles; 2 mM, filled triangles). (Figure 8D) Activation and steady state fast inactivation curves for hNa _v 1.7 in control (filled squares; curve fitting, solid lines) and after application of 2G1 1 (1 mM, filled circles; curve fitting, dotted lines). Curve fitting of the experimental data to Boltzmann sigmoidal curves, allowed the estimation of half effect voltages. (Figure 8E) Summary of the effects of different mAbs on hNa _v 1.7 currents. At the concentration of 1 mM, 2G1 1 was able to significantly reduce the Na _v currents (22.8% ± 3.7; n=14), while 1 G5 did not change the amplitude of the currents (0.77% ± 1.34, n=12). 2G11 at 2 mM reduced the Na _v currents of 35.4% ± 6.1 (n=15). (Figure 8F) Dose-response curve of the effect of 2G1 1 on hNa _v 1.7 currents. Results are provided as the percentage of Na _v 1.7 inhibition as a function of the concentration of the antibody (in mM, logarithmic scale). The IC ₅₀ for 2G1 1 was measured to be 1.06 ± 0.29 mM.

Figures 9A to F show the effect of the anti-hNa _v 1.7 mAb 1 H5 on the amplitude of the Na _v 1.7 currents recorded at resting-closed state using patch-clamp whole-cell technique in HEK293 cells overexpressing the hNa _v 1.7 channels. (Figure 9A) Examples of hNa _v 1.7 current recordings in hNa _v 1.7-HEK293 cells in control and in response to the application of 1 H5 (voltage step to -15 mV). 1 H5 at concentrations of 1 mM reduced the hNa _v 1.7 currents. (Figure 9B) Time course of the effect of 1 H5 (1 mM) on the hNa _v 1.7 currents. The onset time of the mAb 1 H5 effect was estimated by curve-fitting the 1 H5 induced current decay with an exponential function (solid line; T=53 sec). (Figure 9C) Normalized current-voltage (l-V) relationships for hNa _v 1.7 currents in control (filled squares) and after the application of 1 H5 (1 mM filled circles; 2 mM, gray filled circles). (Figure 9D) Activation and steady state fast inactivation curves for hNa _v 1.7 in control (filled squares; curve fitting, solid lines) and after application of 1 H5 (1 mM, filled circles; curve fitting, dotted lines). Curve fitting of the experimental data to Boltzmann sigmoidal curves, allowed the estimation of half effect voltages. (Figure 9E) Summary of the effects of different mAbs on hNa _v 1.7 currents. At the concentration of 1 mM, 1 H5 was able to significantly reduce the Na _v currents (38.4% ± 4.8; n=10), while 1 G5 did not change the amplitude of the currents (0.77% ± 1.34, n=12). 1 H5 at 2 mM reduced the Na _v currents of 36.7% ± 3.2 (n=14). (Figure 9F) Dose-response curve of the effect of 1 H5 on hNa _v 1.7 currents. Results are provided as the percentage of Na _v 1.7 inhibition as a function of the concentration of the antibody (in mM, logarithmic scale). The IC ₅₀ for 1 H5 was measured to be 0.56 ± 0.02 mM.

Figure 10 shows the ability of mAbs 3A8 and 11 E5 to mediate reversal of hyperalgesia in a chronic pain model. Results are shown as the latency in paw withdrawal (in secs, mean ± SEM of 4- 6 animals per group) in function of antibody tested and time (white bars: baseline; black bars: 4 hours after injection).

Figures 11A and B provide a time-course effect of mAb 3A8 and small molecule TC-N1752 on CFA-induced thermal hyperalgesia in rats. TC-N1752 is a selective blocker of human Na _v 1.7 channels (IC ₅₀ values are 0.17, 0.3, 0.4 and 1.1 mM at hNa _v 1.7, hNa _v 1.3, hNa _v 1.4 and hNa _v 1.5 respectively). (Figure 11A) provides the time-course and dose-response effects of an anti-hNa _v 1.7 mAb (3A8, 50 pg (□) and 100 (■) pg), the selective Na _v 1.7 channel inhibitor TC-N1752 (100 pg (D)) or 11 E5 (a negative control (·)) were tested using the Hargreaves Model of Hyperalgesia in rats. Nociceptive thermal behaviours were measured for up to 4 h post injection. Results are shown as the latency in paw withdrawal (in secs, mean ± SEM of 4-6 animals per group) in function of therapeutic tested and time (in hours). (Figure 11 B) provides the percentage of the effect on left paw withdrawal (% Effect) of 3A8 (50 (□) and 100 (■) pg), 11 E5 (a negative control (·)) and TC- N 1752(100 pg (D)) Results are show as the percentage the effect (%Effect) in function of time. The maximus effect for 3A8 50pg was 24.09±3.69 (n=5) and it was reached 2 hours after injection; for 3A8 100pg was 40.55±2.20 (n=5) after 2 hours from injection.

Figures 12A to C provide the time-course of the effect of 2G1 1 (Figure 12A), 1 H5 (Figure 12B) and 1 B6 (Figure 12C) on CFA-induced thermal hyperalgesia in rats. The data are plotted with those obtained with TC-N1752 (positive control) and 1 1 E5 (negative control). On the panels on the left, results are shown as the latency in paw withdrawal (in secs, mean ± SEM of 4-6 animals per group) as a function of therapeutic tested and time (in hours). On the panels on the right, results are expressed as percentage effect on left paw withdrawal (% Effect). Results are shown as mean ± SEM of 4-6 animals per group. The maximum percentages of the effects (%Effect) were 27.98±7.17 for 2G1 1 (n=4, after 1 hour from injection), 35.23±0.88 for 1 H5 (n=4, after 2 hours from injection) and 25.96±3.69 for 1 B6 (n=4, after 4 hours of injection).

Figures 13A and B provide the in vivo effects of mAb 3A8 at a 30 mg/kg dose. (Figure 13A) Time-course effect of mAb 3A8 (□) and a control (PBS, ·) injected by intravenous route on CFA-induced thermal hyperalgesia in rats. (Figure 13B) Effect of 3A8 expressed as percentage of Maximum Possible Effect (%MPE) 4 h after intraplantar injection of test compounds. Results are shown as mean ± SEM of 4 animals per group.

Figures 14A to F show the effects of anti-hNa _v 1.7 mAbs in a mouse model of Na _v 1.7-mediated pain based on subcutaneous injection of OD1. (Figure 14A) Concentration-response effect of subcutaneous injection of OD1 (0.5-1 mM) into the dorsal side of the hind paw of mice. Spontaneous pain behaviours were evidenced by licking, flinching, lifting and shaking of the injected paw. Administration of 100 pg of 3A8 (Figure 14C), 1 H5 (Figure 14D) and 2G11 (Figure 14E) but not 11 E5 (Figure 14B; negative control) or 1 B6 (Figure 14F) reversed spontaneous pain behaviours in mice evoked by OD1. Selective Na _v 1.7 channel inhibitor TC-N1752 (100 pg) was used as positive control. Anti-hNa _v 1.7 mAbs and TC-N1752 were injected 60 minutes prior to OD1 injection. Results are shown as mean ± SEM of 3 animals per group.

Figures 15A to J show the mapping, as tested by ELISA, of the antigen binding domain of the anti-hNa _v 1.7 mAbs 3A8, 1 B6, 2G1 1 and 1 H5. (Figure 15A) Seven overlapping antigen-peptides derived from DIE3IR were generated. (Figure 15B) Binding of mAbs 3A8, 1 B6, 2G1 1 and 1 H5 to peptides FC5DIE3IR, pep#40a (P40a) and pep#40b (P40b). (Figure 15C and Figure 15D) show the mAb 3A8 binding to peptides FC5DIE3IR, pep#1 (P1), pep#2 (P2) and pep#3 (P3). (Figure 15E) shows the mAb 1 B6 binding to peptides FC5DIE3IR, pep#1 (P1), pep#2 (P2) and pep#3 (P3). (Figures 15F and Figure 15G) show the mAb 1 H5 binding to peptides FC5DIE3IR, pep#1 (P1), pep#2 (P2) and pep#3 (P3). (Figures 15H and Figure 151) show the binding of mAbs 3A8, 1 B6 and 2G1 1 to pep#2a (P2a) and pep#2b (P2b), respectively. (Figure 15J shows the summary of the mAb binding properties of 3A8 (with respect to the DIE3IR peptide of SEQ ID NO: 1 , the P40a peptide of SEQ ID NO: 105, the P2 peptide of SEQ ID NO: 5 and the P2a peptide of SEQ ID NO: 103), 1 B6 (with respect to the DIE3IR peptide of SEQ ID NO: 1 , the P40a peptide of SEQ ID NO: 105 and the P2 peptide of SEQ ID NO: 5), 2G1 1 (with respect to the DIE3IR peptide of SEQ ID NO: 1 and the P40a peptide of SEQ ID NO: 105) and 1 H5 (with respect to the DIE3IR peptide of SEQ ID NO: 1 and the P3 peptide of SEQ ID NO: 6). The shaded area represent the peptide sequence in wich the epitope resides per each mAbs.

Figure 16 shows the position of the peptides spanning DIE3IR domain FRKYFY (SEQ ID NO: 107) and NTLESEED (SEQ ID NO :108) on FC5DIE3IR. The rectangles represent the peptides FRKYFY (SEQ ID NO: 107) and NTLESEED (SEQ ID NO: 108). Deuteration was measured across all three time points. Arrows indicate insertion of 70 aa DIE3IR peptide (KHKCFRNSLENNETLESIMNTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPEGYTC VKIG RNPDYGY, SEQ ID NO: 1). A redundancy of 2.3 was achieved with 38 peptides covering 74% of the sequence.

Figures 17A and B display the binding kinetics for peptides spanning DIE3IR domain FRKYFY (SEQ ID NO: 107) and NTLESEED (SEQ ID NO :108), respectively. Normalized deuteration plotted as a function of time for (Figure 17A) Phe129-Tyr134 (FRKYFY; SEQ ID NO: 107) and (Figure 17B) residues Asn121-Asp128 (NTLESEED; SEQ ID NO: 108). HDX-MS results are also shown for FC5DIE31 R unbound (black circles). Deuteration was measured in triplicate. Error bars represent ± 1 SD.

Figures 18A and B show the effects of peptide antigen fragments pep#2 (P2) and pep#3 (P3) on 3A8-mediated reversal of hyperalgesia. (Figure 18A) Peptide fragments raised against the binding epitope of the anti-hNa _v 1.7 mAb 3A8 (P2 (o)) or against an non-related region of 3A8 (P3 (·)) were co-injected with 3A8 by intraplantar route (1 :5, 3A8 to peptide ratio). Reversal of hyperalgesia was also tested with 3A8 alone (75 pg (□)), and with a negative control (11 E5, 50 pg (■)). Results are shown as mean ± SEM. (Figure 18B) Effect of P2 and P3 on 3A8-induced reversal of hyperalgesia expressed as percentage of maximum possible effect (%MPE) 4hs after intraplantar injection of test compounds. Results are shown as mean ± SEM of 2-5 animals per group. Figures 19A and B show the results of Biacore T200 SPR analysis of FC5 (control; Figure 19A) and FC5DIE3IR (Figure 19B) binding at 1 mM to captured mAb 3A8. The sensorgrams showed that 3A8 bound specifically FC5DIE3IR and did not bind FC5.

Figures 20A to H show the results of Surface Plasmon Resonance (SPR) analysis to determine the binding affinity of FC5DIE3IR to Fc-captured mAb 3A8 (Figure 20A and B), 2G11 (Figure 20C and D), 1 B6 (Figure 20E and F) and 1 H5 (Figure 20G and H) using Single Cycle Kinetics (SCK). Fc captured mAb 3A8, 2G11 and 1 B6 showed strong and specific binding to FC5DIE3IR with K _D s (Figure 20B, 20D, 20F and 20H; see values Table 5). The mAb 1 H5 could not be fitted with the 1 :1 model, consequently K _D s, on-rate (ka (1/Ms)) and Rmax values could not be calculated.

Figures 21A and B show the effect of the recombinant chimeric human-mouse mAbs hFc- F233-3A8 (Figure 21 A) and hFc-F236-1 B6 (Figure 21 B) at the concentration of 2 mM on the amplitude of the Na _v 1.7 currents recorded at resting-closed state using patch-clamp whole-cell technique in HEK293 cells overexpressing the hNa _v 1.7 channels.

Figures 22A and B show the expression of hNa _v 1.7 in cancer cell lines. (Figure 22A) Western blot showing expression of hNa _v 1.7 (~ 230 kDa) in the following human cancer cell lines: DU- 145 (model of prostate cancer), Jurkat (model of leukemia, LN18 (model of glioblastoma), PC-3 (model of prostate cancer), SKOV-3 (model of ovarian cancer), U87MG (model of glioblastoma) and U87Mgvlll (model of glioblastoma). HEK293 cells overexpressing hNa _v 1.7 were used as positive control. Note that Na _v 1.7 was absent only in the PC-3 prostate cancer cell line. (Figure 22B) Western blot showing expression of hNa _v 1.7 (~130 kDa) in the human prostate cancer cell lines Capan-1 , Bxpc3, Mia-Paca2 and Panel . Actin was used as reference for protein expression.

Figures 23A and B show hNa _v 1.7 currents recorded using whole cell patch clamp in glioblastoma U87MG and ovarian cancer SKOV-3 cells. (Figure 23A) Recording of hNa _v 1.7 currents in a glioblastoma U87MG cell in control (CTRL) conditions and after block of the Na ⁺ currents by tetrodotoxin (TTX, 0.5 mM). (Figure 23B) Recording of hNa _v 1.7 currents form an ovarian cancer SKOV-3 cell in control (CTRL) and after application of 1 H5 (1 mM). MAb 1 H5 reduces the current of ~50%.

Figures 24A to D provide histrograms representing quantitative analysis of U87MG (Figures 24A and 24B), U8MGvlll (Figure 24C) and SKOV3 (Figure 24D) cell growth in soft agar by cellular metabolic activity measured using Alamar Blue. Cells were subjected to either DME (Control; negative control), TTX (0.5 mM; positive control), 1 B6 (1 mM), 1 H5 (1 mM) or 2G1 1 (1 mM). Each bar represents mean fluorescence units per well ± SEM; n=3. (Figure 24A) In glioblastoma U87MG cells, TTX (0.5 mM) reduced colony formation by 75%, mAb 1 H5 (1 mM) by 37%, while mAb 1 B6 (1 mM) did not affect colony formation. (Figure 24B) In a different experiment using U87MG, TTX (0.5 mM) reduced colony formation by 84% and mAb 2G1 1 (1 mM) by 60%. (Figure 24C) In glioblastoma U87MGvlll cells, TTX (0.5 mM) reduced colony formation of by 55%, 1 B6 (1 mM) by 53% and 1 H5 (1 mM) by 60%. (Figure 24D) In ovarian cancer SKOV-3 cells, TTX (0.5 mM) reduced colony formation by 70%, mAb 1 H5 (1 mM) by 57%, while mAb 1 B6 (1 mM) did not reduce colony formation.

Figures 25A and B provide the result of a phage ELISA of V _h H colonies obtained from a non- immunized llama. (Figure 25A) Phages were rescued from individual colonies, selected from the fourth round of panning and grown overnight at 37°C. Wells of a Nunc microtiter plate were coated with FC5DIE3IR and FC5 proteins at 5 pg/ml in PBS. After blocking, 100 mI of phage supernatants (from clones A-H) were added to the respective wells followed by incubation for 1 h at room temperature and addition of anti-M13-HRP conjugate. Binding was detected with TMB substrate and A ₄₅₀ was measured using an ELISA plate reader. As shown, six positive clones (Dl-A, Dl-B, Dl-C, Dl-D, Dl-E and Dl-H), having a signal of at least 3x background, were identified by phage-ELISA. (Figure 25B) The selected phage D did not bind to the V _h H FC5 or to another extracellular loop Dill of the hNa _v 1.7 channels grafted to FC5 scaffold (protein FC5DIIIDIR) showing its specificity for FC5DIE3IR.

Figures 26A to F show the effect of the application of V _H HS on the amplitude of the Na _v 1.7 currents recorded using patch-clamp whole-cell technique in HEK293 cells overexpressing the hNa _v 1.7 channels. (Figure 26A) HNa _v 1.7 currents recorded in hNa _v 1.7-HEK293 cells in control and in response to the application of the V _H HS Dl-D (left; voltage step to -30mV) and Dl-B (right; voltage step to -20mV) respectively. Dl-D, applied at 1 and 3 mM concentrations reduced hNa _v 1.7 current in a dose dependent manner, whereas Dl-B (1 mM) had no reducing effect. (Figure 26B) Recordings of normalized hNa _v 1.7 currents and effect of the application of 1 mM Dl-D (open circles) and 1 mM Dl-B (filled circles) V _H HS. In a 500 sec recording, the Dl-B did not reduce hNa _v 1.7 current, whereas Dl-D reduced the current to approx. 91 % of control (dashed line). Exponential curve-fitting of the Dl-D induced current decay (solid line) was used to estimate the onset time of the V _h H effect. In this experiment, the onset value was 150 sec. (Figure 26C) Normalized current- voltage (l-V) relationships for hNa _v 1.7 HEK293 expressing cells in control (open circles) and after application of Dl-D at 1 (filled circles) and 2 (open squares) mM showing the dose dependent decrease in currents. (Figure 26D) Activation and steady state fast inactivation curves for hNa _v 1.7 in control (open circles, solid lines) and after application of 1 mM Dl-D (filled circles, dotted lines). Curve fitting of the experimental data to Boltzmann sigmoidal curves, allowed the estimation of half effect voltages. (Figure 26E) Effect of the application of V _H HS on the amplitude of the Na _v 1.7 currents recorded using patch-clamp whole-cell technique in hNa _v 1.7-HEK293 cells. The Na _v 1.7 currents were normalised to 100% in control (absence of V _H HS). All V _H HS were tested at 1 mM. At 1 mM only Dl-D reduced the amplitude of the currents (11.7%±5.8, n=12). Dl-D significantly reduced the currents of 15.0%± 6.9 (n= 4) and 24.1 %± 5.6 (n= 7) at 2 mM and 3 mM respectively. (Figure 26F) Dose-response curve for Dl-D, from this curve, the IC ₅₀ of the effect of Dl-D on the hNa _v 1.7 current can be extrapolated to IC ₅₀ =1.48 ± 0.25 mM.

Figures 27A and B show the effect of the V _h H antibody Dl-D on a CFA-induced thermal hyperalgesia in rats (chronic pain model). (Figure 27A) shows the time-course of the effect of Dl-D (50 pg and 100 pg), 11 E5 (100 pg, negative control) and TC-N1752 (100 pg, positive control) on CFA-induced thermal hyperalgesia in rats. Nociceptive thermal behaviours were measured for up to 4 h post injection. Results are shown as the latency in paw withdrawal (in secs, meant SEM of 4-7 animals per group) as a function of therapeutic tested and time (in hours). (Figure 27B) shows the histogram of the effect of 11 E5, TC-N1752, Dl-D 50 pg and Dl-D 100 pg on hyperalgesia expressed as percentage of maximum possible effect (%MPE) after intraplantar injection of test compounds.

Figures 28A and B show the effects of V _h H Dl-D in a mouse model of Na _v 1.7-mediated pain obtained by plantal subcutaneous injection of OD1. (Figure 28A) shows that administration of 50 pg of Dl-D (·) reversed spontaneous pain behaviours evoked in mice by plantal injection of OD1 (■). (Figure 28B) shows that administration of 50 pg of a V _H HS not acting on the hNa _v 1.7 (A20.1 ; ·) did not have any effect on the spontaneous pain behaviours in mice evoked by OD1 (■). V _H HS were injected 60 minutes prior to OD1 injection. Results are shown as mean ± SEM of 2-3 animals per group.

Figures 29A and B provide the results of Surface Plasmon Resonance (SPR) showing the binding of V _h H Dl-D to immobilized FC5 and FC5DIE3IR. (Figure 29A) Test run at 50 and 250 nM. V _h H Dl-D showed clear binding to immobilized FC5DIE3IR (Figure 29B), with trace amounts of binding observed to the immobilized FC5 (Figure 29B).

Figures 30A and B provide the results of Surface Plasmon Resonance (SPR). (Figure 30A) shows the binding affinity of V _h H Dl-D to immobilized FC5DIE3IR using multiple cycle kinetics (MCK). (Figure 30B) shows that V _h H Dl-D binds to immobilized FC5DIE3IR with a K _D of approximately 2 mM and an observed R _max of approx. 700 RU, indicating a high level of activity of immobilized FC5DIE3IR.

Figures 31A and B provide the results of phage ELISA screening of V _h H antibodies obtained from an immunized llama. Phages were rescued from 48 individual colonies, selected from the fourth round of panning and grown overnight at 37°C. Wells of a Nunc microtiter plate were coated with FC5DIE3IR and FC5 (as a negative control) proteins at 5 pg/ml in PBS. After blocking, 100 pi of phage supernatants (from clones 1-48) were added to the respective wells (FC5 and FC5DIE3IR) followed by incubation for 1 h at room temperature and addition of anti- M13-HRP conjugate. After washing the wells, binding was detected with the addition of TMB substrate and A450 was measured using an ELISA plate reader. Results are provided as OD at 450 nm in function of V _h H colony number.

Figures 32A to D provide Surface Plasmon Resonance (SPR) results showing the binding affinity of V _H HS DI-4 (Figure 32A), DI-16 (Figure 32B), DI-28 (Figure 32C) and DI-48 (Figure 32D) to immobilized V _h H FC5 (left panel), FC5DIE3IR (center panel) and V _h H 2A3-H4 (right panel) were determined using single cycle kinetics (SCK) or steady state analysis.

DETAILED DESCRIPTION

Anti-Na _v 1.7 antibodies

The present disclosure provides for specific antibodies against the Na _v 1.7 polypeptide. The antibodies are considered “specific” to the Na _v 1.7 polypeptide because their affinity for the Na _v 1.7 polypeptide is higher than for other polypeptides (for example other polypeptides from the Na _v family). The antibodies of the present disclosure can be specific for the human Na _v 1.7 polypeptide (as described in GenBank Accession Number NP_002968 or the GeneCard ID Number GC02M166195). In an embodiment, when the antibody is a monoclonal antibody (mAb), its dissociation constant (K _D ) with respect to the Na _v 1.7 polypeptide is in the nanomolar range (1 - 1000 nM). In such embodiment, the monoclonal antibody can have a K _D between about 1 and about 100 nM, between about 1 and about 50 nM, between about 1 and about 25 nM, between about 1 and about 20 nM, between about 5 and about 15 nM or between about 7 nM and 12 nM with respect to the Na _v 1.7 polypeptide. In an embodiment, when the antibody is a single-domain antibody (sdAb), its dissociation constant (K _D ) with respect to the Na _v 1.7 polypeptide is in the nanomolar range (1 - 1000 nM) or the micromolar range (1 - 1000 mM). In such embodiment, the single domain antibody can have a K _D between about 1 and about 100 mM, between about 1 and about 50 pM, between about 1 and about 25 pM, between about 1 and about 5 pM or about 2 pM with respect to the Na _v 1.7 polypeptide. The present invention provides antibodies, including mAbs and sdAbs, that target the Na _v 1.7 channel, and specifically bind a Na _v 1 .7 polypeptide, wherein said polypeptide comprises a peptide in the extracellular loop 3 of domain Dl of the Na _v 1.7 channel, having a nanomolar or micromolar affinity.

The antibodies of the present disclosure are also capable of antagonizing the biological activity of the Na _v 1.7 polypeptide. As indicated above, the Na _v 1.7 polypeptide is a sodium channel involved in nociception (e.g., the sensation of pain). By antagonizing its biological activity, the antibodies of the present disclosure can thus be used to alleviate the symptoms of pain and/or to treat or alleviate the symptoms of an hyperproliferative disease, such as cancer. As shown herein, some the antibodies presented in the Examples are capable of reducing the amplitude of a current associated with the Na _v 1.7 polypeptide which in return is understood to limit or inhibit the flux of sodium ions through the Na _v 1.7 polypeptide. As such, in an embodiment, an antibody capable of reducing the amplitude of a current associated with the Na _v 1.7 polypeptide is considered to also be capable of antagonizing the biological activity of the Na _v 1.7 polypeptide. In an embodiment, the antibodies have a concentration response relationship (IC ₅₀ ) towards the Na _v 1.7 polypeptide in the nanomolar range (1 - 1000 nM) or in the micromolar range (1 - 1000pM). In another embodiment, the antibodies have a degree of maximum inhibition towards the Na _v 1.7 polypeptide between about 5-65% (for example, between 10-20%, between 30-45%, between 45-55% or between 40-65%). In some embodiments, the antibodies have a degree of maximum inhibition towards the Na _v 1.7 polypeptide of at least 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or 65%. In some embodiments, the antibodies have a degree of maximum inhibition of about 10-20%, for example16%, 30-45%, for example 40%, 45-55%, for example 55% or 40-65%, for example 62%. As also shown herein, some of the antibodies presented in the Examples are capable of maintaining or reducing a normalized current associated with the Na _v 1.7 polypeptide which in return is understood to limit or inhibit the flux of sodium ions through the Na _v 1.7 polypeptide. As such, in an embodiment, an antibody capable of maintaining or reducing the normalized current associated with the Na _v 1.7 polypeptide is considered to also be capable of antagonizing the biological activity of the Na _v 1.7 polypeptide. As further shown herein, some of the antibodies presented in the Examples are capable of increasing the latency of paw withdrawal in an hyperalgesia animal model (for example the Rat Hargreaves Model of Hyperalgesia) when compared to control treatment or antibodies. As such, in an embodiment, an antibody capable of increasing the latency of paw withdrawal in an hyperalgesia animal model is considered to also be capable of antagonizing the biological activity of the Na _v 1.7 polypeptide. As also shown herein, some of the antibodies presented in the Examples are capable of achieving a percentage of maximal possible effect of at least 5, 10, 15, 18, 20, 25, 30, 35, 40% or more. Consequently, in such an embodiment, an antibody capable of increasing the percentage of maximal possible effect is considered to also be capable of antagonizing the biological activity of the Na _v 1.7 polypeptide. As shown herein, some of the antibodies of the Examples are capable of reducing the cumulative nociceptive behavior by 5, 10, 15, 20, 25, 30, 35, 40, 45, 50% or more. Consequently, in such embodiment, an antibody capable of decreasing the cumulative nociceptive behavior is also considered to be specific of antagonizing the biological activity of the Na _v 1.7 polypeptide. As further shown herein, some of the antibodies presented in the Examples are capable of reducing colony formation of cancer cells by 5, 10, 15, 20, 25, 30, 35, 40, 45, 50% or more. Consequently, in such an embodiment, an antibody capable of decreasing colony formation of cancer cells is considered to also be capable of antagonizing the biological activity of the Na _v 1.7 polypeptide.

An“antibody”, as used in the context of the present disclosure, refers to an immunoglobulin polypeptide having at least three complementary determining regions (CDRs) and, in some embodiments, up to twelve CDRs. A“complementary determining region” refers to a region of the immunoglobulin polypeptide located in the variable parts of the polypeptide and involved in specifically binding the epitope. The combination of CDRs constitutes the paratope of the antibody.

Some naturally occurring immunoglobulins (for example mouse and human immunoglobulins) have a common core structure in which two identical light chains (about 24 kD) and two identical heavy chains (about 55 or 70 kD) form a tetramer. The amino-terminal portion of each chain is known as the variable (V) region and can be distinguished from the more conserved constant (C) regions of the remainder of each chain. Within the variable region of the light chain is a C- terminal portion known as the J region. Within the variable region of the heavy chain, there is a D region in addition to the J region. Most of the amino acid sequence variation in immunoglobulins is confined to three separate locations in the V regions known as hypervariable regions or complementarity determining regions (CDRs) which, as indicated above are directly involved in antigen binding. Proceeding from the amino-terminus, these regions are designated CDR1 , CDR2 and CDR3, respectively. The CDRs are held in place by more conserved framework regions (FRs). Proceeding from the amino-terminus, these regions are designated FR1 , FR2, FR3, and FR4, respectively. The locations of CDR and FR regions and a numbering system have been defined by Kabat et al.

Antibodies of the present disclosure include monoclonal antibodies. Antibodies which are specific for a single epitope on the the Na _v 1.7 polypeptide are considered as monoclonal antibodies (also referred to as mAbs). In some embodiments, monoclonal antibodies are produced from a single clone of an immune cell. Monoclonal antibodies can be produced by techniques known in the art, such as by using cell culture by fusing a myeloma cell to a spleen cell from a subject (such as a mouse or a human) which has been immunized with an antigen comprising the epitope of the Na _v 1.7 polypeptide (for example the FC5DIE3IR polypeptide as described in the Examples below). Monoclonal antibodies can also be obtained phage display by screening library of monoclonal antibodies using an antigen comprising the epitope of the Na _v 1.7 polypeptide (for example the FC5DIE3IR polypeptide as described in the Examples below). Additional techniques for making monoclonal antibodies include, but are not limited to single B cell culture, single cell amplification from B cell populations. Monoclonal antibodies of the present disclosure can be from various origins (e.g., mouse or human for example) and can include two identical light chains and two identical heavy chains, wherein each chain comprises three CDRs. Monoclonal antibodies can be from any isotype, including, but not limited to immunoglobulin A (IgA), IgD, IgE, IgG (including subtypes IgG 1 , lgG2 or lgG3) or IgM. Monoclonocal antibodies can be, in an embodiment, from the IgG isotype. A single-domain antibody (also referred to as a sdAb or a nanobody) is considered a monoclonal antibody as it is specific for a single epitope. Single-domain antibodies have a single heavy chain or light chain comprising three CDRs. In an embodiment, the single-domain antibody has a single heavy chain comprising three CDRs. Single domain antibodies can be found in nature, for example in camelids (V _h H antibodies) and in cartilaginous fishes (V _NA R fragments). Single domain antibodies can be engineered from fragmenting IgG antibodies (of human or mouse origin for example).

Antibodies of the present disclosure further include antibody derivatives, such as, for example chimeric and humanized antibodies. The expression “chimeric antibody” refers to an immunoglobulin which comprises regions from two different species. The expression “humanized antibody” refers to an immunoglobulin that comprises both a region derived from a human antibody or immunoglobulin and a region derived from a non-human antibody or immunoglobulin. The action of humanizing an antibody consists in substituting a portion of a non-human antibody with a corresponding portion of a human antibody. For example, a humanized antibody as used herein could comprise a non-human region variable region (such as a region derived from a murine antibody) capable of specifically recognizing the Na _v 1 .7 polypeptide and a human constant region derived from a human antibody. In another example, the humanized immunoglobulin can comprise a heavy chain and a light chain, wherein the light chain comprises a complementarity determining region derived from an antibody of non-human origin which binds to the Na _v 1 .7 polypeptide and a framework region derived from a light chain of human origin, and the heavy chain comprises a complementarity determining region derived from an antibody of non-human origin which binds to the Na _v 1 .7 polypeptide and a framework region derived from a heavy chain of human origin.

Antibodies of the present disclosure also include functional antibody fragments which comprise at least three CDRs, recognize the Na _v 1 .7 polypeptide and are capable of limiting the biological activity of the the Na _v 1 .7 polypeptide. As used herein, a“fragment” of an antibody (which can be, for example, a monoclonal antibody) is a portion of an antibody that is capable of specifically recognizing the same epitope as the full version of the antibody. Antibody fragments include, but are not limited to, the antibody light chain, antibody heavy chain, single chain antibodies, Fv, Fab, Fab' and F(ab') ₂ fragments. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For instance, papain or pepsin cleavage can be used to generate Fab or F(ab') ₂ fragments, respectively. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding the heavy chain of an F(ab') ₂ fragment can be designed to include DNA sequences encoding the CH1 domain and hinge region of the heavy chain. The antibodies of the present disclosure are specific for at least one epitope located on the 3 ^rd loop of domain I of the Na _v 1.7 polypeptide (located between amino acid residues 271 to 341 on the sequence shown in the GenBank Accession number NP_002968 or SEQ ID NO: 72). As shown on Figure 1 , the Na _v 1.7 polypeptide comprises of four domains (Dl, Dll, Dill and DIV), each containing six transmembrane helices (S1-S6) and 4 extracellular loops. S4 is known to be the voltage sensor of the channels. The epitope is preferably located on solvent-accessible region of the 3 ^rd loop of domain I of the Na _v 1.7 polypeptide. In an embodiment, the epitope is located on the DIE3IR peptide which comprises or consists essentially of the following amino acid sequence:

KHKCFRNSLENNETLESIMNTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPEGYTCV KIGR

NPDYGY (SEQ ID NO: 1).

In the context of the present disclosure, and especially when referring to the amino acid sequence of the DIE3IR peptide, the expression“consisting essentially of indicates that the epitope is necessarily located on the amino acid sequence of SEQ ID NO: 1 , but that additional, non-essential, amino acid residues can be added at the amino or the carboxyl end of the amino acid sequence of SEQ ID NO: 1 (for example when the DIE3IR peptide is used as an immunogen to identify/generate anti-Na _v 1.7 antibodies). In an embodiment, when the antibody is a monoclonal antibody, its dissociation constant (K _D ) with respect to the DIE3IR peptide is in the nanomolar range (1 - 1000 nM). In such embodiment, the monoclonal antibody can have a K _D between about 1 and about 100 nM, between about 1 and about 50 nM, between about 1 and about 25 nM, between about 5 and about 15 nM or between about 7 and 12 nM with respect to the DIE3IR peptide. In an embodiment, when the antibody is a single-domain antibody, its dissociation constant (K _D ) with respect to the DIE3IR peptide is in the micromolar range (1 - 1000 mM). In such embodiment, the single domain antibody can have a K _D between about 1 and about 100 pM, between about 1 and about 50 pM, between about 1 and about 25 pM, between about 1 and about 5 pM or about 2 pM with respect to the DIE3IR peptide.

In some embodiments, the epitope recognized by the anti-Na _v 1 .7 antibody is located on the peptide #2 which comprises or consists essentially of the following amino acid sequence:

TLESEEDFRKYFYYLEGSKDALLCGFSTDS (SEQ ID NO: 5).

In the context of the present disclosure, and especially when referred to the amino acid sequence of peptide #2, the expression“consisting essentially of indicates that the epitope is necessarily located on the amino acid sequence of SEQ ID NO: 5, but that additional, non- essential, amino acid residues can be added at the amino or the carboxy end of the amino acid sequence of SEQ ID NO: 5. In an embodiment, when the antibody is a monoclonal antibody, its dissociation constant (K _D ) with respect to peptide #2 is in the nanomolar range (1 - 1000 nM). In such embodiment, the monoclonal antibody can have a K _D between about 1 and about 100 nM, between about 1 and about 50 nM, between about 1 and about 25 nM, between about 5 and about 15 nM or between about 7 and 12 nM with respect to peptide #2.

In other embodiments, the epitope recognized by the anti-Na _v 1.7 antibody is located on the peptide #2a which comprises or consists essentially of the following amino acid sequence:

TLESEEDFRKYFYYLEGSKD (SEQ ID NO: 103).

In the context of the present disclosure, and especially when referring to the amino acid sequence of peptide #2a, the expression“consisting essentially of indicates that the epitope is necessarily located on the amino acid sequence of SEQ ID NO: 103, but that additional, non- essential, amino acid residues can be added at the amino or the carboxy end of the amino acid sequence of SEQ ID NO: 103. In an embodiment, when the antibody is a monoclonal antibody, its dissociation constant (K _D ) with respect to peptide #2a is in the nanomolar range (1 - 1000 nM). In such embodiment, the monoclonal antibody can have a K _D between about 1 and about 100 nM, between about 1 and about 50 nM, between about 1 and about 25 nM or between about 5 and about 15 nM with respect to peptide #2a. In other embodiments, the epitope recognized by the anti-Na _v 1.7 antibody is located on the peptide #2b which comprises or consists essentially of the following amino acid sequence:

YFYYLEGSKDALLCGFSTDS (SEQ ID NO: 104).

In the context of the present disclosure, and especially when referring to the amino acid sequence of peptide #2b, the expression“consisting essentially of indicates that the epitope is necessarily located on the amino acid sequence of SEQ ID NO: 104, but that additional, non- essential, amino acid residues can be added at the amino or the carboxy end of the amino acid sequence of SEQ ID NO: 104. In an embodiment, when the antibody is a monoclonal antibody, its dissociation constant (K _D ) with respect to peptide #2b is in the nanomolar range (1 - 1000 nM). In such embodiment, the monoclonal antibody can have a K _D between about 1 and about 100 nM, between about 1 and about 50 nM, between about 1 and about 25 nM or between about 5 and about 15 nM with respect to peptide #2b.

In other embodiments, the epitope recognized by the anti-Na _v 1.7 antibody is located on the peptide #3 which comprises or consists essentially of the following amino acid sequence:

ALLCGFSTDSGQCPEGYTCVKIGRNPDYGY (SEQ ID NO: 6).

In the context of the present disclosure, and especially when referring to the amino acid sequence of peptide #3, the expression“consisting essentially of indicates that the epitope is necessarily located on the amino acid sequence of SEQ ID NO: 6, but that additional, non- essential, amino acid residues can be added at the amino or the carboxy end of the amino acid sequence of SEQ ID NO: 6. In an embodiment, when the antibody is a monoclonal antibody, its dissociation constant (K _D ) with respect to peptide #3 is in the nanomolar range (1 - 1000 nM). In such embodiment, the monoclonal antibody can have a K _D between about 1 and about 100 nM, between about 1 and about 50 nM, between about 1 and about 25 nM or between about 5 and about 15 nM with respect to peptide #3.

In other embodiments, the epitope recognized by the anti-Na _v 1.7 antibody is located on the peptide #40a which comprises or consists essentially of the following amino acid sequence:

KHKCFRNSLENNETLESIMNTLESEEDFRKYFYYLEGSKD (SEQ ID NO: 105).

In the context of the present disclosure, and especially when referring to the amino acid sequence of peptide #40a, the expression“consisting essentially of indicates that the epitope is necessarily located on the amino acid sequence of SEQ ID NO: 105, but that additional, non- essential, amino acid residues can be added at the amino or the carboxy end of the amino acid sequence of SEQ ID NO: 105. In an embodiment, when the antibody is a monoclonal antibody, its dissociation constant (K _D ) with respect to peptide #40a is in the nanomolar range (1 - 1000 nM). In such embodiment, the monoclonal antibody can have a K _D between about 1 and about 100 nM, between about 1 and about 50 nM, between about 1 and about 25 nM or between about 5 and about 15 nM with respect to peptide #40a.

In other embodiments, the epitope recognized by the anti-Na _v 1.7 antibody is located on the peptide #40b which comprises or consists essentially of the following amino acid sequence:

YFYYLEGSKDALLCGFSTDSGQCPEGYTCVKIGRNPDYGY (SEQ ID NO: 106).

In the context of the present disclosure, and especially when referring to the amino acid sequence of peptide #40b, the expression“consisting essentially of indicates that the epitope is necessarily located on the amino acid sequence of SEQ ID NO: 106, but that additional, non- essential, amino acid residues can be added at the amino or the carboxy end of the amino acid sequence of SEQ ID NO: 106. In an embodiment, when the antibody is a monoclonal antibody, its dissociation constant (K _D ) with respect to peptide #40b is in the nanomolar range (1 - 1000 nM). In such embodiment, the monoclonal antibody can have a K _D between about 1 and about 100 nM, between about 1 and about 50 nM, between about 1 and about 25 nM or between about 5 and about 15 nM with respect to peptide #40b.

When the antibody of the present disclosure is a monoclonal antibody, it can include both a heavy and a light chain and can, in some embodiments, comprise two identical copies of the light chain and two identical copies of the heavy chain. Each chain can comprise three CDRs, wherein each CDR is flanked at both the amino terminus and on the carboxyl terminus, by a framework region. When the antibody of the present disclosure is a single-chain antibody, it can include either a heavy or a light chain. The single-domain antibody can comprise three CDRs, wherein each CDR is flanked at both the amino terminus and on the carboxyl terminus, by a framework region.

In an embodiment, the antibody of present invention has at least one complementary determining region comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 7, 8, 9, 13, 14, 15, 39, 40, 41 , 75, 76, 77, 80, 81 , 82, 85, 86, 87, 90, 91 , 92, 95, 96, 97, 100, 101 or 102 variant thereof or fragments thereof. In the context of the present disclosure, and especially when referred to the amino acid sequence of CDR, the expression “consisting essentially of indicates that the CDR necessarily comprises the amino acid sequence of SEQ ID NO: 7, 8, 9, 13, 14, 15, 39, 40, 41 , 75, 76, 77, 80, 81 , 82, 85, 86, 87, 90, 91 , 92, 95, 96, 97, 100, 101 or 102, but that additional, non-essential, amino acid residues can be added at the amino or the carboxyl end of those sequences (as long as these amino acid residues do not substantially modify the affinity of the antibody for the Na _v 1.7 polypeptide or its ability to antagonize the biological activity of the Na _v 1.7 polypeptide).

The antibody of the present disclosure can include a functional variant of a CDR having the amino acid sequence of SEQ ID NO: 7, 8, 9, 13, 14, 15, 39, 40, 41 , 75, 76, 77, 80, 81 , 82, 85, 86, 87, 90, 91 , 92, 95, 96, 97, 100, 101 or 102. A variant CDR comprises at least one amino acid difference when compared to the amino acid sequence of the CDR. As used herein, a variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the antibody (e.g., providing specificity and affinity towards the Na _v 1.7 polypeptide). In some embodiments, the overall charge, structure or hydrophobic-hydrophilic properties of the antibody can be altered without adversely affecting a biological activity. Accordingly, the amino acid sequence of the CDR can be altered, for example to render the antibody more hydrophobic or hydrophilic, without adversely affecting the biological activities of the antibody. The CDR variants have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the CDRs described herein. The term“percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. The level of identity can be determined conventionally using known computer programs. Identity can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignments of the sequences disclosed herein were performed using the Clustal method of alignment (Higgins and Sharp (1989) CAB I OS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PEN ALT Y= 10). Default parameters for pairwise alignments using the Clustal method were KTUPLB 1 , GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The CDR variants may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group. A“variant” of the CDR can be a conservative variant or an allelic variant.

The antibody of the present disclosure can include a functional fragment of a CDR having the amino acid sequence of SEQ ID NO: 7, 8, 9, 13, 14, 15, 39, 40, 41 , 75, 76, 77, 80, 81 , 82, 85, 86, 87, 90, 91 , 92, 95, 96, 97, 100, 101 or 102. A fragment of a CDR comprises at least one less amino acid residue compared to the amino acid sequence of the CDR. The CDR fragments comprise some consecutive amino acid residues of the CDR of amino acid sequence of SEQ ID NO: 7, 8, 9, 13, 14, 15, 39, 40, 41 , 75, 76, 77, 80, 81 , 82, 85, 86, 87, 90, 91 , 92, 95, 96, 97, 100, 101 or 102. The CDR fragments have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the CDRs described herein.

In an embodiment, the antibody of the present disclosure comprises at least one CDR comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 7, 8, 9, 13, 14, 15, 75, 76, 77, 80, 81 , 82, 85, 86, 87, 90, 91 , 92, 95, 96, 97, 100, 101 and/or 102. In another embodiment, the antibody comprises at least two CDRs each comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 7, 8, 9, 13, 14, 15, 75, 76, 77, 80, 81 , 82, 85, 86, 87, 90, 91 , 92, 95, 96, 97, 100, 101 and/or 102, functional variants thereof and functional fragments thereof. In yet another embodiment, the antibody comprises at least three CDRs each comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 7, 8, 9, 13, 14, 15, 75, 76, 77, 80, 81 , 82, 85, 86, 87, 90, 91 , 92, 95, 96, 97, 100, 101 and/or 102, functional variants thereof and functional fragments thereof. In still another embodiment, the antibody comprises at least four CDRs each comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 7, 8, 9, 13, 14, 15, 75, 76, 77, 80, 81 , 82, 85, 86, 87, 90, 91 , 92, 95, 96, 97, 100, 101 and/or 102. In a further embodiment, the antibody comprises at least five CDRs each comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 7, 8, 9, 13, 14, 15, 75, 76, 77, 80, 81 , 82, 85, 86, 87, 90, 91 , 92, 95, 96, 97, 100, 101 and/or 102, functional variants thereof and functional fragments thereof. In still a further embodiment, the antibody comprises six CDRs each comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 7, 8, 9, 13, 14, 15, 75, 76, 77, 80, 81 , 82, 85, 86, 87, 90, 91 , 92, 95, 96, 97, 100, 101 and/or 102, functional variants thereof and functional fragments thereof.

In another embodiment, the antibody comprises a heavy chain and the heavy chain comprises at least one CDR comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 7, 8, 9, 75, 76, 77, 85, 86, 87, 95, 96 and/or 97, functional variants thereof and functional fragments thereof. In still another embodiment the antibody comprises a heavy chain and the heavy chain comprises at least one CDR comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 7, 8, 9, 75, 76, 77, 85, 86, 87, 95, 96 and/or 97, functional variants thereof and functional fragments thereof. In a further embodiment, the heavy chain comprises at least two CDRs each comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 7, 8, 9, 75, 76, 77, 85, 86, 87, 95, 96 or 97, functional variants thereof and functional fragments thereof. In still a further embodiment, the heavy chain comprises three CDRs each comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 7, 8 and 9, functional variants thereof and functional fragments thereof. In another embodiment, the heavy chain comprises or consists essentially of the amino acid sequence of SEQ ID NO: 1 1 or 12, functional variants thereof and functional fragments thereof. In the context of the present disclosure, and especially when referred to the amino acid sequence of heavy chain, the expression“consisting essentially of indicates that the CDR necessarily comprises the amino acid sequence of SEQ ID NO: 1 1 or 12, but that additional, non-essential, amino acid residues can be added at the amino or the carboxyl end of those sequences (as long as these amino acid residues do not substantially modify the affinity of the antibody for the Na _v 1.7 polypeptide or its ability to antagonize the biological activity of the Na _v 1.7 polypeptide). In still a further embodiment, the heavy chain comprises three CDRs each comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 75, 76 and 77, functional variants thereof and functional fragments thereof. In another embodiment, the heavy chain comprises or consists essentially of the amino acid sequence of SEQ ID NO: 74, functional variants thereof and functional fragments thereof. In still a further embodiment, the heavy chain comprises three CDRs each comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 85, 86 and 87, functional variants thereof and functional fragments thereof. In another embodiment, the heavy chain comprises or consists essentially of the amino acid sequence of SEQ ID NO: 84, functional variants thereof and functional fragments thereof. In still a further embodiment, the heavy chain comprises three CDRs each comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 95, 96 and 97, functional variants thereof and functional fragments thereof. In another embodiment, the heavy chain comprises or consists essentially of the amino acid sequence of SEQ ID NO: 94, functional variants thereof and functional fragments thereof. The antibody can be a monoclonal antibody or a single-domain antibody.

In another embodiment, the antibody comprises a light chain and the light chain comprises at least one CDR comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 13, 14, 15, 80, 81 , 82, 90, 91 , 92, 100, 101 and/or 102, functional variants thereof and functional fragments thereof. In still another embodiment the antibody comprises a light chain and the light chain comprises at least one CDR comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 13, 14 and/or 15, functional variants thereof and functional fragments thereof. In a further embodiment, the light chain comprises at least two CDRs each comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 13, 14 and/or 15, functional variants thereof and functional fragments thereof. In still a further embodiment, the light chain comprises three CDRs each comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 13, 14 and/or 15, functional variants thereof and functional fragments thereof. In another embodiment, the light chain comprises or consists essentially of the amino acid sequence of SEQ ID NO: 17 or 18, functional variants thereof and functional fragments thereof. In the context of the present disclosure, and especially when referred to the amino acid sequence of light chain, the expression“consisting essentially of indicates that the CDR necessarily comprises the amino acid sequence of SEQ ID NO: 17 or 18, but that additional, non-essential, amino acid residues can be added at the amino or the carboxyl end of those sequences (as long as these amino acid residues do not substantially modify the affinity of the antibody for the Na _v 1.7 polypeptide or its ability to antagonize the biological activity of the Na _v 1.7 polypeptide). In still a further embodiment, the light chain comprises three CDRs each comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 80, 81 and/or 82, functional variants thereof and functional fragments thereof. In another embodiment, the light chain comprises or consists essentially of the amino acid sequence of SEQ ID NO: 79, functional variants thereof and functional fragments thereof. In still a further embodiment, the light chain comprises three CDRs each comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 90, 91 and/or 92, functional variants thereof and functional fragments thereof. In another embodiment, the light chain comprises or consists essentially of the amino acid sequence of SEQ ID NO: 89, functional variants thereof and functional fragments thereof. In still a further embodiment, the light chain comprises three CDRs each comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 100, 101 and/or 1022, functional variants thereof and functional fragments thereof. In another embodiment, the light chain comprises or consists essentially of the amino acid sequence of SEQ ID NO: 99, functional variants thereof and functional fragments thereof. The antibody can be a monoclonal antibody or a single-domain antibody.

In yet another embodiment, the antibody can comprise both a heavy chain and the light chain. In such embodiment, the heavy chain can comprise at least one, at least two or three CDRs each CDR comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 7, 8 and 9, functional variants thereof and functional fragments thereof. In another embodiment, the heavy chain can comprise or consist essentially of the amino acid sequence of SEQ ID NO: 1 1 or 12, functional variants thereof and functional fragments thereof. In addition, the light chain can comprise at least one, at least two or three CDRs each CDR comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 13, 14 and 15, functional variants thereof and functional fragments thereof. In some embodiment, the light chain can comprise or consist essentially of the amino acid sequence of SEQ ID NO: 17 or 18, functional variants thereof and functional fragments thereof. In some embodiments, the antibody can be a monoclonal antibody, such as, for example, from the IgG isotype and, in some embodiments, from the lgG1 subtype.

In yet another embodiment, the antibody can comprise both a heavy chain and the light chain. In such embodiment, the heavy chain can comprise at least one, at least two or three CDRs each CDR comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 75, 76 and 77, functional variants thereof and functional fragments thereof. In another embodiment, the heavy chain can comprise or consist essentially of the amino acid sequence of SEQ ID NO: 74, functional variants thereof and functional fragments thereof. In addition, the light chain can comprise at least one, at least two or three CDRs each CDR comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 80, 81 and 82, functional variants thereof and functional fragments thereof. In some embodiment, the light chain can comprise or consist essentially of the amino acid sequence of SEQ ID NO: 79, functional variants thereof and functional fragments thereof. In some embodiments, the antibody can be a monoclonal antibody, such as, for example, from the IgG isotype and, in some embodiments, from the lgG2a subtype.

In yet another embodiment, the antibody can comprise both a heavy chain and the light chain. In such embodiment, the heavy chain can comprise at least one, at least two or three CDRs each CDR comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 85, 86 and 87, functional variants thereof and functional fragments thereof. In another embodiment, the heavy chain can comprise or consist essentially of the amino acid sequence of SEQ ID NO: 84, functional variants thereof and functional fragments thereof. In addition, the light chain can comprise at least one, at least two or three CDRs each CDR comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 90, 91 and 92, functional variants thereof and functional fragments thereof. In some embodiment, the light chain can comprise or consist essentially of the amino acid sequence of SEQ ID NO: 89, functional variants thereof and functional fragments thereof. In some embodiments, the antibody can be a monoclonal antibody, such as, for example, from the IgG isotype and, in some embodiments, from the IgG 1 subtype.

In yet another embodiment, the antibody can comprise both a heavy chain and the light chain. In such embodiment, the heavy chain can comprise at least one, at least two or three CDRs each CDR comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 95, 96 and 97, functional variants thereof and functional fragments thereof. In another embodiment, the heavy chain can comprise or consist essentially of the amino acid sequence of SEQ ID NO: 94, functional variants thereof and functional fragments thereof. In addition, the light chain can comprise at least one, at least two or three CDRs each CDR comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 100, 101 and 102, functional variants thereof and functional fragments thereof. In some embodiment, the light chain can comprise or consist essentially of the amino acid sequence of SEQ ID NO: 99, functional variants thereof and functional fragments thereof. In some embodiments, the antibody can be a monoclonal antibody, such as, for example, from the IgG isotype and, in some embodiments, from the IgG 1 subtype.

In an embodiment, the antibody of the present disclosure comprises at least one CDR comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 13, 14 and/or 15. In another embodiment, the antibody comprises at least two CDRs each comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 13, 14 and/or 15, functional variants thereof and functional fragments thereof. In yet another embodiment, the antibody comprises the three CDRs each comprising or consisting essentially of a distinct amino acid sequence from the following: SEQ ID NO: 13, 14 and/or 15, functional variants thereof and functional fragments thereof. In some embodiments, the antibody can comprise or consist essentially of the amino acid sequence of SEQ ID NO: 38, functional variants thereof and functional fragments thereof. In an embodiment, the CDRs having the amino acid sequence of SEQ ID NO: 13, 14 and/or 15 can be located on a heavy chain of a single-domain antibody. In some embodiment, the single-domain antibody can be a V _h H camelid antibody.

In some embodiments, the antibody of the present disclosure can include a light chain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 17, 18, 1 10, 79, 1 15, 1 16, 89, 1 19, 120, 99, 123, 124, functional variants and/or functional fragments thereof. The light chain of the antibodies of the present disclosure can include a leader sequence (such as, for example, the examplary leader sequence shown in Table 7 or any other suitable leader sequence) to facilitate its extracellular expression.

In some embodiments, the antibody of the present disclosure can include a heavy chain comprising or consisting essentialy of the amino acid sequence of SEQ ID NO: 1 1 , 12, 109, 38, 74, 1 13, 1 14, 84, 1 17, 1 18, 94, 121 , 122, functional variants and/or functional fragments thereof. The heavy chain of the antibodies of the present disclosure can include a leader sequence (such as, for example, the examplary leader sequence shown in Table 7 or any other suitable leader sequence) to facilitate its extracellular expression.

In some embodiment, the antibodies of the present disclosure may be further designed to cross the blood-brain barrier to reach the spinal cord thereby allowing the antibody to mediate its therapeutic actions of a treated subject. In one embodiment, it is possible to link an antibody disclosed herein to another antibody or antibody fragment capable of transmigrating the blood brain barrier. For example, said another antibody or antibody fragement may be a further entity (e.g., an antibody fragment) which is capable of recognizing an epitope of a human cerebromicrovascular endothelial cell as described in US Patent Serial Number 8,715,659 or to a blood-barrier antigen found on the surface of a mammalian cell as described in US Patent Serial Number 8,383,107 (collectively referred to as antibody fragment allowing the migration across the blood-brain barrier). This can be achieved by providing an antibody fragment of the antibody of the present disclosure with an antibody fragment allowing the migration across the blood-brain barrier. This can also be achieved by adding an antibody fragment allowing the migration across the blood-brain barrier to the antibody (thereby forming a chimeric protein, or a fusion protein). The present disclosure thus provides a chimeric protein comprising the antibody of the present disclosure comprising an antibody fragment moiety which allows/facilitates the migration across the blood-brain barrier.

This chimeric protein can take the form of antibody-drug-conjugate (ADC) or bi-specific antibodies in which one antibody binding specificity recognizes a BBB receptor that undergoes receptor- mediated transcytosis (RMT) from the brain into the CNS, and the second binding specificity recognizes a therapeutic target i.e. Na _v 1.7 within the CNS. The development of BBB-crossing bispecific antibodies requires targeted antibody engineering to optimize multiple characteristics of “BBB carrier” and therapeutic arms, as well as other antibody properties impacting pharmacokinetics and effector function. It is widely recognised now that the preclinical models of chronic pain do not translate well to the clinic with many failures of compounds that had good efficacy in the animal models. The possibility to target an additional CNS site may provide the additional efficacy required to be a successful analgesic. With respect to a potential cancer indications, the development of a bi-specific Ab targeting both the tumour cells and Na _v 1.7 could ensure concentration of the therapeutic Ab in the tumour. If CNS access is required for efficacy in humans then a BBB-crossing mAb targeting Na _v 1.7 may have such an advantage. With respect to a cancer indication a bi-specific Ab targeting both the tumour cell and Na _v 1.7 may have an advantage in allowing lower systemic exposure due to concentration within the tumour and thus avoiding side-effects associated with inhibiting the normal functioning of the Na _v 1.7 channel.

The present disclosure also provides nucleotide molecules encoding the antibodies described herein. The nucleotide molecules can be provided in an isolated form and may be derived from a variety of sources including DNA, cDNA, synthetic DNA, synthetic RNA, derivatives, mimetics or combinations thereof. Such sequences may comprise genomic DNA, which may or may not include naturally occurring introns, genic regions, nongenic regions, and regulatory regions. Moreover, such genomic DNA may be obtained in association with promoter regions or poly (A) sequences. The sequences, genomic DNA, or cDNA may be obtained in any of several ways. Genomic DNA can be extracted and purified from suitable cells by means well known in the art. Alternatively, mRNA can be isolated from a cell and used to produce cDNA by reverse transcription or other means. The nucleotide molecules described herein are used in certain embodiments of the methods of the present disclosure for production of RNA, proteins or polypeptides, through incorporation into host cells, tissues, or organisms. The nucleotide molecules can include any nucleic acid sequence described in Table 7. In an embodiment, the nucleotide molecules can be codon-optimized for expression in a particular host. The nucleotide molecules can include, in some embodiments, one or more promoter sequence and/or one or more terminator sequence. The nucleotide molecules can be included in a vector for expression in a recombinant host.

Methods of using the anti-Na _v U antibodies

Due to their high specificity towards the Na _v 1.7 polypeptide, the antibodies of the present disclosure can be used to substantially purify the Na _v 1.7 polypeptide from a mixture suspected of comprising the Na _v 1.7 polypeptide. In such embodiment, the antibodies of the present disclosure can be provided in association with a solid support (e.g., a bead, a resin, a plate for example) for binding to the Na _v 1.7 polypeptide and allowing the (partial or complete) removal of the other components of the mixture.

The antibodies of the present disclosure can be used to detect, and in some embodiments, localize or quantify the amounts of the Na _v 1.7 polypeptide either in vitro (in immunological assays, such as, for example, ELISA, immunological staining and flow cytometry) or in vivo (in imaging techniques). In such embodiment, the antibodies of the present disclosure can be associated (coupled or physically linked) to a detectable label and used in combination with a method for detecting, localizing and/or quantifying the amount of the Na _v 1.7 polypeptide by determining the presence, absence, location, amount of the detactable label. Examples of detectable labels include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials and radioactive materials. Examples of suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include, but are not limited to, streptavidin/biotin and avidin/biotin. Examples of suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Examples of a luminescent material include, but are not limited to, luminol. Examples of bioluminescent materials include, but are not limited to, luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include, but are not limited to, ¹²⁵ l, ¹³¹ 1 , ³⁵ S, ³² P or ³ H.

Due to their ability to antagonize the biological activity of the Na _v 1 .7 polypeptide, the antibodies of the present disclosure can also be used as a medicine for the alleviation of the symptoms of pain. As used herein, the expression“alleviation of symptoms of pain” refer to the ability of the antibody to limit the symptomology of pain in a subject in need thereof (e.g., experiencing pain). In some embodiments, the antibodies can be used to treat chronic pain. In other embodiments, the antibodies can be used to treat neuropathic pain. Symptoms associated with pain include, but are not limited to: nociception, nausea, headache, swelling, weakness, lack of energy, mood changes, trouble sleeping and/or decreased appetite. Type of pain: nociceptive, inflammatory and neuropathic. In some embodiments, the pain is a chronic pain caused by cancer, inflammation or degeneration of joints; a neuropathic pain caused by injury to nerves, phantom limbs; a pain caused by tissue injury (trauma, burns, etc.) and/or a post-surgery pain.

Because some of the embodiments of the antibodies of the present disclosure are capable of reducing colony formation of cancerous cells, the antibodies can also be used for the treatment or the alleviation of symptoms associated with an hyperproliferative disease. The expressions “treatment or alleviation of symptoms” refer to the ability of a method or an antibody to limit the development, progression and/or symptomology of an hyperproliferative disease. Broadly, the treatment and/or alleviation of symptoms can encompass the reduction of proliferation of the cells (e.g. , by reducing the total number of cells in an hyperproliferative state and/or by reducing the pace of proliferation of cells). Symptoms associated with proliferation-associated disorder include, but are not limited to: local symptoms which are associated with the site of the primary cancer (such as lumps or swelling (tumor), hemorrhage, ulceration and pain), metastatic symptoms which are associated to the spread of cancer to other locations in the body. (such as enlarged lymph nodes, hepatomegaly, splenomegaly, pain, fracture of affected bones, and neurological symptoms), and systemic symptoms (such as weight loss, fatigue, excessive sweating, anemia and paraneoplastic phenomena). Hyperproliferative diseases form a class of diseases where cells proliferate more rapidly, and usually not in an ordered fashion. The proliferation of cells cause an hyperproliferative state that may lead to biological dysfunctions, such as the formation of tumors (malignant or benign). One of the hyperproliferative disease is cancer. Also known medically as a malignant neoplasm, cancer is a term for a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the body through the lymphatic system or bloodstream. In another embodiment, the cancer is associated with the expression and, in some embodiments overexpression, of the Na _v 1 .7 polypeptide. In some embodiments, the antibodies can be used in combination with other chemotherapeutic agents.

As indicated above, the antibody can be provided in a chimeric form when it is intended that the antibody cross the blood brain barrier to provide its therapeutic benefits. For some therapeutic or preventive applications, the antibody of the present disclosure can also be coupled to a chemotherapeutic agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), a radioactive isotope (i.e., a radioconjugate). Exemplary toxins include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha- sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII , and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.

In some embodiments, the antibodies and the chimeric proteins of the present disclosure can be provided as pharmaceutical compositions. As used herein, “pharmaceutical composition” means therapeutically effective amounts (dose) of the antibody/chimeric protein together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions can be liquid or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCI, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, and detergents (e.g., Tween 20™, Tween 80™, Pluronic F68™, bile acid salts). The pharmaceutical composition can comprise pharmaceutically acceptable solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens), bulking substances, amino acids or tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. Controlled or sustained release compositions include formulation in lipophilic depots ( e.g ., fatty acids, waxes, oils). Also comprehended by the invention are particulate compositions coated with polymers (e.g., poloxamers or poloxamines).

The antibodies of the present disclosure may be used individually or in combination. For example, a pharmaceutical composition of the present invention may comprise one or more than one antibody defined herein. For example, antibodies 3A8, 1 B6, 1 H5, 2G1 1 and Dl-D may be used individually or in a combination of two or more antibodies. In some further embodiments, the antibodies can be used with other therapeutic agents for managing pain and/or cancer.

The antibodies and the chimeric proteins of the present disclosure (which can be included in a pharmaceutical composition) can be formulated or intended to be administered by various routes, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. The antibodies and chimeric proteins of the present disclosure may be administered, either orally or parenterally, systemically or locally. In an embodiment, the antibodies and chimeric proteins are formulated/intended for intravenous administration, such as, by injection or infusion. The effective dosage can be chosen from the range of 0.01 mg to 100 mg per kg of body weight per administration. Alternatively, the dosage is a fixed dosage and is in the range of 1 to 1000 mg, preferably 5 to 50 mg per patient may be chosen. In some embodiments, the effective dosage can be at least 10 mg/kg and at most 100 mg/kg.

The antibodies and chimeric proteins of the present disclosure are formulated/intended to be administered at a therapeutically effective amount. A“therapeutically effective amount” as used herein refers to that amount which provides a therapeutic effect (e.g., alleviation of the symptoms of pain) for a given condition and administration regimen. The antibodies and chimeric proteins can be used alone or in combination with other therapeutic agents for the treatment of pain. When used in combination with other therapeutic agents for the treatment of pain, the antibodies and chimeric protein can be formulated/intended to be administered sequentially, subsequently or simultaneously.

The antibodies and the chimeric protein of the present disclosure can be used or administered to various subjects, including humans and non-human mammals (such as cats and dogs for example). Single-domain antibody-based immunogen

The present disclosure also provides immunogens as well as methods for making antibodies specific to a pepitde epitope with the immunogens. As shown in the Examples below, an immungen (FC5DIE3IR) was used to immunize an animal and/or select an antibody from a library of antibodies in order to obtain or identify an antibody specific for a peptide epitope found on the Na _v 1.7 polypeptide. As also shown in the Examples below, the antibodies obtained were specific for the Na _v 1.7 polypeptide. Consequently, the present disclosure provides an immunogen for making additional antibodies, including additional anti-Na _v 1.7 polypeptide antibodies, based on a similar approach.

As used in the context of the present disclosure, an“immunogen” is a polypeptide which is capable of either eliciting an immune reaction (e.g., production of antibodies) upon the administration in a host as well as being able to select, from a library of antibodies, antibodies specific for the epitope. The immunogen of the present disclosure is intended to be expressed in a recombinant fashion in a host, as such, the epitope it bears is constituted of a contiguous stretch of amino acid residues which form an epitope in the immunogen.

The immunogen of the present disclosure comprises or consists of a single-domain antibody (sdAb) in which the epitope is introduced in one complementary determining region (and in some embodiments, in a single complementary determining region of the sdAb). Single-domain antibodies, such a camelid V _h H antibodies, usually have three CDRs (from amino to carboxyl terminus, CDR1 , CDR2 and CDR3) and the peptide epitope can be introduced in any one of the three CDRs. In a specific embodiment, the peptide epitope is introduced in the CDR3 of the sdAb. Preferably, the epitope is introduced in the single-domain antibody in such a way that the overall tridimensional structure of the framework regions remains substantially similar to the overall tridimensional structure of the original single-domain antibody that does not bear the peptide epitope. This can be achieved, for example, by deleting one or more amino acid residues in one location (for example the CDR) intended to receive the peptide epitope and introducing the peptide epitope at the location of the deletion. In some embodiments, the entire CDR is removed and replaced by the peptide epitope. In other embodiments, only parts of the CDR (which may be a contiguous strecth of amino acids) is removed and replaced by the peptide epitope.

The peptide epitope that can be introduced in the CDR of the immunogen can be of any length, for example between 2 and 500 amino acid residues. In an embodiment, the peptide epitope is between 2 and 400, 2 and 300, 2 and 200, 2 and 100, 5 and 100, 10 and 100, 20 and 100, 30 and 100, 40 and 100 or 50 and 100 amino acid residues. In a specific embodiment, the peptide epitope is 70 amino acid residue-long. The immunogen can comprise any sdAb and, in some embodiments, the immunogen is derived from a camelid V _h H antibody, the FC5 antibody (as described in US Patent Serial Number 8,715,659 and US Patent Serial Number 8,383,107). When the immunogen is derived from the FC5 antibody, it can comprise or consist essentially of the polypeptide of formula (I):

NH ₂ - SS-FR1 -CDR1 -FR2-CDR2-FR3-CDR3-FR4 - COOH (I).

In the polypeptides of formula (I), NH ₂ refers to the amino terminus of the polypeptide and COOH refers to the carboxyl terminus of the polypeptide. Since the polypeptide of formula (I) are intended to be produced from a recombinant host, the moieties it comprises are comprised of stretch of contiguous amino acid residues linked to one another by an amide bond.

The moiety SS is an optional component of the polypeptide of formula (I). The abbreviation“SS” refers to a “signal sequence” which can allow targeting the polypeptide of formula (I) for secretion from the recombinant host producing the immunogen. The SS is usually absent for the secreted form of the polypeptide. As such, the SS is usually present on the polypeptides of formula (I) when they are located inside the recombinant host and usually absent on the polypeptides of formula (I) when they are secreted. When present, the carboxyl terminus of AA is bound (direclty or indirectly) to the amino terminus of the FR1 moiety. If present, the SS can have or consist essentially of the amino acid sequence of SEQ ID NO: 69. However, the person skilled in the art will appreciate that other SS can be used to achieve extracellular expression of the immunogen.

The moieties FR1 , FR2, FR3 and FR4 refer to the first, second, third and fourth framework regions of the sdAb, when starting from the amino terminus of the polypeptide of formula (I). The moieties CDR1 , CDR2 and CDR3 refer to the first, second and third complementary determining regions of the sdAb, when strarting from the amino terminus of the polypeptide of formula (i). When the SS moiety is present, the carboxyl terminus of the SS moiety is bound (directly or indirectly) to the amino terminus of the FR1 moiety. The carboxyl terminus of the FR1 moiety is bound (directly or indirectly) to the amino terminus of the CDR1 moiety. The carboxyl terminus of the CDR1 moiety is bound (directly or indirectly) to the amino terminus of the FR2 moiety. The carboxyl terminus of the FR2 moiety is bound (directly or indirectly) to the amino terminus of the CDR2 moiety. The carboxyl terminus of the CDR2 moiety is bound (directly or indirectly) to the amino terminus of the FR3 moiety. The carboxyl terminus of the FR3 moiety is bound (directly or indirectly) to the amino terminus of the CDR3 moiety. The carboxyl terminus of the CDR3 moiety is bound (directly or indirectly) to the amino terminus of the FR4 moiety.

When the immunogen is derived from the FC5 antibody, the FR1 moiety has the amino acid sequence of SEQ ID NO: 60, the FR2 moiety has the amino acid sequence of SEQ ID NO: 61 , the FR3 moiety has the amino acid sequence of SEQ ID NO: 62 and the FR4 moiety has the amino acid sequence of SEQ ID NO 63.

In an embodiment, the peptide epitope is included in the CDR1 moiety. In such embodiment, the polypeptide of formula (I) can have a CDR2 moiety comprising the amino acid sequence of SEQ ID NO: 65 and a CDR3 moiety comprising the amino acid sequence of SEQ ID NO: 66.

In another embodiment, the peptide epitope is included in the CDR2 moiety. In such embodiment, the polypeptide of formula (I) can have a CDR1 moiety comprising the amino acid sequence of SEQ ID NO: 64 and a CDR3 moiety comprising the amino acid sequence of SEQ ID NO: 66.

In yet another embodiment, the peptide epitope is included in the CDR3 moiety. In such embodiment, the polypeptide of formula (I) can have a CDR1 moiety comprising the amino acid sequence of SEQ ID NO: 64 and a CDR2 moiety comprising the amino acid sequence of SEQ ID NO: 65. In such embodiment, the polypeptide of formula (I) can have the amino acid sequence of SEQ ID NO: 70 (including a signal sequence) or 71 (with no signal sequence) in which the residue identified with“Xaa” is the location of the peptide epitope. When the peptide epitope is the DIE3IR peptide, the polypeptide of formula (I) can have the amino acid sequence of SEQ ID NO: 2 or 67 (including a signal sequence) or 3 or 68 (with no signal sequence).

In an embodiment, the immunogen comprises an antibody or antibody fragment wherein the antibody comprises the peptide of SEQ ID NO: 2, 3, 67, 68, 103, 104, 105 or 106. The peptide is intended to replace a portion of the antibody, preferably a CDR region of the antibody. Wherein the antibody may comprise a peptide selected from the group consisting of SEQ ID NO: 2, 3, 67, 68, 103, 104, 105 or 106 wherein the modified immunogen is structurally equivalent to the original (unmodified) antibody. When the original antibody has been modified to comprise a peptide of SEQ ID NO: 2, 3, 67, 68, 103, 104, 105 or 106, the modified antibody can be used as immunogen capable of yielding anti-Na _v 1.7 antibodies. An immunogen of the present invention may comprise, but is not limited to, an antibody comprising the amino acid sequence of SEQ ID NO: 2, 3, 67, 68, 103, 104, 105 or 106.

The present disclosure comprises using the immunogen for making or selecting an antibody specific to the peptide epitope. The immunogen can be used in an animal for eliciting an immune reaction and the production of antibodies which may be further screened for their ability to specifically bind to the peptide antibody or the target polypeptide comprising the peptide epitope. The immunogen can also be used to screen a library of antibodies to select those having specificity towards the peptide antibody or the target polypeptide comprising the peptide epitope. The immunogen of the present disclosure can be used in combination with another control sdAb which corresponds to the sdAb used to make the immunogen but which lacks the peptide epitope.

The present disclosure comprises a method of making/selecting an antibody (which can be, for example, a monoclonal antibody or a single-domain antibody) specific for the peptide epitope. The method comprises a step of selecting an antibody specific for the peptide epitope from a library of antibodies or a population of antibody-producing cells by contacting the immunogen with the library or the population and selecting the antibodies/cells producing antibodies which binding specifically to the immunogen. The method can comprise a step of contacting a control sdAb (which corresponds to the sdAb used to make the immunogen but which lacks the peptide epitope) with the library/population and discarding the antibodies/cells producing antibodies which bind specifically to the control sdAb. In some embodiments, the method can also comprise a step of immunizing an animal (such as, for example, a mouse or a llama) with the immunogen (alone or in combination with an adjuvant, once or multiple times) and generating antibody-producing cell lines or clones from an organ of the immunized animal. In such embodiment, the method can also comprising fusing the antibody-producing cell obtained from the animal to a cancer cell to make an hybridoma.

The affinity of the antibodies selected using the immunogen of the present disclosure for the peptide epitope or the target polypeptide comprising the peptide epitope can be determined by various techniques known in the art including an immunological assay (such as, for example, an ELISA) and/or surface plasmon resonance (SPR).

The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

EXAMPLE I - MATERIALS AND METHODS

Epitope selection. Na _v 1.7 is comprised of four domains (Dl, Dll, Dill and DIV), each containing six transmembrane helices (S1-S6) and 4 extracellular loops (Figure 1). S4 is the voltage sensor of the channels. Studies of mutant Na _v 1.7 in native neuron (mutations in the gene coding for the Na _v 1.7, SCN9, which cause a channelopathy-associated insensitivity to pain, CIP), sequence and structural homology of human Na _v 1.7 sodium channel to the solved crystal structures of the bacterial sodium channel Na _v Ab (Payandeh et al., 201 1) and of eukaryotic ion channel Na _v PaS (Shen et al., 2017) were used in order to select a solvent-accessible region of 70 amino-acid peptide (referred to as DIE3IR and provided as SEQ ID NO: 1) from the extracellular loop 3 of the domain Dl of the wild-type human Na _v 1.7, which also corresponds to the sequence that contains mutations linked to CIP. DIE3IR was then grafted within the CDR3 (Kabat definition) in the V _h H FC5, thus producing the recombinant protein FC5DIE3IR (corresponding to SEQ ID NO: 2 or 67 with the signal peptide and to SEQ ID NO: 3 or 68 without the signal peptide).

In the design of this recombinant protein construct, six amino acid residues in the middle of the CDR3 of FC5 were removed and replaced by the DIE3IR sequence of 70 amino-acid residues. Seven amino-acid residues from the original CDR3 of FC5 were retained, three on the N-terminal side and four on the C-terminal side of the grafted DIE3IR peptide, in an attempt to maximize its accessibility for antibody recognition and to minimize possible alterations of its native folding due to proximity to the FC5 structure. A molecular model of the FC5 V _h H with the retained regions of the CDR3 (between which the DIE3IR peptide is to be grafted) is shown in Figure 2A. The DIE3IR extracellular segment is predicted to form a well-folded globular domain stabilized by two disulphide bonds and to have its N- and C-termini in close proximity (Figure 2B), based on its high homology to the recent cryo-EM structures of eukaryotic sodium channels. This solvent accessible extracellular domain appears to be prone to interactions with immunoglobulin (Ig) folded structures, based on the crystal structure of eel Na _v 1.4 complexed with the Ig-like b-1 subunit (Figure 2C), which we found to be relevant for our immunization attempts.

FC5DIE3IR protein production. The codon-optimized sequence encoding the human DIE3IR domain engrafted within the FC5 in place of CDR3 region was synthesized (GenScript, Piscataway, NJ, USA) and cloned into the pTT5 vector. The Chinese hamster ovary cell line expressing a truncated EBNA1 protein (CHO-3E7; Raymond et al., 2015) were grown in suspension in serum-free Freestyle™ F17 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 0.1 % Kolliphor P188 (Sigma-Aldrich, St. Louis, Missouri, USA) and 4 mM Glutamine (Sigma- Aldrich, St. Louis, Missouri, USA). Cultures were maintained in 125 ml Erlenmeyer ventilated flasks shaken at 120 rpm in a humidified incubator at 37°C with 5% C0 ₂ . Cell density and viability were determined using the Cedex Innovatis automated cell counter Cedex Analyzer (Roche, Basel, Switzerland) based on the trypan blue exclusion method.

Linear deacylated polyethylenimine Max (PEImax) was obtained from Polysciences (Warrington, PA, USA). Stock solution (3 mg/ml) was prepared in ultrapure water, sterilized by filtration (0.2 pm), aliquoted and stored a +4°C. Cells were diluted one day before transfection in fresh medium at 0.7 x 10 ⁶ cells/ml. They were then transfected the following day with viability greater than 99% at densities between 2.0 and 2.5 x 10 ⁶ cells/ml with 1 pg of plasmid DNA per ml of CHO culture. To that effect, plasmid DNA and PEImax were separately prepared in complete F17 medium. Plasmid DNA (pTT5- FC5DIE3IR) was diluted at 20 pg/ml in F17 medium, and an equivalent volume of F17 medium containing 100 pg/ml of 25 kDa PEImax was added. The polyplexes mixture was immediately vortexed and incubated 5 min at room temperature prior to addition to the cells. Twenty four hours post-transfection (hpt), cells were fed with peptone TN1 (0.5% w/v final), and the culture temperature was shifted to 32°C. Cell culture was centrifuged 20 min at 3000g 10-12 days post-transfection (dpt) at a viability > 60%. The supernatant was collected and loaded on a 5 ml MabSelect™ SuRe™ column (GE Healthcare) equilibrated in PBS. The column was washed with PBS and FC5DIE3IR was eluted with 100 mM citrate buffer pH 3.6. The fractions containing FC5DIE3IR were pooled and the citrate buffer was exchanged against PBS on Econo-Pac® 10DG columns (Bio-Rad, cat# 732-2010). Purified FC5DIE3IR was concentrated on Amicon Ultra 3kDa and sterilized by passing through 0.2 pm filters, aliquoted, and stored at -80°C.

As indicated in Examples II, III and IV, this recombinant protein has been used as antigen/immunogen to immunize mice and generate monoclonal antibodies (mAbs), isolate V _H HS against hNa _v 1.7 from a phage-displayed naive V _h H library generated from llama, alpaca and camel, immunize llamas to obtain a FC5DIE3IR llama immune library, isolate V _H HS against hNa _v 1.7 from a phage-displayed V _h H library generated from immunizing llamas with FC5DIE3IR.

Generation of hNa _v 1.7-HEK293 cell line. Anti-hNa _v 1.7 mAbs and V _H HS were tested on hNa _v 1.7 currents recorded with patch-clamp whole-cell technique in HEK293 cells overexpressing hNa _v 1.7 channels (hNa _v 1.7-HEK293 cells). HEK-293 cells (ATCC CRL1573) were transfected with human Na _v 1.7 plasmid (SC398916; OriGene Technologies, Rockville, MD, USA) as per Lipofectamine 2000’s protocol (Invitrogen Life Technologies P/N 52887). One day before transfection, HEK293 cells were seeded in 3 wells of a 6 well plate at a density of 8 x 10 ⁵ cells/well in growth medium (EMEM supplemented with 10% FBS). On the morning of transfection, the growth medium was replaced and the DNA/Lipofectamine solution was prepared as follows: for each well, 125 pi of OptiMEME containing 3 pg of DNA was mixed with 125 pi of OptiMEM pre-incubated with 9 pi of Lipofectamine 2000 at room temperature for 5 minutes. The DNA/Lipofectamine solution was then incubated for 20 minutes prior to adding it to the cells. After 5 h of transfection, the growth medium was replaced. Two days post transfection, cells were combined, plated in 6 10 cm ² dishes, and grown in the presence 300 pg/ml G418 sulfate, an antibiotic selective for transfected cells (400-130-IG, Wisent Bio Products, Saint- Jean-Baptiste, QC, Canada). Growth medium containing G418 sulfate was replaced every 2 days. When colonies became visible, they were selected and plated on cover slips for screening.

Western blot. Western blot was performed to determine the expression of the hNa _v 1.7 polypeptide in the transfected cells. Proteins (10 ng for each cell type, determined using the Bradford’s reagent) were isolated, loaded and run on a precast Biorad Mini Protean TGX 4%- 20% gel. The proteins of the gel were transferred to a membrane and incubated with an Alomone ACS-008 rabbit antibody (for Na _v 1.7, expected size 226 kDa, dilution 1 :200) or a Millipore MAB374 mouse antibody (for GAPDH, expected size 35 kDa, dilution 1 :500). The membranes were washed and incubated with a secondary antibody labelled with horseradish peroxidase. The presence of each protein was determined using enhanced chemiluminescence. Figure 3 shows the results of the Western Blot demonstrating a higher level of hNa _v 1.7 protein in hNa _v 1.7-HEK293 cells compared to non-transfected cells.

Antibody Binding Assays (Protein-ELISA). FC5DIE3IR binding of V _h H monomers was assessed by direct protein ELISA. Briefly, wells of a NUNC MaxiSorp microtiter plate were coated overnight at 4°C with 0.5 pg of the recombinant protein (FC5DIE3IR) in 100 pi PBS, pH 7.4. As negative control, the parental FC5 V _h H was used for immobilization as described above. After blocking with Starting block for 2 h, monomeric V _H HS (Dl-A, Dl-B, Dl-C, Dl-D, Dl-E, Dl-F, Dl-G and Dl-H) were added to the corresponding wells (FC5DIE3IR and FC5, respectively) and incubated for 2 h at room temperature. Wells were washed with PBST (0.05% v/v Tween-20) and further incubated with 100 pi rabbit anti-His IgG conjugated to HRP (1 :5000 in PBS) (Bethyl Laboratories) for 1 hour at room temperature. Binding was detected with TMB substrate (Kirkegaard and Perry Laboratories) and the reaction was stopped with 1 M H P0 and A was measured using an ELISA plate reader as described (Hussack et al., 2012).

Cellular preparations for the patch-clamp assays. To prepare the cells for a patch-clamp whole-cell experiment, a vial of frozen hNa _v 1.7-HEK293 cells was thawed in a 37.0°C water bath for ~ 2 min or until completely thawed. Cells were then transferred to a 15 ml centrifuge tube and brought up to a volume of 5 ml with complete media (EMEM + 10% FBS + G418). Cells were centrifuged at 1000g for 3 min and the supernatant aspirated from pellet. The pellet was then re-suspended in 4 ml of complete media. The pellet was gently triturated several times with a 5 ml pipette to break the cell pellet apart and remove clumps. A complete media was added to the cell suspension to obtain a final volume of 12 ml. Cell suspension was then added to a labelled T-75 flask which was rocked back and forth a few times to ensure homogeneous cell distribution prior to placing into incubator. Cells were maintained in a 37.0 °C / 5% C0 incubator and fed every 2 days with complete media. Cells were split when confluency reached ~ 70-80%.

At time of cell splitting, a dilute cell suspension was prepared in complete media (100 mI of cells plus 2.4 ml of media). 500 mI of dilute cell suspension was added to each well of a 24 well plate containing poly-l-lysine (0.025 mg/ml; Sigma-Aldrich, St. Louis, Missouri, USA) coated 13 mm plastic coverslips (Thermanox Plastic coverslips, Thermo Fisher, Waltham, Massachusetts, USA).

Patch-clamp whole-cell recordings. Human Na _v 1.7-HEK293 cells were further evaluated using patch-clamp whole-cell recordings to determine the functionality of the hNa _v 1.7 channels overexpressed in the HEK293 cells. Whole-cell patch-clamp recordings were obtained with a Multiclamp 700B amplifier (Molecular Devices, Carlsbad, CA, USA) controlled with pCIamp (v 10.2) software (Molecular Devices, Carlsbad, CA) used in combination with a Digidata 1440A A/D converter (Molecular Devices, Carlsbad, CA). Data were acquired at 20 KHz and filtered at 2 KHz. Patch-clamp experiments were conducted on transfected cells within 24-48 hours of plating. All recordings were done in the patch-clamp whole-cell configuration and voltage-clamp mode. Voltage-clamp experiments were performed with borosilicate pipettes filled with a solution (pipette solution) containing (in mM): CsF (140), NaCI (10), EGTA (1), HEPES (10), ATP (2), GTP (0.2). The pH was corrected to 7.3 using CsOH. The osmolarity of the pipette solution was adjusted to 280-290 mOsm. Recording electrodes were fire-polished with a microforge (MF-830, Narishige, Japan) and when filled with the pipette solution had a resistance of 1.5-3 MW. Whole-cell access resistances measured in voltage-clamp ranged from 5 to 10 MW. The access resistance was routinely monitored throughout each experiment. To record Na ⁺ currents, cells were maintained in an extracellular recording solution contained (in mM): NaCI (140), KCI (3), CaCI ₂ (1), MgCI ₂ (1), HEPES (10). The pH was corrected to 7.3 using NaOH, and the osmolarity adjusted to ~ 300 mOsm. Over-expression of hNa _v 1.7 was analyzed by plotting current-voltage (l-V) relationships of evoked inward currents in response to step protocols (voltage steps from -85 to +60 mV in 5mV increments) and raw traces of inward current, and by analyzing half-maximal activation (g/g _M Ax) and inactivation (I/I _M AX) potential (Figure 4). For reference, HEK293 cells express an endogenous Na _v 1.7 current which peaks between 200-500 pA.

Patch-clamp whole-cell assays for determining functionality of mAbs in vitro. Stock solutions of each mAb to be tested, were prepared in extracellular recording solution the day of the experiment. The concentration of the mAb was 20 times the concentration to be tested. This was done to consistently add 50 pi of mAb solution to the recording chamber containing the cells. The 50 pi of mAb solution was statically added to 650 mI of extracellular recording solution present in the recording chamber.

Whole-cell patch-clamp recordings were obtained with a Multiclamp 700B amplifier (Molecular Devices, Carlsbad, CA, USA) controlled with a pCIamp (v10.2) software (Molecular Devices, Carlsbad, CA) used in combination with a Digidata 1320A A/D converter (Molecular Devices). Data were acquired at 20 KHz and filtered at 2 KHz. Borosilicate pipettes had a resistance between 1.5 and 32MW when filled with a solution containing (mM): CsF (140), NaCI (10), MgCI ₂ (1), HEPES (10), ATP-Mg ²⁺ (2), GTP-Mg ²⁺ (0.2). The pH was adjusted to 7.3 with CsOH. Pipettes were pulled from borosilicate glass using a P-97 Flaming-Brown type micropipette puller (Sutter Instrument, Novato, CA, USA) and fire-polished with a microforge (MF-830; Narishige, Japan).

To record Na ⁺ currents, the cells were maintained in an extracellular solution containing (in mM): NaCI (150), CsCI (3), CaCI ₂ (1), MgCI ₂ (1), HEPES (10). The pH was adjusted to 7.3 with NaOH). The stimulus protocol to evoke Na ⁺ currents consisted of 1 Hz frequency, and 20 ms duration voltage steps elicited from the resting-state of hNa _v 1.7 channels (-120 mV) to a potential that evoke maximal inward Na ⁺ current. The test potential was determined by stimulating the cell to various potential levels around the peak activation potential of hNa _v 1.7 (from -40 mV to -20 mV). The stimulus potential that generated the greatest magnitude of inward current was used for subsequent experimental protocols. Cells were allowed to stabilize for up to 5 minutes following initial breaking of the membrane into the whole-cell configuration.

Following observation of a stable baseline, the experimental protocol was initiated. Two minutes baseline recordings were followed by recordings during mAb exposure. mAbs were applied through static bath application. The effect of the mAbs was assessed comparing the amplitudes of the currents in control and in the presence of the mAb. Analyses were performed off-line with the software IGOR (WaveMetrics Inc., Portland, Oregon, USA) and/or Origin 2016 (OriginLab Corporation, Northampton, MA, USA). Statistical significance of the results was determined with paired Student’s t-tests (two-tailed). All values are expressed as means ± SEM, and a p-value of <0.05 is considered significant.

The current-voltage (l-V) relationships were obtained by plotting the amplitudes of the evoked inward currents in response to voltage steps versus the potentials of the steps (voltage steps from -85 to +60 mV in 5 mV increments). To calculate fast inactivation, the cells were held at -120 mV, 500 ms voltage steps (from -140 to -35 mV; 5 mV increments) were followed by a depolarization step of the duration of 20 msec at 0 mV.

Hargreaves Model of Hyperalgesia in rats. Rats aged 4-6 weeks (weight range, 180-220 g) were used for intraplantar (ipl) administrations of various antibodies and evaluation of their efficacy in the Hargreaves model of inflammatory pain. Chronic inflammatory pain is induced by injecting a low volume (50 pi) of complete Freund’s adjuvant (CFA; heat-killed M. Tuberculosis - Sigma, St. Louis, MO- suspended in oilisaline 2:1 emulsion) into the right hind paw. The paw withdrawal latency in response to the application of a radiant stimulus onto the plantar surface of both right and left paw was measured using the plantar Analgesia Meter equipment for paw stimulation (IITC Life Science, Woodland Hills, CA). The time taken by the animal to respond by licking or flicking its paw is interpreted as positive response (paw withdrawal latency). After 48 h of the CFA injection, the baseline for latency paw withdrawal was measured and the mAbs tested. Test compound were injected by intraplantar route (50 pg diluted in 50 pi) with Hamilton (glass) syringe. A cut-off time (20 sec) is established at the end of which the heat source shuts off automatically to avoid tissue damage. Animals were kept (randomized) one per cage and staff performing pain experiments are blinded to the content of injectable compounds. Percentage of maximum possible effect (%MPE) was calculated using the formula %MPE = (Test Latency - Baseline Latency) x 100 / (Cut-off - Baseline Latency).

OD1 Mouse Pain Model. OD1 model was performed as previously described (Deuis et al., 2016) with minor modifications. CD-1 mice aged 4-6 weeks (weight range, 25-30g) were kept under 12-h light-dark cycles and had standard rodent chow and water ad libitum. Scorpion a- toxin OD1 , isolated from the venom of the Iranian yellow scorpion, is a C-terminally amidated polypeptide of 65 amino acids. It is shown to be a potent modulator of Na _v 1.7 (EC ₅₀ 4.5 nM), with little effect on Na _v 1.3 and Na _v 1.5 (EC ₅₀ > 1 mM) and no effect on Na _v 1.2 and Na _v 1.8. OD1 (Bristol, United Kingdom) was diluted in phosphate-buffered saline/0.1 % BSA and administered by shallow subcutaneous injection into the dorsal side of the left hind paw of mice in a volume of 30 pl_ under isoflurane anesthesia. Mice were then placed into polyvinyl boxes (10 x 10 x 10 cm), and spontaneous pain behavior (licks and flinches) was recorded and counted by blinded investigator. Test compounds were injected subcutaneous injection into the dorsal side of the left hind paw 60 minutes before OD1 injection.

Manufacturing of peptides #1-3, #2a, #20a, #40a and #40b. The peptides were custom- synthesized (Biomatik) and tested for antibody binding by ELISA. For ELISA, peptides #1-3, #20a, #20b, #40a and #40b were coated on ELISA plates at various doses (100-500 ng/well) overnight at 4°C in PBS. The wells were blocked with 1 % BSA in TBS-T for 30min and then incubated with 1 :1000 dilution of anti-hNa _v 1.7 mAb at room temperature (RT) for one hr. Following three TBS-T washes (3 x 5 min), mAb binding to peptides was detected by incubating with HRP-conjugated anti-mouse Fc antibody for 90 min at RT in TBS-T. The bound antibody was detected with SureBlue™ TMB reagent kit (KPL) by colorimetric measurement at 450 nm according to manufacturer’s instructions.

To verify a possible cross reactivity among members of the Na _v family, the epitope sequences of the mAbs were searched in the database“non-redundant protein sequences” (nr) using Basic Local Alignment Search Tool (BLAST) 2.8.1 (protein-protein BLAST; Altschul et at, 1997). It was found that, the highest homology that pep#2 has with other members of the human Na _v family is 78% homology with the structure of the hNa _v 1.4 in complex with b1 (E value = 3e-07) and 72% with the hNa _v 1.2 (E value = 1 e-05), while, the pep#2a has a 78% homology with hNa _v 1.4 (E value =4.7). The highest homology that peptide pep#3 has is 80% with the hNa _v 1.2 (E value =8e-17). Overall this analysis suggests that the probability that 3A8, 1 H5, 2G1 1 and 1 B6 acts on others subtypes of the Na _v family other than the Na _v 1.7 is low.

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS). FC5DIE3IR and purified mAbs (3A8, 1 H5, 2G1 1 , 1 B6, 1 G5) were diluted to 40 and 20 mM, respectively, and mixed at a 1 : 1 ratio and kept on at 25°C. For the unbound control, FC5DIE3IR was diluted to 20 mM with PBS. Labelling reactions were initiated by adding 3 pL of IGF1 R complexes to 3 pL of 90% D ₂ 0 at 25°C. Labelling was quenched after 1 , 10, and 60 min by the addition of 39 pL ice-cold 8M urea/500 mM TCEP/200 mM Gly-HCI, pH 3.0, and samples were allowed to reduce for 2 min on ice. The sample was loaded into a 20 pL loop and manually injected in a custom valve cooler. This was followed by digestion at room temperature with a Poroszyme Immobilized Pepsin Cartridge (2.1 x 30 mm, Thermo Scientific) at 50 pL/min for 1.5 min, followed by washing/trapping onto a C18 PepMapl OO cartridge kept at 1 °C (5 pm, 1 x 5mm, Thermo Scientific) at 400 pL/min for 1 min in mobile phase A (0.23% formic acid in water). Peptides were eluted at 1 °C from a Biobasic-18 (5 pm, 0.32 x 50mm, Thermo Scientific) analytical column at 10 pL/min with a 10-35% mobile phase B gradient (0.23% formic acid in acetonitrile) over 6 min. LC-MS analysis was performed with an Agilent 1260 Infinity pump coupled to a Q- Tof Ultima API mass spectrometer (Water). Peptides were identified with Mascot and validated using an unlabelled sample injection. Data was collected in triplicate and analyzed with MS studio, and mass shifts were considered significant if greater than > 3 SD with a p-value of 0.05 (Rey et ai, 2016).

Surface Plasmon Resonance of mAbs. FC5 and FC5DIE3IR proteins were SEC purified using a Superdex 75 column (GE Healthcare) connected to an AKTAPurifier (GE Healthcare) in HBS-EP+ running buffer (10 mM HEPES, pH 7.4, containing 150 mM NaCI, 3 mM EDTA and 0.05% P20) at a flow rate of 1 ml/min. Monomeric eluted peaks were used to flow over Fc-capture 3A8 mAb surfaces. To prepare the 3A8 mAb surfaces, approximately 6000 RUs of anti-mouse IgG (GE Healthcare) was first immobilized on CM5 Series S sensor chips (GE Healthcare) at pH 5.0, using standard amine coupling methods previously reported (Hussack et ai, 2011) and according to the manufacturer’s instructions (GE Healthcare). HBS-EP running buffer used for SPR experiments. The mAb 3A8 was captured on the anti-mouse IgG surface using 10 pg/ml of mAb, 30 sec contact time and flow rate of 10 mI/min. IMAC-purified Dl-D V _h H was passed through a Superdex 75 (GE Healthcare) column for SEC purification in HBS-EP buffer (10 mM HEPES, pH 7.4, containing 150 mM NaCI, 3 mM EDTA and 0.005% P20), connected to an AKTAPurifier (GE Healthcare) at a flow rate of 1 ml/min. Test binding experiments of FC5 control and FC5DIE3IR involved flowing 1 mM concentrations of SEC-pure material over the mAb surface at a flow rate of 30 mI/min for 5 min. Surfaces could be regenerated using 10 mM glycine, pH 1.7, with a 120 sec pulse at 20 mI/min. All SPR experiments were evaluated with Biacore T200 Software v 3.0. For kinetic analysis, single cycle kinetic analysis was used by flowing a range of FC5DIE3IR concentrations (320 nM - 20 nM) at a flow rate of 30 mI/min, with 600 sec contact time and 1200 sec dissociation. Regeneration conditions were the same as above. Data were fit to a 1 :1 binding model and analyzed using the Biacore T200 Software v3.0.

For affinity determination, various concentrations of the antibodies (10-5000 nM) were injected over FC5DIE3IR, FC5 and 2A3-H4 surfaces, using an ethanolamine blocked surface as a reference, at a flow rate of 40 mI/min with 60 s contact time and 180 s of dissociation. Surfaces were regenerated by washing with running buffer (HBS-EP). Data were analyzed with BIAevaluation 4.1 software as described (Hussack et ai, 201 1) and affinities determined using the steady state binding model. Surface Plasmon Resonance for V _h H antibodies. For SPR experiments, IMAC-purified V _H HS Dl- D, DI-4, DI-16, DI-28 and DI-48 were passed through a Superdex 75 (GE Healthcare) column for SEC purification in HBS-EP+ buffer (10mM HEPES, pH 7.4, containing 150 mM NaCI, 3 mM EDTA and 0.05% P20), connected to an AKTAPurifier (GE Healthcare) at a flow rate of 1 ml/min. Monomeric V _h H fractions were collected and protein concentration determined by measuring A ₂₈ O measurement. Using a Biacore T200 instrument (GE Healthcare), approximately 500-1000 RUs of FC5DIE3IR, FC5 and an irrelevant V _h H (2A3-H4) were immobilized on a CM5 Series S sensor chip (GE Healthcare) at pH 4.0 using standard amine coupling according to the manufacturer’s instructions (GE Healthcare). For affinity determination, various concentrations of the antibodies (312 nM - 5000 nM for DI-4, DI-48 and Dl-D; 2.5 nM - 500 nM for DI-16 and DI-28) were injected over FC5DIE3IR, FC5 and 2A3-H4 surfaces, using an ethanolamine blocked surface as a reference, at a flow rate of 30 mI/min with 180 s contact time and 600 s of dissociation. Surfaces were regenerated by washing with a 120 s pulse of 10 mM glycine pH 1.5. Data were analyzed with Biacore T200 Software v 3.0 and affinities determined using steady state binding or a 1 :1 binding model.

Soft Agar Assay Method. The anchorage-independent cell growth of two glioblastoma cell lines (U87MG and U87MGvlll) and an ovarian cell line (SKOV-3) was evaluated in semi-solid agar in the absence or presence of either TTX (0.5 mM) or 1 B6 (1 mM), 1 H5 (1 mM) or 2G1 1 (1 mM) as described previously (Moreno et al., 2006). Approximately 1800 cells ± treatment were resuspended in 150 mI of growth medium (DMEM + 10% FBS) containing either 0.6% agar (U87MG and U87MGvlll) or 0.45% agar (SKOV3) and seeded into a well of a 24-well plate previously layered with 250 mI of 0.6% agar. The solidified cell layer was covered with 50 mI of DMEM ± treatment that was replaced every 3 days over a 14-day period. Phase contrast images (six fields per well) were captured using a digital video camera (Olympus U-CMT) and analyzed with Northern Eclipse v.5.0 software. To measure cell viability, at the end of the experiment, 50 mI of Alamar Blue™ (Cedarlane, Burlington, Ontario) was added to each well and fluorescence readings (530 nm excitation and 590 nm emission) were performed every 10 minutes for a period of 180 minutes.

Western Blot Method. Cell pellets from DU-145, Jurkat, LN18, PC-3, SKOV-3, U87MG, U87MG and Nav1.7-overexpressing HEK cells were lysed in RIPA buffer (BioRad) with Complete protease inhibitor cocktail (Roche) and protein concentrations determined using BioRad Reagent. Protein samples (30pg) were prepared with loading buffer (BioRad), boiled for 5 minutes in a boiling water bath, and were loaded on a Mini-protean TGX 4-15% gel (BioRad). Proteins and Precision Plus Dual Colored Standard (BioRad) were separated at 100V then electrotransfered to nitrocellulose at 80V for 1 h. The nitrocellulose membrane was blocked for 1 h in 1X TBST with 5% Carnation skim milk. Rabbit polyclonal anti-Na _v 1.7 (Alomone labs, ASC- 008) was diluted 1 :200 in the same buffer and incubated on the blots overnight at 4°C on a rocking shaker. The blots were washed 3 times for 10 minutes with TBST, and incubated with anti-rabbit-HRP antibody (Invitrogen) at 1 :5000 in TBST/milk for 1 h at room temperature. The blots were washed 3 times for 10 minutes as above, incubated for 5 minutes with Clarity ECL substrate (BioRad) and exposed to film. Film was developed on a Minolta SRX101A film developer and scanned on a Sharp MX-41 1 1 N copier. The membrane was washed for 30 minutes with TBST and incubated for 1 h with anti-pactin-HRP (Sigma). The blot was washed 3 times for 10 minutes with TBST and exposed to film. Images were analyzed using Adobe Photoshop 7.0.1.

Western Blot Method (alternative method). Cell pellets from pancreatic tumor cell lines, Capan-1 , Bxpc3, Mia-Paca2 and Panel , were lysed in RIPA buffer (BioRad) with Complete protease inhibitor cocktail (Roche) and protein concentrations determined using the Quantipro BCA Assay Kit (Sigma). The gel was run using the 12-230kDA separation module (ProteinSimple), and the rabbit detection module (ProteinSimple). The samples (0.8mg/ml) were prepared by combining the Master Mix to sample in a 1 :4 ratio. The samples and a biotinylated ladder were heated in a Accublock digital dry bath at 95°C for 5 minutes. The samples were cooled to room temperature, vortexed to mix and centrifuged in a Mandel mini microfuge. The biotinylated ladder (5 pi) was loaded in the first well of row A1. The samples (4 mI) were loaded in the remaining wells of row A. An antibody diluent (10 mI) was added to row B and the first well of row C. A rabbit polyclonal anti-Na _v 1.7 (Alomone labs, ASC-008, 1 :20) and an anti-pactin-HRP (Sigma, 1 :100) were combined in antibody diluent and added to the remaining wells of row C. Streptavidin-HRP (10 mI) was added to the first well of row D. Anti-rabbit secondary antibodies were added to the other wells of row D. Luminol and peroxide were mixed (200 mI of each) and 15 mI was added to each well of row E. The plate was covered with a lid and centrifuged at 1000g for 5 minutes in a Eppendorf 581 OR centrifuge. A wash buffer (500 mI) was added to three rows of wells on the plate. The foil was removed from the separation reagents and the plate and capillaries were placed in the Wes™ (automated western blot system; ProteinSimple, San Jose, CA, USA) for analisys.

EXAMPLE II - ANTI-NA _V 7 MOUSE MONOCLONAL ANTIBODIES

Animal immunization. Four six-week old female A/J mice (The Jackson Laboratory, Bar Harbor, ME) were bled (pre-immune serum) and injected intraperitoneally and subcutaneously with 100 pg of FC5DIE3IR antigen emulsified in Titermax adjuvant (Cedarlane Labs, Burlington, ON) at day 0 and in PBS without adjuvant at day 26. Blood was collected in microvette CB 300Z (Sarstedt, Montreal, QC) at day 33, and serum was stored at -20°C until further use.

ELISA (serum titer determination). Pre- and post-immune sera titer of animals immunized with FC5DIE3IR antigen were assessed by ELISA. Unless otherwise stated, all incubations were performed at room temperature. Briefly, half-area 96-well plates (Costar #3690) were coated with 25 pi per well of FC5DIE3IR at 5 pg/ml in PBS and incubated overnight at 4°C. Microplates were washed three times in PBS and blocked for 30 min with PBS containing 1 % bovine serum albumin (BSA, Sigma Cat#A7030). Blocking buffer was removed and 25 mI of serial dilutions of sera samples were added. After a 2 h incubation, microplates were washed 4 times with PBS- Tween 20 0.05% and 25 mI of a 1/5.000 dilution of alkaline phosphatase conjugated goat antimouse IgG (H+L) (#1 15-056-062, Jackson Immunoresearch, Cedarlane, Burlington, ON) in blocking buffer was added. After a 1 h incubation, microplates were washed 4 times and 25 mI of p-nitrophenyl phosphate (pNPP) substrate (Sigma-Aldrich Canada Co., Oakville, ON) at 1 mg/ml in carbonate buffer at pH 9.6 was added and further incubated for 30 min. Absorbance was read at 405 nm using a SpectraMax plate reader (Molecular Devices, Sunnyvale, CA). All pre-immune bleeds were negative and all post-immune bleeds were very strong (above 1/12800) on FC5DIE3IR. After 2-3 months, an i.p. booster injection (100 pg of FC5DIE3IR protein in PBS) was done 3/4 days prior to fusion experiment.

Fusion of the harvested spleen cells. All manipulations were done under sterile conditions. Spleen cells were harvested in Iscove’s Modified Dulbecco’s medium (IMDM, Gibco Cat. #31980-030) and fused to NS0 myeloma cell line using polyethylene glycol (PEG, F233) or electrofusion (F236) protocols.

Fusion with PEG. Spleen cells and myeloma cells were washed in IMDM, counted in RBC lysing buffer (Sigma, Cat#7757-100ML) and mixed together at a 5:1 ratio. Pelleted cells were fused together by adding 1 ml of a 50% solution of PEG 4000 (EMD-Millipore Cat#9727-2) in PBS preheated at 37°C drop-wise over one minute, and incubated at 37°C for an additional 90 sec. The reaction was stopped by addition of 30 ml of IMDM at 22°C over a period of 2 min. After a 10 min incubation, freshly fused cells were spun at 233g for 10min. Cells were washed once in IMDM supplemented with 10% heat inactivated FBS (Sigma Cat #F1051).

Electrofusion protocol. Spleen cells and myeloma cells were washed separately in IMDM. Red blood cells from splenocytes preparation were lyzed with RBC lysing buffer. Cells were washed in Isoosmolar buffer (Eppendorf cat#4308070536), then in Cytofusion Medium C (BTX cat#47- 0001). Myeloma and lymphocytes were mixed together at a 2:1 ratio and fused using an ECM 2001 Cell Fusion System (BTX, Harvard Bioscience Inc.) following manufacturer’s instructions.

Following fusion, cells were suspended at a concentration of 2 x 10 ⁵ input myeloma cells per ml in HAT selection medium (IMDM containing 20% heat inactivated FBS, penicillin-streptomycin (Sigma Cat#P7539), 1 ng/ml mouse IL-6 (Biolegend Cat#575706), HAT media supplement (Sigma Cat#H0262) and L-glutamine (Hy-Clone Cat#SH30034.01) and incubated at 37°C, 5% C0 ₂ . The next day, hybridoma cells were washed and suspended at a concentration of 2-3X10 ⁵ input myeloma cells per ml in semi-solid medium D (StemCell Technologies Cat.#03804) supplemented with 5% heat inactivated FBS, 1 ng/ml mouse IL-6 and 10 pg/ml FITC- F(ab’) ₂ Goat anti-mouse IgG (Jackson # 1 15-096-071 for F233 or 1 15-096-062 for F236). The cell mixture was plated in Omnitray dish (Nunc cat#24281 1) and further incubated for 6-7 days at 37°C, 5% C0 ₂ . Fluorescent secretor clones were then transferred using a mammalian cell clone picker (ClonepixFL, Molecular Devices) into sterile 96-w plates (Costar #3595) containing 200 pi of IMDM supplemented with 20% heat inactivated FBS, penicillin-streptomycin, 1 ng/ml mouse IL-6, HT media supplement (Sigma Cat# H0137) and L-glutamine and incubated for 2-3 days at 37°C, 5% C0 ₂ .

Screening. Hybridoma supernatants were screened by ELISA to detect specific binders. To this end, 96-well half-area plates (Costar #3690) were coated with 25 pi of FC5DIE3IR or FC5-V _h H or FC5-hFc-1X0 at 5 mg/ml in PBS and incubated overnight at 4°C. Microplates were washed 3 times with PBS, blocked with PBS-BSA 1 %, and 25 mI of hybridoma supernatants were added and incubated at 37°C, 5% C0 ₂ for 2 hours. Plates were washed 4 times with PBS-Tween 20 0.05% and incubated for one hour at 37°C, 5% C0 ₂ with 25 mI of secondary antibody alkaline phosphatase conjugated F(ab’) ₂ goat anti-mouse IgG Fc-gamma specific (Jackson Immunoresearch # 1 15-056-071) diluted 1/3000 in blocking buffer. After 4 washes with PBS- Tween 20 0.05%, 25 mI of a 1 mg/ml pNPP substrate solution was added and further incubated for one hour at 37°C. OD ₄ o _5nm measurements were done using a microplate reader (Spectramax 340 PC, Molecular Devices).

From the two fusions of the mouse spleen cells, 31 mAbs specific for FC5DIE3IR were identified from which supernatant was collected and evaluated for binding to CHO expressing human Na _v 1.7 by ELISA.

Cell-ELISA. Sterile 96-well plates (Costar #3595) were pre-treated with 50 mI/well of branched polyethyleneimine (PEI, Sigma Cat#408727) at 25 pg/ml in PBS for 30 min at room temperature. PEI was removed and CHO-3E7 or hNA _v 1.7-CHO (ChanTest cat#CT4003) were seeded at 6 X 10 ⁵ cells per well in Freestyle F17 Expression Medium (Gibco Cat#A1383501) and incubated overnight at 37°C, 5% C0 ₂ . Microplates were blocked with Starting block (Thermofisher Cat#37538) for 30 min at room temperature, and 50 pi of hybridoma supernatants were added and incubated at 4°C for 3 h. Plates were washed 5 times with PBS and incubated for one hour at 4°C with 50 pi of secondary antibody HRP-conjugated F(ab’) ₂ goat anti-mouse IgG (H+L) (Jackson Immunoresearch # 1 15-036-062) diluted 1/5000 in blocking buffer. After 10 washes with PBS, 50mI of TMB substrate solution (Pierce Cat#34021) was added and further incubated for 60 min at room temperature. Twenty-five mI/well of stop solution (H2S04 1 M) was added and OD ₄₅ o _nm measurements were done using a microplate reader (Spectramax 340 PC, Molecular Devices). Summary ELISA results for the top five clones are shown in Table 1.

Table 1. Evaluation of the supernatant collected from mAb-producing hybridomas by ELISA

*Not determined

All mAbs were then purified using Protein G Mag Sepharose (GE Cat#28-9670-70) and desalted using Zeba-spin desalting columns (Pierce cat#89889) equilibrated in PBS. The final concentration of the antibody solutions was determined using a Nano-drop 1000 (ThermoFisher), using IgG as sample type.

Evaluation of apparent affinity in ELISA. Purified antibodies were assessed for their binding activity by ELISA in a dose-dependent binding curve. Serial 1/3 dilutions of purified mAbs starting at 120 nM were applied onto wells and ELISA was performed as described above (screening section). The data were analyzed with GraphPad Prism software using one-site specific binding-with Hill slope non-linear regression curve fit model to determine B _max (maximum specific binding) and Kd _app (concentration needed to achieve a half-maximum binding at equilibrium) for each mAb tested. As shown in Figure 5, the apparent K _D of the 3A8 clone (1.9 nM) is approximately 2.5 times lower than for the 1 G5 clone (4.8 nM). Without wishing to be bound to theory, the difference in B _max observed in Figure 5 (3.2 vs 1.7) may be due to the use of polyclonal secondary antibodies that may bind more efficiently to lgG2a (3A8) vs IgG 1 (1 G5).

Re-cloning of hybridomas. Selected hybridomas were re-cloned by limiting dilution to ensure their monoclonality.

In vitro identification of functional monoclonal antibody (mAbs). To determine the functional effect of anti-hNa _v 1.7 mAbs on hNa _v 1.7 channels, Na _v currents were evoked using whole-cell patch-clamp technique in hNa _v 1.7-HEK293 cells as described in Example I. The hNa _v 1.7- HEK293 cells were prepared from frozen vials and plated on poly-l-lysine coated 13 mm plastic coverslips. The coverslips were then mounted on a recording chamber and placed under an inverted microscope to perform the patch-clamp experiments. Recordings of Na _v currents were obtained as described in Example I.

As shown in Figure 6, the mAb 3A8 was able to reduce the amplitude of the Na _v currents by 8.2% ± 1.7 (n= 7) and 16.0% ± 2.1 (n= 5) at the concentration of 1 mM and 2 pM, respectively, while the mAb 1 G5 did not change the amplitude of the currents (0.77% ± 1.34, n=12 Figures 6A, B and E). The mAb 3A8 had an effect onset (time constants of the block of the Na _v 1.7 currents) of 184 ± 39 sec (n=4) when applied at the concentration 1 pM and 105 ± 6 sec (n= 2) when applied at the concentrations of 2 pM (Figure 6B). These data suggest that 3A8 is functional in inhibiting the flux of Na ⁺ thought the hNa _v 1.7 channels. Based upon the results presented in Figure 6, the mAb 3A8 concentration response relationship (IC ₅₀ ) and efficacy (degree of maximum inhibition) were 0.9 ± 0.2 pM and 16.0 ± 2.1 %, respectively (Figure 6F).

Next, it was evaluated if the anti-hNa _v 1.7 3A8 mAb had an effect on the kinetics and voltage dependence properties of the hNa _v 1.7 channels. To do this, the effect of 3A8 on the current- voltage (l-V) relationship, the voltage-dependence of activation and steady-state fast inactivation of hNa _v 1.7 channels were examined in hNa _v 1.7-HEK293 cells (Figures 6C, 6D). The mAb 3A8 shifted the voltage-dependence of activation of Na _v 1.7 currents to more hyperpolarized membrane potentials (V _1/2 activation: D = -6.0 ± 1.0 mV at 1 pM; n= 7), suggesting a gating modifier activity (Figure 6D). A minor effect was also observed on the voltage-dependence of the steady-state fast inactivation (V _1/2 inactivation: D = -7.1 ±1.7 mV at 1 pM; n=4). However, these shifts towards hyperpolarizing voltages of activation and steady-state inactivation curves may not be entirely due to mAb 3A8. In fact, it has been reported that changes in activation and inactivation of Na _v channels may be caused by perturbations of the membrane-cytoskeleton interaction due to the application of the antibody. In addition, negative- shifts of the same order were observed also in control experiment in which an equivalent volume of extracellular solution containing no antibody was added instead of the antibody solution. To further investigate the effects of 3A8 on hNa _v 1.7 kinetics of activation and fast inactivation, the time-to-peak and the time constants of fast inactivation of hNa _v 1.7 channels were calculated. The mAb 3A8 (1 mM) did not significantly change the times-to-peak measured at -20 mV (control, 0.91 ± 0.06 ms; 3A8, 0.88 ± 0.07 ms; n= 7; p>0.10, paired f-Test). It also did not change the inactivation time constants at -20 mV (control 0.80 ± 0.12 ms; 3A8 0.72 ± 0.08 ms; p >0.10, paired f-Test).

As shown in Figure 7, the mAb 1 B6 was able to reduce the amplitude of the Na _v currents by 27.7% ± 2.9 (n= 20) and 40.3% ± 5.7 (n=12) at the concentration of 1 mM and 2 mM, respectively (Figures 7A, 7C and 7E; Table 2). The mAb 1 B6 had an effect onset (time constants of the block of the Na _v 1.7 currents) of 416 ± 126 sec (n=5) when applied at the concentration 1 mM and 84 ± 6 sec (n=4) when applied at the concentrations of 2 mM (Figure 7B). 1 B6 concentration response relationship (IC ₅₀ ) and efficacy (degree of maximum inhibition) was 1.02 ± 0.19 mM and 54.3% ± 3.7, respectively (Figure 7F; Table 2).

As shown in Figure 8, the mAb 2G1 1 was able to reduce the amplitude of the Na _v currents by 22.8% ± 3.7 (n=14) and 35.4% ± 6.1 (n=15) at the concentration of 1 mM and 2 mM, respectively (Figures 8A, 8C and 8E; Table 2). The mAb 2G1 1 had an effect onset (time constants of the block of the Na _v 1.7 currents) of 68.8 ± 0.8 sec (n= 2) when applied at the concentration 1 mM and 39 ± 9 sec (n=3) when applied at the concentrations of 2 mM (Figure 8B). 2G1 1 concentration response relationship (IC ₅₀ ) and efficacy (degree of maximum inhibition) was 1.06 ± 0.29 mM and 50.5% ±.03, respectively (Figure 8F; Table 2).

As shown in Figure 9, the mAb 1 H5 was able to reduce the amplitude of the Na _v currents by 38.4% ± 4.8 (n=10) and 36.7% ± 3.2 (n=14) at the concentration of 1 mM and 2 mM, respectively(Figures 9A, 9C and 9E; Table 2). The mAb 1 H5 had an effect onset (time constants of the block of the Na _v 1.7 currents) of 78 ± 15 sec (n=4) when applied at the concentration 1 mM and 149 ± 46 sec (n=4) when applied at the concentrations of 2 mM (Figure 9B). 1 H5 concentration response relationship (IC ₅₀ ) and efficacy (degree of maximum inhibition) was 0.56 ± 0.02 mM and 38.4% ± 4.4, respectively (Figure 9F; Table 2).

These data suggest that also and 1 B6, 2G 1 1 and 1 H5 are functional in inhibiting the flux of Na ⁺ thought the hNa _v 1.7 channels. Values for the effect of anti-hNa _v 1.7 1 B6 (Figure 7D) 2G1 1 (Figure 8D), 1 H5 (Figure 9D) on the kinetics and voltage dependence properties of the hNa _v 1.7 channels were calculated in the same way as for 3A8 (see above) and are listed in Table 2.

Overall, these data suggest that the main functional effect of 3A8, 1 B6, 2G1 1 and 1 H5 on Na _v 1.7 is on the Na ⁺ permeation pathway, which reduces the amplitude of the current through the channel. Table 2 below summarizes the characteristics of the monoclonal antibodies obtained.

Table 2. Values for the effect of anti-hNa _v 1.7 3A8, 1 G5, 1 H5, 2G11 , 1 B6 on the kinetics and voltage dependence properties of the hNa _v 1.7 channels in the resting/closed state

In vivo Validation of the mAh 3A8: Rat Hargreaves Model of Hyperalgesia. To evaluate if the functional in vitro effect of anti hNa _v 1.7 mAbs translated in a functional in vivo effect against pain, the mAbs were tested in rats using a Hargreaves model of hyperalgesia. The anti-hNa _v 1.7 mAb 3A8 was tested along with a negative control anti-GFP mAb 11 E5. As previously demonstrated, CFA induced hyperalgesia to a thermal stimulus 48 h post injection. Baseline latency paw withdrawal was reduced to 4.33 ± 0.22 sec (n=12) in ipsilateral paw versus 19.89 ± 0.06 sec (n=12) of contralateral paw (see Figure 10). The mAb effect on the latency paw withdrawal was measured 4 hours after administration. Intraplantar injection of 3A8 (8.50 ± 0.94sec, n=3) but not 11 E5 (5.13 ± 0.19 sec, n=3) significantly increased latency paw withdrawal (p< 0.05 vs baseline;

Figure 10). Taken together, these results suggest that targeting Na _v 1.7 successfully promoted the reversal of hyperalgesia in a chronic pain model.

To further evaluate the effect of 3A8, a CFA-induced thermal hyperalgesia in rats was used to compared the mAb with the selective blocker for hNa _v 1.7 channels TC-N1752 (Tocris, Bristol, United Kingdom). Figure 1 1 shows the time-course effect and dose-response effects of anti- hNa _v 1.7 mAb 3A8 at 50 pg and 100 pg, and TC-N1752 (100 pg) injected by intraplantar route. The anti-hNa _v 1.7 mAb 3A8 was able to increase the latency paw withdrawal time when injected at 50 pg (6.08 ± 0.70 sec, 7.38 ± 0.67 sec, 6.79 ± 0.50 sec at 1 , 2 and 4 h respectively; n=3) and at 100 pg (9.56 ± 0.70 sec, 10.58 ± 0.90 sec, 10.28 ± 1.20 sec at 1 , 2 and 4 h respectively; n=4). In addition, 3A8 at 100 pg had an effect similar to that of TC-N1752 (100 pg; 8.15 ± 1.6 sec, 1 1.17 ± 1.19 sec, 1 1.90 ± 0.99 sec at 1 , 2 and 4 hours respectively; n=3). The effect of 3A8 (50 and 100 pg), 1 1 E5 (negative control) and TC-N1752 (100 pg) were also expressed as percentage of the effect on left paw withdrawal (% Effect, Figure 1 1 B). Table 3 below summarizes the values of % Effect for 1 1 E5, 3A8 and TC-N1752. From these values the %MPE is extrapolated. Overall these data suggest that anti-hNa _v 1.7 mAb 3A8 functionally mediated reversal of hyperalgesia in a chronic pain model.

Table 3. Values of the effect of 1 1 E5, 3A8 and TC-N1752 expressed as percentage of the effect on left paw withdrawal.

To further evaluate the effect of the anti-hNa _v 1.7 mAbs 2G11, 1H5 and 1B6, and the selective blocker for hNa _v 1.7 channels TC-N1752 (Tocris, Bristol, United Kingdom) a CFA-induced thermal hyperalgesia in rats was used. Figure 12 shows the time-course effect and dose- response effects of anti-hNa _v 1.7 mAbs 2G11, 1H5, 1B6 at 100 pg, and TC-N1752 (100 pg) injected by intraplantar route. Intraplantar injection of 2G11, 1H5 or 1B6 (8.45 ± 0.44 sec, n=4; 9.43 ± 0.72 sec, n=4; 8.66 ± 0.45 sec, n=4) but not 11E5 (5.12 ± 0.19 sec, n=2) significantly increased latency paw withdrawal (p< 0.05 vs baseline). Taken together, these results suggest that targeting Na _v 1.7 successfully promoted the reversal of hyperalgesia in a chronic pain model.

To further evaluate the effect of 2G11, 1B6 and 1H5, they were tested on CFA-induced thermal hyperalgesia in rats and compared it with the selective blocker for hNa _v 1.7 channels TC-N1752 (Tocris, Bristol, United Kingdom). Figure 12 right shows the time-course effect of anti-hNa _v 1.7 2G11, 1H5and 1B6 and TC-N1752 at 100 pg injected by intraplantar route.2G11, 1H5 and 1B6 were able to increase the latency paw withdrawal time when injected at 100 pg (2G11: 8.80 ± 1.02 sec, n=4; 7.86 ± 0.33 sec, n=4; 8.45 ± 0.44 sec at 1, 2 and 4 hours respectively; n=4; 1H5: 7.97 ± 0.61 sec, n=4; 10.03 ± 0.11 sec, n=4; 9.43 ± 0.72 sec at 1, 2 and 4 hours respectively; n=4; 1B6: 7.79 ± 0.56 sec, n=4; 8.08 ± 0.46 sec, n=4; 8.66 ± 0.45 sec at 1, 2 and 4 hours respectively; n=4;) similar to that of TC-N1752 (8.15 ± 1.6sec, 11.17 ± 1.19sec, 11.90 ± 0.99sec at 1, 2 and 4 hours respectively; n=3). Overall these data suggest that each of 2G11, 1H5 and 1B6 functionally mediated reversal of hyperalgesia in a chronic pain model.

The effect of 2G11, 1H5 and 1B6 were also expressed as percentage of the effect on left paw withdrawal (% Effect). Table 4 below summarizes the values of % Effect for 1 H5, 2G11 and and 1B6. From these values the %MPE was extrapolated (2G11, 27.98 ± 7.17 (n=4) after 1 hour from injection; 1H5, 35.23 ± 0.88 (n=4) after 2 hours from injection; 1B6, 25.96 ± 3.69 (n=4) after 4 hours of injection).

Table 4. Values of the effect of 1H5, 2G11 and 1B6 expressed as percentage of the effect on left paw withdrawal.

It was next evaluated the effects of 3A8 injected by intravenous route. Figure 13A shows that 3A8 at a dose of 30 mg/kg through the tail vein was able to reverse CFA-induced hyperalgesia. Effects are observed one hour post injection and lasts for up to 24 h. Figure 13B shows the response at 4 h expressed as percentage of the maximum possible effect (PBS, 5.50 ± 3.4, n=3 and 3A8, 26.60 ± 3.26, n=4).

To further evaluate the functionality of the anti-Na _v 1.7 mAbs and target engagement, they were tested on a Na _v 1.7 pain model; OD1 pain model. The intraplantar administration of OD1 in mice induces spontaneous pain behaviors as evidenced by licking, flinching, lifting and shaking of the injected hind paw. These behaviors are dose dependent (Figure 14A) and develop rapidly, occurring soon after injection, and persists for up to 40 min after injection. One major advantage of the intraplantar route of administration is that it delivers the peptide directly to the terminals of peripheral sensory neurons in the skin, allowing simple quantification of unilateral pain behaviors while avoiding systemic adverse effects. Different concentrations of OD1 were tested (Figure 14A). The injection of 30 pi of OD1 at the concentration of 0.75 mM evoked a response of 200 sec after 15 minutes from the injection (Figure 14A), similar to what reported in literature (Deuis et al. , 2016). Thus this concentration was selected to test our to test the antibodies. The effect of 100 pg of mAb injection was tested and compared it with the injection of saline (saline plus OD1). The pain behavior was considered at 100%, 20 min after the injection. Twenty min after the injection, 100 pg of 3A8 (Figure 14C), 1 H5 (Figure 14D), 2G1 1 (Figure 14E) caused a significant reduction of the time spent in pain behaviors (3A8; 73.76% ± 5.38; 1 H5, 73.84% ± 16.78; 2G1 1 63.47 ± 4.78) whereas 1 B6 (Figure 14F; 94.13% ± 28.63) was not different from the negative control 1 1 E5 (Figure 14B; 93.40% ± 8.67). TC-N5172 was 51.62% ± 4.85.

Identification of the hNa _v 1.7 epitope for mAb 3A8, 1B6 and 1H5. To identify the epitope for mAb 3A8 on DIE3IR, seven overlapping antigen-peptides were generated (Figure 15A) and an ELISA was performed to determine the binding between these peptides and the various mAb (Figures 15C to 151). The maximum binding for 3A8 was observed with pep#40a (Figure 15B), pep#2 (Figure 15C and 15D) and pep#2a (Figure 15H). Similarly, the maximum binding for 1 B6 was observed with pep#40a (Figure 15B), pep #2 (Figure 15E), but 1 B6 showed no binding whith pep#2a (Figure 15F) and pep#2b (Figure 151). The mAb 2G1 1 showed binding for only the pep#40a (Figure 15B), The mAb 1 H5 showed low binding with pep#40a (Figure 15B) and pep#3 (Figure 15F and 15G). Figure 15J show a summary of the region of binding between mAbs and peptides. This clearly indicates that 3A8 and 1 B6 binding motif resides in pep #2a and pep #2, respectively. The maximum binding for 1 H5 was observed with Pep #3, while Pep #1 and Pep # 2 showed low binding, clearly indicating that 1 H5 binding motif resides in Pep #3.

Differential HDX-MS was performed to obtain a snapshot of the changes in conformational stability imparted by binding events and as a result to map antibody based-interactions (Shaolong et al., 2018). Differential HDX-MS was performed by comparing the extent of deuteration between bound and unbound forms of FC5DIE3IR. Overall, deuteration was measured for 38 peptides covering 74% of the sequence of the entire FC5-DIE3IR, including 94% coverage of the grafted DIE3IR domain (Figure 16). Full HDX-MS kinetics plots can be found in Figure 17. As expected, no effect on conformational stability was observed for the negative binding control, 1 G5. No significant changes in deuteration were observed outside of the DIE3IR domain for any of the mAbs, demonstrating that the mAbs are only binding the grafted DIE3IR domain. Stabilization across residues Asn121-Tyr134 (NTLESEED; SEQ ID NO: 108) was observed in response to 3A8 and 2G1 1 binding (Figures 17A and 17B). Stabilization in response to 1 B6 binding was located to residues Phe129-Tyr134 (FRKYFY; SEQ ID NO: 107; Figure 17A). In each of these cases, stabilization is centered on the DIE3IR a-helix spanning residues Ser125-Lys131 , providing additional evidence that the epitopes are at least partially conformational in nature. Unlike 2G1 1 and 1 B6, the deuteration of 3A8 did not converge with that of the unbound control, particularly in residues Phe129-Tyr134. This may be a result of 3A8 inducing a stronger stabilization at the aforementioned helix, however the functional impact of this observation is unclear. Finally, no stabilization was observed in the presence of 1 H5; this is likely a result of its challenging binding kinetics (Table 5).

In vivo validation of the 3A8 epitope. To verify if in the sequence of the peptide antigen fragment Pep#2 (P2) resides the binding motif of 3A8, the effect of P2 and Pep#3 (P3) on 3A8-mediated reversal of hyperalgesia was tested. P2 (375 pg) or P3 (375 pg) were co-injected with 3A8 (75 pg) by intraplantar route in rats Hargreaves model and their effect compared to the reversal of hyperalgesia caused by 3A8 (75 pg) and 11 E5 (negative control, 50 pg) (Figure 18A). When coinjected with 3A8, P2 was able to reduce (6.76 ± 0.32 sec, n=5) the latency paw withdrawal time observed with 3A8 alone (10.59 ± 0.87 sec, n=3), while P3 did not had any effect on 3A8 ability to reduce the latency paw withdrawal time (10.17 ± 0.65 sec; n=4; Figure 18B). The 3A8-induced reversal of hyperalgesia (35.35% ± 4.44; n=3) was significantly reduced by P2 (13.49% ± 2.62; n=5), while was not affected by P3 (34.63% ± 4.71 , n= 5; Figure 18B). These data strongly support 3A8 binding motif residing in the P2 sequence.

Surface Plasmon Resonance (SPR) for monoclonal antibodies. To determine the binding affinity of 3A8, 2G1 1 , 1 B6 and 1 H5 for the recombinant protein FC5DIE3IR, Surface Plasmon Resonance (SPR) analyses were performed as described in Example I. Pure monomeric fractions of target protein FC5DIE3IR and control V _h H FC5 were obtained using Size Exclusion Chromatography purification (SEC). To examine the binding of 3A8, 2G1 1 , 1 B6 and 1 H5 to the recombinant protein FC5DIE3IR, two SPR assay orientations were used:

• Fc-captured mAb 3A8, 2G1 1 , 1 B6 and 1 H5, and flow of FC5DIE3IR and FC5 control, and

Immobilized FC5DIE3IR and FC5 control, and flow mAb 3A8, 2G1 1 , 1 B6 and 1 H5 In both orientations 3A8, 2G1 1 and 1 B6 strongly and specifically bound to FC5DIE3IR and did not bind to FC5 control (see Table 5). At 1 mM concentration of FC5 and FC5DIE3IR flowed over the captured 3A8 (Figure 19), specific and high affinity binding of FC5DIE3IR is evident. The results also showed that 3A8 bind to the immobilized FC5DIE3IR with an extremely slow off rate. The binding affinity of FC5DIE3IR to Fc-captured 3A8. 2G21 1 , 1 B6 and 1 H5 was determined using Single Cycle Kinetics (SCK; Figure 20) analysis, using a range of FC5DIE3IR concentrations from 320 nM - 20 nM. The captured 3A8, 2G1 1 and 1 B6 showed strong and specific binding to FC5DIE3IR. Values of K _D s, on-rate (ka (1/Ms)) and R _max are shown in Table 5. Rmax indicates that the captured mAbs possesses high activity (theoretical R _max ~120 RU). The mAb 1 H5 could not be fitted with the 1 :1 model, consequently KDs, on-rate (ka (1/Ms)) and Rmax values could not be calculated.

Table 5. Affinity and binding kinetics for mAbs 2G11 , 1 B6, 1 H5, 3A8 and 1G5 obtained with SPR.

Recombinant Chimeric Human-Mouse mAbs. To validate the sequences of the mAbs (3A8, 1 B6; Table 7), recombinant chimeric human-mouse mAbs (hFc-F233-3A8, hFc-F236-1 B6) have been production and purified (for sequences see Table 7).

In vitro Validation of Recombinant Chimeric Human-Mouse mAbs. To determine if the recombinant chimeric human-mouse mAbs hFc-F233-3A8 and hFc-F236-1 B6 were fuctional in reducing the hNa _v 1.7 currents, Na _v currents were evoked using whole-cell patch-clamp technique in hNa _v 1.7-HEK293 cells as described in Example I. The hNa _v 1.7-HEK293 cells were prepared from frozen vials and plated on poly-l-lysine coated 13 mm plastic coverslips. The coverslips were then mounted on a recording chamber and placed under an inverted microscope to perform the patch-clamp experiments. Recordings of Na _v currents were obtained as described in Example I.

At the concentration of 2 mM, hFc-F233-3A8 and hFc-F236-1 B6 were able to reduce significantly the amplitude of the Na _v currents (Figures 21 A and 21 B) by 28.6% ± 6.0 (n=9; p= 0.005 ) and 29.7% ± 5.4 (n=1 1 ; p=0.0005), respectively. These data validate the antibodies sequences.

Effect of mAbs Application on Cancer models. The four monoclonal antibodies targeting Na _v 1.7 were tested on a number cancer cell lines expressing Na _v 1.7. Expression of Na _v 1.7 in these cell lines was first assessed with western blot analysis. Figures 22A and 22B show the results of western blot analysis performed on 1 1 cancer cell lines: DU-145 (model of prostate cancer), Jurkat (model of leukemia, LN18 (model of glioblastoma), PC-3 (model of prostate cancer), SKOV-3 (model of ovarian cancer), U87MG and U87Mgvlll (models of glioblastoma), Capan-1 , Bxpc3, Mia-Paca2 and Panel (all models of pancreatic cancer). Expression of the Na _v 1.7 protein was found in all cell lines, except the prostate cancer PC-3 cells. The functionality of Na _v 1.7 on these cell lines was also studied in electrophysiology experiments. Figures 23A and 23B show recordings of Na _v 1.7 currents measured in the U87MG glioblastoma cells and the SKOV-3 ovarian cancer cells, respectively. Application of tetrodotoxin (TTX), a potent inhibitor of voltage gated sodium channels, successfully and completely blocked Na _v 1.7 currents in these cells (Figure 23A), and application of monoclonal antibodies provoked a partial inhibition of Na _v 1.7 curents, which was consistent with the degree on inhibition observed in HEK293 cells overexpressing Na _v 1.7 (Figure 23B). Both TTX and the monoclonal antibodies were then applied in functional malignancy in vitro experiments, in which the capacity of the cancer cells lines to form colonies in a soft agar substrate was measured. TTX was used as positive control and reduced colony formatin by 55% to 84%, depending of the cell line studied. The antibodies 1 H5, 1 B6 and 2G1 1 were also tested on the two glioblastoma cells lines U87MG and U87MGvlll (which differs from U87MG in a mutation on the EGF receptor) and the ovarian cancer cell line SKOV-3 (Figure 24D). The antibody 1 H5 reduced colony formation in all three cell lines (37%, 60% and 57% respectively; Figures 24A, 24C and 24D); 2G1 1 was only teset on the U87MG cells and it was efficacious in inhibiting colony formation by 60% (Figure 24B); 1 B6 had no inhibitory effect on U87MG and SKOV-3 cells (Figures 24A and 24D) but interestingly reduced colony formation in the mutation bearing U87MGvlll glioblastoma cell line by 53% (Figure 24C).

EXAMPLE III - ANTI-NA .7 V _h H ANTIBODIES FROM A LAC-M NA ^' iVE LIBRARY

V _H HS from a phage-displayed LAC-M naive library. The FC5DIE3IR recombinant protein (described in Example I) was used in panning experiment to isolate V _H HS from a phage- displayed LAC-M naive library (Kumaran et al., 2012). Before the panning, rescued phages were pre-incubated with FC5 protein in a blocking buffer with slow rotation overnight at 4°C. After centrifugation in a microfuge for 10 min, pre-adsorbed phages were added onto subtraction wells coated with FC5 V _h H protein and a blocking buffer to remove/reduce the phage population binding to the parental FC5 V _h H protein and plastic surface/blocking buffer. Thereafter, the pre-adsorbed phages were exposed to the target well(s) coated with FC5DIE3IR recombinant protein. Panning was performed for a total of four rounds against the FC5DIE3IR antigen. 5x10 ¹⁰ cells of LAC-M library were grown in 500 ml of 2YT-Tet (12.5 pg/ml) at 220 rpm, 30°C overnight. The culture was then centrifuged at 5,000 rpm, 4°C for 15 min and filtered through 0.2 pm filter unit. The phage supernatant was precipitated with 1/5 volume of PEG-NaCI (500 ml +100 ml), incubated on ice for 1 h and then centrifuged at 10,000 rpm, 4°C, for 15 min. The pellet was re-suspended in 1 ml PBS and a serial dilution was done to determine the phage titer. To start the panning, 100 pi of 0.2-1 mg FC5DIE3IR/ml PBS was added to one well of a NUNC MaxiSorp™ ELISA plate (eBioscience, San Diego, CA). Another well (Blank) was coated with the same amount of FC5 V _h H protein. The wells were sealed with parafilm and incubated overnight at 4°C. The rescued phages (from LAC-M library) were also pre-incubated with FC5 V _h H + StartingBlock (Thermo Scinetific, USA) (1 :1 ratio) with slow rotation overnight at 4°C. On Day 2, the wells were rinsed twice and blocked with 200 pi of StartingBlock at room temperature (RT) for 1-2 h. After one hour, 100 pi of pre-adsorb phage (10 ¹² ) was added to the blank well and continued the incubation for another hour. This step will further pre-adsorb undesired phage binders. Subsequently, 100 pi of pre-adsorbed phages were transferred from the Blank well to the DIE3IR-coated well and incubated for 2 h at RT. The supernatant containing the unbound phages was discarded and the wells were rinsed 5 times with PBS - 0.1 % (v/v) Tween and 5 times with PBS (the washing times were increased to 7 times, 10 times, and 12 times on the 2 ^nd , 3 ^rd and 4 ^th rounds of panning, respectively). The bound phages were eluted by adding 100 pl/well of 100 mM trimethylamine (TEA) and incubated at RT for exactly 10 min. The content of the well (100 pi) were transferred into a microcentrifuge tube containing 50 pi of 1 M Tris-HCI (pH 7.4) and vortexed to neutralize the TEA. TG1 cells were infected with the neutralized phages and incubated at 37°C for 15-30 min. A serial dilution (10 ¹ , 10 ² and 10 ³ ) was prepared with 10 pi of the infected cells in 150 pi of TG1 cells. The cells were then plated on 2YT-Tet plates and Incubated at 37°C O/N. The remaining infected TG1 cells were spun at 3500 rpm for 3-5 min at 4°C. The pellet was suspended in 200 pi of 2YT-Tet, spread on a large 2YT-Tet plate, and incubated at 37°C O/N. On day 3, the bacterial cells were scraped off from the large plate using 50 ml of 2YT-Tet 5 ml of the cells were diluted with 5 ml of fresh 2YT-Tet broth and the culture were grown at 30°C at 220 rpm for 5 h. The culture was then centrifuged at 4100 rpm at 4°C for 30 min and the phage supernatant was filtered through 0.2 pm filter unit. The phage supernatant was precipitated as described above and the phage pellet was re suspended in 200 pi starting block. The phage concentration (at 10 ² dilution) was measured on Nanovue and the physical phage titer was determined using the following formula:

pfu/ml=A ₂₆ oXl 0 ¹ °x100x22.14.

Additional rounds (up to 3) of panning were performed by coating the ELISA plate with the same proteins and using the amplified phages after each round of panning as described above. After four rounds of panning, 77 randomly picked colonies were grown and subjected to monoclonal phage ELISA screening as described previously (Arbabi-Ghahroudi et al., 2009) except that 5 pg/ml of FC5DIE3IR and FC5 V _h H (the negative control) were coated onto a microtiter plate. All positive clones specifically binding to FC5DIE3IR were sequenced and unique V _h H sequences were selected for sub-cloning, large-scale expression, and purification. A total of 6 V _H HS specific to FC5DIE3IR were isolated. Six V _H H clones specific to FC5DIE3R (Dl-A, Dl-B, Dl-C, Dl-D, Dl-E and Dl-H) were identified by phage ELISA using FC5 parental FC5 V _h H protein as negative control (Figures 25A and B). The sequencing data revealed 2 enriched and 4 unique V _h H sequences (Table 7).

Expression and Purification of Soluble Monomeric V _H HS. DNA constructs (see Table 7 for a description of the nucleotide sequence) encoding the isolated V _H HS were synthesized commercially by GeneArt (Life Technologies, Carlsbad, CA) as described (Baral et al., 2013). 6xHis- and c-Myc-tagged V _h H monomers were expressed in E. coli TG1 , extracted from periplasmic space by osmotic shock and purified by immobilized metal affinity chromatography (IMAC) using a HisTrap HP column (GE Healthcare, Piscataway, NJ) as described in Baral et al., 2013 and Arbabi-Ghahroudi et al. , 2009. The integrity and aggregation status of soluble V _h H monomers were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE), Western blotting and size exclusion chromatography (SEC) as described in Baral et al. , 2013 and Arbabi-Ghahroudi et a!., 2009.

It was determined, by performing an protein-ELISA assay that all V _h H antibodies showed specific binding to the FC5DIE3R protein with little or no cross- reactivity to FC5 V _h H (data not shown).

In vitro Identification of Functional V _H HS. TO determine the functional effect of the isolated V _H HS on hNa _v 1.7 channels, Na _v currents were evoked using the whole-cell patch-clamp technique in hNa _v 1.7-HEK293 cells as described in Example I. Five V _H HS (Dl-A, Dl-B, Dl-C, Dl-D, and Dl-H) were tested using patch-clamp whole-cell. The V _h H Dl-E protein was not stable and consequently not tested. V _H HS were tested at an initial concentration of 1 mM. At this concentration, Dl-D reduced the amplitude of the Na _v 1.7 currents of 1 1.7%±5.8 (n=12), while Dl-A, Dl-B, Dl-C, Dl-H had no effect (Dl-A, 2.73% ± 10.52, n=4; Dl-B, 1.73% ± 1.96, n= 6; Dl-C, 0.46% ± 2.07, n=4; Dl-H, 1.72% ± 1.32, n= 3; Figure 26E). At 2 pM and 3 pM, Dl-D significantly reduced the currents by 15.0% ± 6.9 (n=4) and 24.1 % ± 5.6 (n= 7), respectively (Figures 26A, E and F). The V _h H Dl-D had a slow onset of effect, with time constants of block of 1 13 ± 1 1 sec (n=4), and 200 ± 45 sec (n=4) for concentrations of 1 pM and 2 pM, respectively (Figure 26B). These data suggest that Dl-D is functional in inhibiting the flux of Na ⁺ thought the hNa _v 1.7 channels. Based upon the results presented in Figure 26F, the Dl-D concentration response relationship (IC ₅ o) and efficacy (degree of maximum inhibition) were 1.48 ± 0.25 mM and 27.4 ± 5.% inhibition, respectively.

Next, it was evaluated if the V _h H Dl-D had an effect on the kinetics and voltage dependence properties of the hNa _v 1.7 channels. To do this, the effect of Dl-D on the current-voltage (l-V) relationship as well as the voltage-dependence of activation and steady-state fast inactivation of hNa _v 1.7 channels in hNa _v 1.7-HEK293 cells were examined (Figures 26C and D). The Dl-D shifted the voltage-dependence of activation to more hyperpolarized membrane potentials (V _1/2 activation: D = -6.1 ± 1.0 mV at 1 mM; n=5), suggesting a gating modifier activity (Figure 26C). Minor effect was observed for Dl-D on the voltage-dependence of steady-state fast inactivation for Na _v 1.7 (V _1/2 inactivation: D = -6.8 ± 1.5 mV at 1 mM; n=5). However, like in the case of mAbs, these shifts towards hyperpolarizing voltages of activation and steady-state inactivation curves may be not significant. In fact, it has been previously reported in literature that these changes in activation and inactivation of voltage gated sodium channels may derive from perturbations of the cellular membrane resulting from the application of the V _H H . In addition, negative-shifts of the same order were observed also in control experiment in which an equivalent volume of extracellular solution containing no V _h H was added to the patch-clamp chamber. In order to further investigate the effects of Dl-D on hNa _v 1.7 kinetics of activation and fast inactivation, time-to-peak and the time constants of fast inactivation of hNa _v 1.7 were measured. The V _h H Dl- D (1 mM) did not significantly change the times-to-peak which were 1.76 ± 0.28 ms and 1.64 ± 0.37 ms (measured at -20 mV, n=4) in control and after Dl-D application respectively (p > 0.10, paired f-Test). It also did not change the inactivation time constants which were 1.32 ± 0.33 ms and 1.29 ± 0.30 ms (measured at -20 mV, n=4) in control and with 1 mM Dl-D, respectively (p > 0.10, paired f-Test). These data suggest that the main functional effect of Dl-D on the activity of Na _v 1.7 is on the sodium ion permeation pathway, which reduces the amplitude of the current through the channel, and that the observed effects on Na _v 1.7 kinetic properties likely derive from the perturbation to the membrane-cytoskeleton interactions provoked by the V _h H application.

In vivo validation of the V _H H: rat Hargreaves model of hyperalgesia. To evaluate if the functional in vitro effect of the V _h H Dl-D translated in a functional in vivo effect against pain, Dl-D was tested on CFA-induced thermal hyperalgesia in rats and compared it with the selective blocker for hNa _v 1.7 channels TC-N1752 (Tocris, Bristol, United Kingdom). The V _h H Dl-D was tested also along with a negative control anti-GFP mAb 11 E5. As previously demonstrated, CFA induced hyperalgesia to a thermal stimulus 48 h post injection (Webster et al., 2016). Baseline latency paw withdrawal was reduced to 4.98 ± 0.15 sec (n=18) in ipsilateral paw versus 19.98±0.19 sec (n=18) of contralateral paw (see Figure 7). The Dl-D effect on the latency paw withdrawal was measured 4 h after administration. Intraplantar injection of 50 pg of Dl-D (8.36 ± 0.53 sec, n= 7), 100 pg of Dl-D (11.78 ± 0.50 sec, n= 7) and but not 11 E5 (5.92 ± 0.14 sec, n=4) significantly increased latency paw withdrawal (p< 0.05 vs baseline; Figure 27 A). In addition, Dl-D at 100 pg (13.35 ± 0.44 sec, 11.40 ± 0.69 sec, 11.78 ± 0.50 sec at 1 , 2 and 4 hours respectively; n= 7) had an effect similar to that of TC- N1752 (100 pg; 8.15 ± 2.60 sec, 11.17 ± 1.19 sec, 1 1.90 ± 0.99 sec at 1 , 2 and 4 hours respectively; n=3). Figure 27B shows a histogram of the effect of 11 E5 (5.78% ± 1.08, n=4), TC- N1752 (46.47% ± 7.14, n= 3), Dl-D 50 pg (21.59% ± 4.05, n= 7) and Dl-D 100 pg (45.19% ± 4.40, n= 7) on hyperalgesia expressed as percentage of maximum possible effect (%MPE) 4 h after intraplantar injection of test compounds.

To further evaluate the functionality of the V _h H Dl-D and target engagement, the V _h H was tested on the OD1 pain model. The intraplantar administration of OD1 in mice induces spontaneous pain behaviors as evidenced by licking, flinching, lifting and shaking of the injected hind paw. These behaviors are dose dependent (Figure 14A) and develop rapidly, occurring soon after injection, and persists for up to 40 min after injection. The effect of 50 pg of VHH injection was tested and compared it with the injection of saline (saline plus OD1 30 pi). The pain behavior was considered at 100%, 20 min after the injection. Twenty min after the injection, 50 pg of V _h H Dl-D (Figure 28A), but not V _h H A20.1 (Figure 28B) significantly reduced the time spent in pain behaviors (Dl-D; 44.33% ± 8.8; n=2). Surface Plasmon Resonance (SPR). To determine the binding affinity of the V _h H Dl-D with the recombinant protein FC5DIE3IR used to isolate the V _H H, SPR binding assays were used as provided in Example I. Before starting, pure monomeric fractions of the V _H HS Dl-D and FC5 control, and FC5DIE3IR target protein were obtained using SEC. To determine the binding the V _h H Dl-D with the recombinant protein FC5DIE3IR, FC5DIE3IR and FC5 surfaces were prepared. SPR analysis showed that Dl-D binds specifically to FC5DIE3IR (Figures 29A and 29B) with a K _D of 2 pM (Figures 30A and 30B) and an observed R _max of approx. 700 RU, indicating a high level of activity of immobilized FC5DIE3IR. Weak/residual binding to FC5 was observed by Dl-D, although an affinity could not be determined (data not shown).

EXAMPLE IV - ANTI-NA _V 1.7 V _h H ANTIBODIES FROM A LAC-M NA ^' iVE LIBRARY

V _H HS from a FC5DIE3IR llama immune library. The FC5DIE3IR recombinant protein (100 pg, described in Example I) was used to subcutaneously immunize a young male llama five times at two-week intervals. Briefly, a male llama ( Lama glama) was immunized five times subcutaneously with 100 pg of FC5DIE3IR protein mixed with an equal volume of either complete (day 1) or incomplete (days 14, 28, 42, 49) Freund’s adjuvant (Sigma). Pre-immune blood (15-20 ml) was collected before the first injection and on days 35 and 56. The specific immune responses were analyzed by ELISA using total pre-immune and immune sera. Briefly, microtitre plates (Maxisorp™ plates) (Nalge Nunc International, Rochester, NY) were coated overnight at 4°C with 5 pg/ml of FC5DIE3IR antigen in PBS. As negative control, FC5 protein was coated on each second row on the same plate. Wells were rinsed and blocked with 200 mI of 1 % casein. Serially diluted pre-immune and immune sera were added to the wells and incubated at room temperature for 1.5 h. Wells were washed with PBST (0.05% v/v Tween-20), and incubated with goat anti-llama IgG-HRP (H+L) (1 :1 ,000 in PBS) (Bethyl Laboratories, Montgomery, TX). Signal was detected by adding 100 mI/well TMB peroxidase substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA). Reactions were stopped by adding 100 mI of 1 M phosphoric acid and A450 was measured using a standard ELISA plate reader.

The V _h H library was constructed as described previously (Baral et al., 2013). In brief, total RNA was isolated from approximately 1 X 10 ⁷ lymphocytes collected on day 56 post-immunization using QIAamp™ RNA blood mini kit (Qiagen, Mississauga, Ontario, Canada). First-strand cDNA was synthesized with oligo (dT) primer using 5 pg total RNA as template according to manufacturer’s recommendations (GE Healthcare). Variable and part of the constant domains DNAs were amplified using oligonucleotides MJ1-3 (sense) and two CH2 domain antisense primers CH2 and CH2b3 (for primer sequences see (Baral et al., 2013) and heavy chain fragments (550-650 bp in length) were gel-purified using QIA quick gel extraction kit (Qiagene). The variable regions of heavy chain antibodies (lgG2 and lgG3) were re-amplified in a second PCR reaction using MJ7 and MJ8 primers (for primer sequences see Baral et al. , 2013). The amplified PCR products were purified with a QIAquick PCR purification kit (Qiagene), digested with Sfil (New England BioLabs, Pickering, Ontario, Canada), and re-purified using the same kit. Twelve micrograms of digested V _h H fragments were ligated with 40 pg (3:1 molar ratio, respectively) Sfi- digested pMED1 phagemid vector using LigaFast Rapid DNA ligation system and its protocol (Promega, Madison, Wl), transformed into commercial electrocompetent TG1 E. coli cells (Stratagene, La Jolla, CA) as described previously (Baral et al., 2013) and a library size of 5 X 10 ⁷ transformants was obtained. The V _h H fragments from 30 colonies were PCR- amplified and sequenced to analyze the complexity of the library; all clones had inserts of expected sizes and were different from each other at their CDR regions as determined by sequencing of their encoding V _h H fragments. The library was grown for 3-4 h at 37°C, 250 rpm in 2 X YT/Amp-Glucose (2% w/v) medium. The bacterial cells were pelleted, resuspended in the same medium and stored as glycerol stock at -80°C as described previously (Baral et al., 2013).

Panning experiments was essentially performed as described in Baral et al., 2013. Panning was performed for a total of four rounds against the FC5DIE3IR antigen. Two milliliters of the library stock (5 x 10 ¹⁰ cells) was grown in for 1-2 hours at 37°C, 250 rpm in 2 X YT/Amp-Glucose (2% w/v) medium (A ₆₀₀ = 0.4-0.5), infected with M13K07 helperphage (New England Biolobas) for 1 h at 37 °C. After centrifugation of the culture at 4°C, the infected cell pellets were re-suspended in 200 ml of 2 X YT/Amp with 50 pg/ml kanamycin and incubated overnight at 37°C and 250 rpm. The phage particles in culture supernatant were PEG-precipitated as described previously (Baral et al., 2013) and the phage pellets were re-suspended in 2 ml of sterile PBS and the phage titration was determined. During the panning process, rescued phages from the library were first incubated with FC5 protein in a blocking buffer overnight at 4°C on a rotating shaker. The phage solution was centrifuged at top speed for 10 min and the phage supernatant was added onto subtraction wells coated with FC5 V _h H protein and a blocking buffer to remove/reduce the phage population binding to the parental FC5 V _h H protein and plastic surface/blocking buffer. Thereafter, the pre-adsorbed phages were exposed to the target well(s) coated with FC5DIE3IR recombinant protein. Panning was performed for a total of four rounds against the FC5DIE3IR antigen. To start the panning, 100 pi of 0.4 mg/ml of FC5DIE3IR in PBS was added to one well of a NUNC MaxiSorp™ ELISA plate (eBioscience, San Diego, CA). Another well (blank) was coated with the same amount of FC5 V _h H protein. The wells were sealed with parafilm and incubated overnight at 4°C. The rescued phages (from immune library) were also pre-incubated with FC5 V _h H + StartingBlock (Thermo Scinetific, USA) (1 :1 ratio) with slow rotation overnight at 4°C. On Day 2, the wells were rinsed twice and blocked with 200 pi of StartingBlock at room temperature (RT) for 1-2 h. After one hour, 100 mI of pre-adsorb phage (10 ¹² ) was added to the blank well and continued the incubation for another hour. This step will further pre-adsorb undesired phage binders. Subsequently, 100 mI of pre-adsorbed phages were transferred from the blank well to the FC5DIE3IR-coated well and incubated for 2 h at room temperature. The supernatant containing the unbound phages was discarded and the wells were rinsed 5 times with PBS - 0.1 % (v/v) Tween and 5 times with PBS (the washing times were increased to 7 times, 10 times, and 12 times on the 2 ^nd , 3 ^rd and 4 ^th rounds of panning, respectively). The bound phages were eluted with 0.1 M triethylamine, neutralized with 1 M Tris-HCL, PH 7.4 and incubated with exponentially growing TG1 cells. After 30 min incubation at 37°C, the cells were superinfected with M13K07 for additional 15 min and grown in 2X YT-Amp-Kan overnight at 37°C. Panning was continued for three more rounds following the same conditions except that antigen concentration was reduced to 20, 15, and 10 pg/well and washing was increased 7, 10 and 12X with PBS-T and PBS for the second, third and fourth rounds of panning, respectively. After four rounds of panning, 48 randomly picked colonies were grown and subjected to phage ELISA screening as described previously (Baral et al., 2013) except that 5 pg/ml of FC5DIE3IR and FC5 were coated, respectively, onto each row of the microtiter plates as positive and negative antigens.

In total, four rounds of solid-phase panning were performed and 48 colonies were screened by phage-ELISA as described previously (Arbabi-Ghahroudi et al., 2009) except that 5 pg/ml of FC5DIE3IR and FC5 V _h H (the negative control) were coated onto a microtiter plate. Four V _h H clones specific to FC5DIE3IR (DI-4, DI-16, DI-28, DI-48) were identified by phage ELISA using FC5 parental V _h H protein as negative control (Figures 31 A and 31 B). Additional phage-ELISA screening (on 48 colonies) and sequencing revealed that in total four unique V _h H sequences, DI-4, DI-16, DI-28, and DI-48, were enriched 3x, 3x, 21x, 5x, respectively (Table 7). Unique V _h H sequences were gene-synthesized and cloned into the pSJF2 expression vector for large-scale expression, and purification.

Expression and Purification of Soluble Monomeric V _H HS. DNA cconstructs encoding the isolated V _H HS (DI-4, DI16, DI-28 and DI-48; Table 7) were synthesized and cloned into the pSJF2H expression vector commercially by GeneArt (Life Technologies, Carlsbad, CA) as described (Baral et al., 2013). The final construct included an OmpA leader sequence for secretion of sdAb proteins to the periplasmic space of E. coli, and a cmyc and His ₆ tags for ease of purification, at the C-terminus of the sdAb. Large-scale protein expression and purification was performed essentially as previously described (Baral et al., 2013). Briefly, individual colonies were grown (25 ml LB-Amp overnight at 37°C. The pre-culture was used to seed 1 liter 2xYT- Amp culture and grown at 37°C with vigorous shaking. At OD ₆₀₀ = 0.8, the cultures were induced by the addition of IPTG to a final concentration of 1 mM. The culture was grown overnight at 32°C. The bacteria were pelleted by centrifuging at 6,000 rpm for 12 min, and then submitted to cold osmotic shock to extract sdAb from the periplasmic space. The pellet was resuspended in 30 ml of cold TES buffer (0.2M Tris-CI pH 8.0, 20% sucrose, 0.5mM EDTA). The suspension was incubated on ice and vortexed every 10 min for 1 h. Then 40 ml of cold TES (1/8 volume of total volume) was added and immediately vortexed for 1 min and for 15 sec every 10 min thereafter for 1 h to extract the protein from the periplasm. The periplasmic extracts containing V _H HS were dialyzed overnight against buffer (20 mM phosphate buffer (pH 7.4), 0.5M NaCI, 10 mM imidazole) at 4°C then filtered through a 0.45 pm membrane (EMD Millipore, Canada). Filtered supernatant was loaded onto nickel-charged 5 ml HisTrap™ FF column (GE Healthcare, Canada) and fractionation was performed on an AKTA FPLC purification system (GE Healthcare). The V _H HS were eluted using gradient elution (buffer containing 500 mM imidazole); the fractions were pooled and dialyzed against PBS. V _h H concentrations were determined by absorbance measurements at 280 nm using theoretical MW and extinction coefficients calculated with the ExPASy ProtParam Tool (expasy.org/tools/protparam.html).

Antibody Binding Assays (Protein-ELISA). A phage ELISA assay was performed and four positive clones (DI-4, DI-1 1 , DI-16, DI-28) (having a signal of at least 3x background) were identified and further characterized. Sequencing data showed that clones DI-1 1 and DI-16 have identical sequences and the difference in ELISA signal could be due to lower phage titer for clone DI-1 1. Additional rounds of screening (data not shown) identified a new V _h H clone, named DI-48. Surface Plasmon Resonance (SPR). To determine the binding affinity of the V _H HS DI-4, DI-16, Dl- 28 and DI-48 with the recombinant protein FC5DIE3IR used to isolate the V _H HS, SPR binding assays were performed. Before starting, pure monomeric fractions of the V _H HS DI-4, DI-16, DI-28, DI-48, FC5 control, and FC5DIE3IR target protein were obtained using SEC (data not shown). To determine the binding affinity of DI-4, DI-16, DI-28 and DI-48 for recombinant FC5DIE3IR, FC5DIE3IR, FC5 (negative control) and V _h H 2A3-H4 (negative control) surfaces were prepared. As shown in Table 6, SPR analysis showed that DI-4, DI-16, DI-28 and DI-48 bind specifically to FC5DIE3IR (Figures 32A to 32D) with a K _D of 2.25 mM, 4.3 nM, 4.4 nM and 8.4 pM, with observed R _max s ranging from 318 - 470 RUs (Table 6). These results indicate a high level of activity of immobilized FC5DIE3IR and V _H HS. NO binding to FC5 and to 2A3-H4 was observed for any of the V _H HS tested. DI-4 and DI-48 had similar affinities for FC5DIE3IR as Dl-D (K _D ~2 pM), while DI-16 and DI-48 had affinities 1000 times lower.

Table 6. Affinity and binding kinetics for DI-4, DI-16, DI-28, DI-48 and Dl-D obtained with SPR

Table 7. Nucleotide and amino acid sequences. CDR determination were done using the IMGT website.

While the invention has been described in connection with specific embodiments thereof, it will be understood that the scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole. REFERENCES

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