Patent Application
20210206842
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference in the specification. The name of the text file containing the Sequence Listing is USPTO_Sequence_Listing. The text fie is 126 KB, was created on Nov. 17, 2020 and is being submitted electronically via EFS-Web.
The present disclosure concerns antibodies and compositions comprising antibodies for antagonizing the human Nav1.7 (HNav1.7) channel for various indications including treatment and/or alleviation of symptoms of pain and cancer.
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 (Nav) 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.
Nav 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. Nav are totally absent in non excitable cells, however, in various cancers (e.g. breast, prostate, lung) de-novo functional expression of Nav channels has been found and linked to malignant phenotypes (Fraser et al. 2005; Diss et al., 2001; Roger et al., 2007).
The Nav family has nine members (nine types of pore-forming a subunits), which in mammals are numerically named from Nav1.1 to 1.9. These isoforms share a common overall structural motif and each Nav type has a different function and expression profile. Nav1.1, Nav1.2, Nav1.3, and Nav1.6 are mainly expressed in the central nervous system (CNS). Nav1.7, Nav1.8, and Nav1.9 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. Nav1.4 is the muscle sodium channel and Nav1.5 is the predominant cardiac myocyte channel.
The Nav1.7 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 Nays dependent action potentials. Nav1.7 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 Nav1.7 that causes pain. Genetic, structural and functional studies have shown that Nav1.7 regulates sensory neuron excitability which is a major contributor to several sensory modalities, and have established the contribution of Nav1.7 to human pain disorders. In particular, genetic studies show that mutations in SCN9A gene which encode for Nav1.7 produce familial pain disorders. These mutations can be described as gain of function mutations (increase activity of Nav1.7) which cause paroxysmal extreme pain disorder (PEPD) or loss of function mutations (reduction of Nav1.7 activity) which are linked to complete insensitivity to pain (CIP). Importantly, people totally lacking Nav1.7 have minimal cognitive, cardiac, motor and sensory deficits, supporting Nav1.7 as a valid and indeed attractive target for development of drug against pain.
De novo functional expression of Nav1.7 has been documented in prostate, non small cell lung cancer and leukemia. The expression of Nav1.7 in these cancers is reported to promote cell invasion. It has been shown that in vitro application of tetrodotoxin (TTX), a potent Nays 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 Nav1.7 is a valid target to develop a drug against cancer malignancy.
The first generations of molecules developed against Nays were analgesics. These drugs were clinically effective, but, due to their non-selective Nav activity, their analgesic potential was limited by numerous side effects (dizziness, sedation, convulsions, and cardio-toxicity). For example: lidocaine (as well as other Nav 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 Nav 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 Nav1.7 represents a challenge due to the high degree of amino acid sequence homology among the different Nav subtypes. In addition, Nays 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 Nav 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.
Biologics 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-II a 30 amino acid peptide which is a highly selective Nav1.7 inhibitor does not bind the Nav1.7 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 Nav1.7-mediated antinociception is obtained via both peripheral and central mechanisms. However, since the majority of therapeutics, biologics 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 Nav1.7 inhibitors that not only bind to the Nav1.7 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 Nav1.7 inhibitors which are capable of pene
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):
NH2-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 Nav1.7 polypeptide. The antibody of the present disclosure is specific for an epitope located on a loop 3 of a domain I (DI) of the Nav1.7 polypeptide. The antibody is also capable of antagonizing the biological activity of the Nav1.7 polypeptide. In an embodiment, the Nav1.7 polypeptide is a human Nav1.7 polypeptide and as such, the antibody specifically recognizes the human Nav1.7 polypeptide. In another embodiment, the epitope is located on a first peptide comprising or consisting essentially of the following amino acid sequence:
In a further embodiment, the antibody is a monoclonal antibody which can have, for example, a dissociation constant (KD) 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 (KD) of about 1 nM to about 1000 μM. In still another embodiment, the epitope is located on a second peptide comprising or consisting essentially of the following amino acid sequence:
In still another embodiment, the epitope is located on a second peptide comprising or consisting essentially of the following amino acid sequence:
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 113, 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 115, 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: 113, a variant thereof or a fragment thereof and a light chain of amino acid sequence of SEQ ID NO: 115, 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 117, 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 119, 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: 117, a variant thereof or a fragment thereof and a light chain of amino acid sequence of SEQ ID NO: 119, 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 Nav1.7 polypeptide can be measured as the amplitude of current associated with the Nav1.7 polypeptide and the antibody can be capable of substantially reducing the amplitude of current associated with the Nav1.7 polypeptide. In a further embodiment, the biological activity of the Nav1.7 polypeptide can be measured as a normalized current associated with the Nav1.7 polypeptide and the antibody can be capable of substantially maintaining or reducing the normalized current associated with the Nav1.7 polypeptide. In still a further embodiment, the biological activity of the Nav1.7 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 seventh 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 eighth 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 Nav1.7 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 Nav1.7 polypeptide. In an embodiment, the subject is a human subject. In another embodiment, the subject is an animal subject.
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:
), 1B6 (▾) and 2G11 (♦). Results are provided as the OD at 405 nm in function of the antibody concentration (provided on a logarithmic scale in nM).
The present disclosure provides for specific antibodies against the Nav1.7 polypeptide. The antibodies are considered “specific” to the Nav1.7 polypeptide because their affinity for the Nav1.7 polypeptide is higher than for other polypeptides (for example other polypeptides from the Nav family). The antibodies of the present disclosure can be specific for the human Nav1.7 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 (KD) with respect to the Nav1.7 polypeptide is in the nanomolar range (1-1000 nM). In such embodiment, the monoclonal antibody can have a KD 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 Nav1.7 polypeptide. In an embodiment, when the antibody is a single-domain antibody (sdAb), its dissociation constant (KD) with respect to the Nav1.7 polypeptide is in the nanomolar range (1-1000 nM) or the micromolar range (1-1000 μM). In such embodiment, the single domain antibody can have a KD between about 1 and about 100 μM, between about 1 and about 50 μM, between about 1 and about 25 μM, between about 1 and about 5 μM or about 2 μM with respect to the Nav1.7 polypeptide. The present invention provides antibodies, including mAbs and sdAbs, that target the Nav1.7 channel, and specifically bind a Nav1.7 polypeptide, wherein said polypeptide comprises a peptide in the extracellular loop 3 of domain DI of the Nav1.7 channel, having a nanomolar or micromolar affinity.
The antibodies of the present disclosure are also capable of antagonizing the biological activity of the Nav1.7 polypeptide. As indicated above, the Nav1.7 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 Nav1.7 polypeptide which in return is understood to limit or inhibit the flux of sodium ions through the Nav1.7 polypeptide. As such, in an embodiment, an antibody capable of reducing the amplitude of a current associated with the Nav1.7 polypeptide is considered to also be capable of antagonizing the biological activity of the Nav1.7 polypeptide. In an embodiment, the antibodies have a concentration response relationship (IC50) towards the Nav1.7 polypeptide in the nanomolar range (1-1000 nM) or in the micromolar range (1-1000 μM). In another embodiment, the antibodies have a degree of maximum inhibition towards the Nav1.7 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 Nav1.7 polypeptide of at least 5, 6, 7, 8, 9, 10, 11, 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 example 16%, 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 Nav1.7 polypeptide which in return is understood to limit or inhibit the flux of sodium ions through the Nav1.7 polypeptide. As such, in an embodiment, an antibody capable of maintaining or reducing the normalized current associated with the Nav1.7 polypeptide is considered to also be capable of antagonizing the biological activity of the Nav1.7 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 Nav1.7 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 Nav1.7 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 Nav1.7 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 Nav1.7 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 Nav1.7 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 Nav1.7 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 Nav1.7 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 IgG1, IgG2 or IgG3) 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 (VHH antibodies) and in cartilaginous fishes (VNAR 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 Nav1.7 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 Nav1.7 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 Nav1.7 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 Nav1.7 polypeptide and are capable of limiting the biological activity of the Nav1.7 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 3rd loop of domain I of the Nav1.7 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
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-Nav1.7 antibodies). In an embodiment, when the antibody is a monoclonal antibody, its dissociation constant (KD) with respect to the DIE3IR peptide is in the nanomolar range (1-1000 nM). In such embodiment, the monoclonal antibody can have a KID 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 (KD) with respect to the DIE3IR peptide is in the micromolar range (1-1000 μM). In such embodiment, the single domain antibody can have a KD between about 1 and about 100 μM, between about 1 and about 50 μM, between about 1 and about 25 μM, between about 1 and about 5 μM or about 2 μM with respect to the DIE3IR peptide.
In some embodiments, the epitope recognized by the anti-Nav1.7 antibody is located on the peptide #2 which comprises or consists essentially of the following amino acid sequence:
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 (KD) with respect to peptide #2 is in the nanomolar range (1-1000 nM). In such embodiment, the monoclonal antibody can have a KD 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-Nav1.7 antibody is located on the peptide #2a which comprises or consists essentially of the following amino acid sequence:
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 (KD) with respect to peptide #2a is in the nanomolar range (1-1000 nM). In such embodiment, the monoclonal antibody can have a KD 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-Nav1.7 antibody is located on the peptide #2b which comprises or consists essentially of the following amino acid sequence:
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 (KD) with respect to peptide #2b is in the nanomolar range (1-1000 nM). In such embodiment, the monoclonal antibody can have a KD 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-Nav1.7 antibody is located on the peptide #3 which comprises or consists essentially of the following amino acid sequence:
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 (KD) with respect to peptide #3 is in the nanomolar range (1-1000 nM). In such embodiment, the monoclonal antibody can have a KD 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-Nav1.7 antibody is located on the peptide #40a which comprises or consists essentially of the following amino acid sequence:
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 (KD) with respect to peptide #40a is in the nanomolar range (1-1000 nM). In such embodiment, the monoclonal antibody can have a KD 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-Nav1.7 antibody is located on the peptide #40b which comprises or consists essentially of the following amino acid sequence:
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 (KD) with respect to peptide #40b is in the nanomolar range (1-1000 nM). In such embodiment, the monoclonal antibody can have a KD 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 Nav1.7 polypeptide or its ability to antagonize the biological activity of the Nav1.7 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 Nav1.7 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) CABIOS. 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: 11 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: 11 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 Nav1.7 polypeptide or its ability to antagonize the biological activity of the Nav1.7 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 Nav1.7 polypeptide or its ability to antagonize the biological activity of the Nav1.7 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: 11 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 IgG1 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 IgG2a 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 IgG1 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 IgG1 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 VHH 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, 110, 79, 115, 116, 89, 119, 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 exemplary 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 essentially of the amino acid sequence of SEQ ID NO: 11, 12, 109, 38, 74, 113, 114, 84, 117, 118, 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 exemplary 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 fragment 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 U.S. Pat. No. 8,715,659 or to a blood-barrier antigen found on the surface of a mammalian cell as described in U.S. Pat. No. 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. Nav1.7 within the CNS. The development of BBB-crossing bi-specific 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 Nav1.7 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 Nav1.7 may have such an advantage. With respect to a cancer indication a bi-specific Ab targeting both the tumour cell and Nav1.7 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 Nav1.7 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.
Due to their high specificity towards the Nav1.7 polypeptide, the antibodies of the present disclosure can be used to substantially purify the Nav1.7 polypeptide from a mixture suspected of comprising the Nav1.7 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 Nav1.7 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 Nav1.7 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 Nav1.7 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, 125I, 131I, 35S, 32P or 3H.
Due to their ability to antagonize the biological activity of the Nav1.7 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 Nav1.7 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-HCl, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, and detergents (e.g., Tween20™, Tween80™, 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, 1B6, 1H5, 2G11 and DI-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).
The present disclosure also provides immunogens as well as methods for making antibodies specific to a peptide epitope with the immunogens. As shown in the Examples below, an immungen (FC5DIE31R) 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 Nav1.7 polypeptide. As also shown in the Examples below, the antibodies obtained were specific for the Nav1.7 polypeptide. Consequently, the present disclosure provides an immunogen for making additional antibodies, including additional anti-Nav1.7 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 VHH 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 stretch 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 VHH antibody, the FC5 antibody (as described in U.S. Pat. Nos. 8,715,659 and 8,383,107). When the immunogen is derived from the FC5 antibody, it can comprise or consist essentially of the polypeptide of formula (I):
NH2-SS-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-COOH (I).
In the polypeptides of formula (I), NH2 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 (directly 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 starting 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-Nav1.7 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.
Epitope selection. Nav1.7 is comprised of four domains (DI, DII, DIII and DIV), each containing six transmembrane helices (S1-S6) and 4 extracellular loops (
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 VHH with the retained regions of the CDR3 (between which the DIE3IR peptide is to be grafted) is shown in
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, N.J., 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, Calif., USA) supplemented with 0.1% Kolliphor P188 (Sigma-Aldrich, St. Louis, Mo., USA) and 4 mM Glutamine (Sigma-Aldrich, St. Louis, Mo., USA). Cultures were maintained in 125 ml Erlenmeyer ventilated flasks shaken at 120 rpm in a humidified incubator at 37° C. with 5% CO2. 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 μm), aliquoted and stored à +4° C. Cells were diluted one day before transfection in fresh medium at 0.7×106 cells/ml. They were then transfected the following day with viability greater than 99% at densities between 2.0 and 2.5×106 cells/ml with 1 μg 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-FC5DIE31R) was diluted at 20 μg/ml in F17 medium, and an equivalent volume of F17 medium containing 100 μg/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 3000 g 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 3 kDa and sterilized by passing through 0.2 μm 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 VHHs against hNav1.7 from a phage-displayed naïve VHH library generated from llama, alpaca and camel, immunize llamas to obtain a FC5DIE3IR llama immune library, isolate VHHs against hNav1.7 from a phage-displayed VHH library generated from immunizing llamas with FC5DIE3IR.
Generation of hNav1.7-HEK293 cell line. Anti-hNav1.7 mAbs and VHHs were tested on hNav1.7 currents recorded with patch-clamp whole-cell technique in HEK293 cells overexpressing hNav1.7 channels (hNav1.7-HEK293 cells). HEK-293 cells (ATCC CRL1573) were transfected with human Nav1.7 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 8×105 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 μl of OptiMEME containing 3 μg of DNA was mixed with 125 μl of OptiMEM pre-incubated with 9 μl 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 cm2 dishes, and grown in the presence 300 μg/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 hNav1.7 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 Nav1.7, 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.
Antibody Binding Assays (Protein-ELISA). FC5DIE3IR binding of VHH monomers was assessed by direct protein ELISA. Briefly, wells of a NUNC MaxiSorp microtiter plate were coated overnight at 4° C. with 0.5 μg of the recombinant protein (FC5DIE31R) in 100 μl PBS, pH 7.4. As negative control, the parental FC5 VHH was used for immobilization as described above. After blocking with Starting block for 2 h, monomeric VHHs (DI-A, DI-B, DI-C, DI-D, DI-E, DI-F, DI-G and DI-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 μl rabbit anti-His6 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 1M H3PO4 and A450 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 hNav1.7-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 1000 g 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% CO2 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 μl of cells plus 2.4 ml of media). 500 μl of dilute cell suspension was added to each well of a 24 well plate containing poly-1-lysine (0.025 mg/ml; Sigma-Aldrich, St. Louis, Mo., USA) coated 13 mm plastic coverslips (Thermanox Plastic coverslips, Thermo Fisher, Waltham, Mass., USA).
Patch-clamp whole-cell recordings. Human Nav1.7-HEK293 cells were further evaluated using patch-clamp whole-cell recordings to determine the functionality of the hNav1.7 channels over-expressed in the HEK293 cells. Whole-cell patch-clamp recordings were obtained with a Multiclamp 700B amplifier (Molecular Devices, Carlsbad, Calif., USA) controlled with pClamp (v 10.2) software (Molecular Devices, Carlsbad, Calif.) used in combination with a Digidata 1440A A/D converter (Molecular Devices, Carlsbad, Calif.). 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), NaCl (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 MΩ. Whole-cell access resistances measured in voltage-clamp ranged from 5 to 10 MO. The access resistance was routinely monitored throughout each experiment. To record Na+ currents, cells were maintained in an extracellular recording solution contained (in mM): NaCl (140), KCl (3), CaCl2 (1), MgCl2 (1), HEPES (10). The pH was corrected to 7.3 using NaOH, and the osmolarity adjusted to ˜300 mOsm. Over-expression of hNav1.7 was analyzed by plotting current-voltage (I-V) relationships of evoked inward currents in response to step protocols (voltage steps from ˜85 to +60 mV in 5 mV increments) and raw traces of inward current, and by analyzing half-maximal activation (g/gMAX) and inactivation (I/IMAX) potential (
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 μl of mAb solution to the recording chamber containing the cells. The 50 μl of mAb solution was statically added to 650 μl of extracellular recording solution present in the recording chamber.
Whole-cell patch-clamp recordings were obtained with a Multiclamp 700B amplifier (Molecular Devices, Carlsbad, Calif., USA) controlled with a pClamp (v10.2) software (Molecular Devices, Carlsbad, Calif.) 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 32MΩ when filled with a solution containing (mM): CsF (140), NaCl (10), MgCl2 (1), HEPES (10), ATP-Mg2+ (2), GTP-Mg2+ (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, Calif., 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): NaCl (150), CsCl (3), CaCl2 (1), MgCl2 (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 hNav1.7 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 hNav1.7 (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, Oreg., USA) and/or Origin 2016 (OriginLab Corporation, Northampton, Mass., 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 (I-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 my; 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 μl) of complete Freund's adjuvant (CFA; heat-killed M.Tuberculosis—Sigma, St. Louis, Mo.—suspended in oil:saline 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, Calif.). 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 μg diluted in 50 μl) 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)×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, 25-30 g) were kept under 12-h light-dark cycles and had standard rodent chow and water ad libitum. Scorpion α-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 Nav1.7 (EC50 4.5 nM), with little effect on Nav1.3 and Nav1.5 (EC50>1 μM) and no effect on Nav1.2 and Nav1.8. 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 μL under isoflurane anesthesia. Mice were then placed into polyvinyl boxes (10×10×10 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 30 min and then incubated with 1:1000 dilution of anti-hNav1.7 mAb at room temperature (RT) for one hr. Following three TBS-T washes (3×5 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 Nav 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 al., 1997). It was found that, the highest homology that pep #2 has with other members of the human Nav family is 78% homology with the structure of the hNav1.4 in complex with β1 (E value=3e-07) and 72% with the hNav1.2 (E value=le-05), while, the pep #2a has a 78% homology with hNav1.4 (E value=4.7). The highest homology that peptide pep #3 has is 80% with the hNav1.2 (E value=8e-17). Overall this analysis suggests that the probability that 3A8, 1H5, 2G11 and 1B6 acts on others subtypes of the Nav family other than the Nav1.7 is low.
Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS). FC5DIE3IR and purified mAbs (3A8, 1H5, 2G11, 1B6, 1G5) were diluted to 40 and 20 μM, respectively, and mixed at a 1:1 ratio and kept on at 25° C. For the unbound control, FC5DIE3IR was diluted to 20 μM with PBS. Labelling reactions were initiated by adding 3 μL of IGF1R complexes to 3 μL of 90% D2O at 25° C. Labelling was quenched after 1, 10, and 60 min by the addition of 39 μL ice-cold 8M urea/500 mM TCEP/200 mM Gly-HCl, pH 3.0, and samples were allowed to reduce for 2 min on ice. The sample was loaded into a 20 μL loop and manually injected in a custom valve cooler. This was followed by digestion at room temperature with a Poroszyme Immobilized Pepsin Cartridge (2.1×30 mm, Thermo Scientific) at 50 μL/min for 1.5 min, followed by washing/trapping onto a C18 PepMap100 cartridge kept at 1° C. (5 μm, 1×5 mm, Thermo Scientific) at 400 μL/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 μm, 0.32×50 mm, Thermo Scientific) analytical column at 10 μL/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 al., 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 NaCl, 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 al., 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 μg/ml of mAb, 30 sec contact time and flow rate of 10 μl/min. IMAC-purified DI-D VHH 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 NaCl, 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 μM concentrations of SEC-pure material over the mAb surface at a flow rate of 30 μl/min for 5 min. Surfaces could be regenerated using 10 mM glycine, pH 1.7, with a 120 sec pulse at 20 μl/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 μl/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 μl/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 al., 2011) and affinities determined using the steady state binding model.
Surface Plasmon Resonance for VHH antibodies. For SPR experiments, IMAC-purified VHHs DI-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 (10 mM HEPES, pH 7.4, containing 150 mM NaCl, 3 mM EDTA and 0.05% P20), connected to an AKTAPurifier (GE Healthcare) at a flow rate of 1 ml/min. Monomeric VHH fractions were collected and protein concentration determined by measuring A280 measurement. Using a Biacore T200 instrument (GE Healthcare), approximately 500-1000 RUs of FC5DIE3IR, FC5 and an irrelevant VHH (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 DI-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 μl/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 U87MGvIII) and an ovarian cell line (SKOV-3) was evaluated in semi-solid agar in the absence or presence of either TTX (0.5 μM) or 1B6 (1 μM), 1H5 (1 μM) or 2G11 (1 μM) as described previously (Moreno et al., 2006). Approximately 1800 cells±treatment were resuspended in 150 μl of growth medium (DMEM+10% FBS) containing either 0.6% agar (U87MG and U87MGvIII) or 0.45% agar (SKOV3) and seeded into a well of a 24-well plate previously layered with 250 μl of 0.6% agar. The solidified cell layer was covered with 50 μl 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 μl 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 (30 μg) 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 1×TBST with 5% Carnation skim milk. Rabbit polyclonal anti-Nav1.7 (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-4111N copier. The membrane was washed for 30 minutes with TBST and incubated for 1 h with anti-βactin-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 Pant 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 12-230 kDA separation module (ProteinSimple), and the rabbit detection module (ProteinSimple). The samples (0.8 mg/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 μl) was loaded in the first well of row A1. The samples (4 μl) were loaded in the remaining wells of row A. An antibody diluent (10 μl) was added to row B and the first well of row C. A rabbit polyclonal anti-Nav1.7 (Alomone labs, ASC-008, 1:20) and an anti-βactin-HRP (Sigma, 1:100) were combined in antibody diluent and added to the remaining wells of row C. Streptavidin-HRP (10 μl) 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 μl of each) and 15 μl was added to each well of row E. The plate was covered with a lid and centrifuged at 1000 g for 5 minutes in a Eppendorf 5810R centrifuge. A wash buffer (500 μl) 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, Calif., USA) for analisys.
Animal immunization. Four six-week old female NJ mice (The Jackson Laboratory, Bar Harbor, Me.) were bled (pre-immune serum) and injected intraperitoneally and subcutaneously with 100 μg 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 μl per well of FC5DIE3IR at 5 μg/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 μl 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 μl of a 1/5.000 dilution of alkaline phosphatase conjugated goat anti-mouse IgG (H+L) (#115-056-062, Jackson Immunoresearch, Cedarlane, Burlington, ON) in blocking buffer was added. After a 1 h incubation, microplates were washed 4 times and 25 μl 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, Calif.). 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 μg 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 233 g for 10 min. 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 2×105 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% CO2. The next day, hybridoma cells were washed and suspended at a concentration of 2-3X105 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 μg/ml FITC-F(ab′)2 Goat anti-mouse IgG (Jackson #115-096-071 for F233 or 115-096-062 for F236). The cell mixture was plated in Omnitray dish (Nunc cat #242811) and further incubated for 6-7 days at 37° C., 5% CO2. Fluorescent secretor clones were then transferred using a mammalian cell clone picker (ClonepixFL, Molecular Devices) into sterile 96-w plates (Costar #3595) containing 200 μl 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% CO2.
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 μl of FC5DIE3IR or FC5-VHH or FC5-hFc-1X0 at 5 μg/ml in PBS and incubated overnight at 4° C. Microplates were washed 3 times with PBS, blocked with PBS-BSA 1%, and 25 μl of hybridoma supernatants were added and incubated at 37° C., 5% CO2 for 2 hours. Plates were washed 4 times with PBS-Tween 20 0.05% and incubated for one hour at 37° C., 5% CO2 with 25 μl of secondary antibody alkaline phosphatase conjugated F(ab′)2 goat anti-mouse IgG Fc-gamma specific (Jackson Immunoresearch #115-056-071) diluted 1/3000 in blocking buffer. After 4 washes with PBS-Tween 20 0.05%, 25 μl of a 1 mg/ml pNPP substrate solution was added and further incubated for one hour at 37° C. OD405 nm 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 Nav1.7 by ELISA.
Cell-ELISA. Sterile 96-well plates (Costar #3595) were pre-treated with 50 μl/well of branched polyethyleneimine (PEI, Sigma Cat #408727) at 25 μg/ml in PBS for 30 min at room temperature. PEI was removed and CHO-3E7 or hNAv1.7-CHO (ChanTest cat #CT4003) were seeded at 6×105 cells per well in FreeStyle F17 Expression Medium (Gibco Cat #A1383501) and incubated overnight at 37° C., 5% CO2. Microplates were blocked with Starting block (Thermofisher Cat #37538) for 30 min at room temperature, and 50 μl 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 μl of secondary antibody HRP-conjugated F(ab′)2 goat anti-mouse IgG (H+L) (Jackson Immunoresearch #115-036-062) diluted 1/5000 in blocking buffer. After 10 washes with PBS, 50 μl of TMB substrate solution (Pierce Cat #34021) was added and further incubated for 60 min at room temperature. Twenty-five μl/well of stop solution (H2504 1M) was added and OD450 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.
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 Bmax (maximum specific binding) and Kdapp (concentration needed to achieve a half-maximum binding at equilibrium) for each mAb tested. As shown in
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-hNav1.7 mAbs on hNav1.7 channels, Nav currents were evoked using whole-cell patch-clamp technique in hNav1.7-HEK293 cells as described in Example I. The hNav1.7-HEK293 cells were prepared from frozen vials and plated on poly-1-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 Nav currents were obtained as described in Example I.
As shown in
Next, it was evaluated if the anti-hNav1.7 3 A8 mAb had an effect on the kinetics and voltage dependence properties of the hNav1.7 channels. To do this, the effect of 3A8 on the current-voltage (I-V) relationship, the voltage-dependence of activation and steady-state fast inactivation of hNav1.7 channels were examined in hNav1.7-HEK293 cells (
To further investigate the effects of 3A8 on hNav1.7 kinetics of activation and fast inactivation, the time-to-peak and the time constants of fast inactivation of hNav1.7 channels were calculated. The mAb 3A8 (1 μM) 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 t-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 t-Test).
As shown in
As shown in
As shown in
These data suggest that also and 1B6, 2G11 and 1H5 are functional in inhibiting the flux of Na+ thought the hNav1.7 channels. Values for the effect of anti-hNav1.7 1B6 (
Overall, these data suggest that the main functional effect of 3A8, 1B6, 2G11 and 1H5 on Nav1.7 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.
In vivo Validation of the mAb 3A8: Rat Hargreaves Model of Hyperalgesia. To evaluate if the functional in vitro effect of anti hNav1.7 mAbs translated in a functional in vivo effect against pain, the mAbs were tested in rats using a Hargreaves model of hyperalgesia. The anti-hNav1.7 mAb 3A8 was tested along with a negative control anti-GFP mAb 11E5. 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
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 hNav1.7 channels TC-N1752 (Tocris, Bristol, United Kingdom).
To further evaluate the effect of the anti-hNav1.7 mAbs 2G11, 1H5 and 1B6, and the selective blocker for hNav1.7 channels TC-N1752 (Tocris, Bristol, United Kingdom) a CFA-induced thermal hyperalgesia in rats was used.
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 hNav1.7 channels TC-N1752 (Tocris, Bristol, United Kingdom).
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 1H5, 2G11 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).
It was next evaluated the effects of 3A8 injected by intravenous route.
To further evaluate the functionality of the anti-Nav1.7 mAbs and target engagement, they were tested on a Nav1.7 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 (
Identification of the hNav1.7 epitope for mAb 3A8, 186 and 1H5. To identify the epitope for mAb 3A8 on DIE3IR, seven overlapping antigen-peptides were generated (
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 (
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 μg) or P3 (375 μg) were co-injected with 3A8 (75 μg) by intraplantar route in rats Hargreaves model and their effect compared to the reversal of hyperalgesia caused by 3A8 (75 μg) and 11E5 (negative control, 50 μg) (
Surface Plasmon Resonance (SPR) for monoclonal antibodies. To determine the binding affinity of 3A8, 2G11, 1B6 and 1H5 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 VHH FC5 were obtained using Size Exclusion Chromatography purification (SEC). To examine the binding of 3A8, 2G11, 1B6 and 1H5 to the recombinant protein FC5DIE3IR, two SPR assay orientations were used:
In both orientations 3A8, 2G11 and 1B6 strongly and specifically bound to FC5DIE3IR and did not bind to FC5 control (see Table 5). At 1 μM concentration of FC5 and FC5DIE3IR flowed over the captured 3A8 (
Recombinant Chimeric Human-Mouse mAbs. To validate the sequences of the mAbs (3A8, 1B6; Table 7), recombinant chimeric human-mouse mAbs (hFc-F233-3A8, hFc-F236-1B6) 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-F236-1B6 were functional in reducing the hNav1.7 currents, Nav currents were evoked using whole-cell patch-clamp technique in hNav1.7-HEK293 cells as described in Example I. The hNav1.7-HEK293 cells were prepared from frozen vials and plated on poly-1-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 Nav currents were obtained as described in Example I.
At the concentration of 2 μM, hFc-F233-3A8 and hFc-F236-1B6 were able to reduce significantly the amplitude of the Nav currents (
Effect of mAbs Application on Cancer models. The four monoclonal antibodies targeting Nav1.7 were tested on a number cancer cell lines expressing Nav1.7. Expression of Nav1.7 in these cell lines was first assessed with western blot analysis.
VHHs from a phage-displayed LAC-M nave library. The FC5DIE3IR recombinant protein (described in Example I) was used in panning experiment to isolate VHHs from a phage-displayed LAC-M naïve 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 VHH protein and a blocking buffer to remove/reduce the phage population binding to the parental FC5 VHH 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. 5×1010 cells of LAC-M library were grown in 500 ml of 2YT-Tet (12.5 μg/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 μm filter unit. The phage supernatant was precipitated with 1/5 volume of PEG-NaCl (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 μl of 0.2-1 mg FC5DIE3IR/m1 PBS was added to one well of a NUNC MaxiSorp™ ELISA plate (eBioscience, San Diego, Calif.). Another well (Blank) was coated with the same amount of FC5 VHH 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 VHH+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 μl of StartingBlock at room temperature (RT) for 1-2 h. After one hour, 100 μl of pre-adsorb phage (1012) was added to the blank well and continued the incubation for another hour. This step will further pre-adsorb undesired phage binders. Subsequently, 100 μl 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 2nd, 3rd and 4th rounds of panning, respectively). The bound phages were eluted by adding 100 μl/well of 100 mM trimethylamine (TEA) and incubated at RT for exactly 10 min. The content of the well (100 μl) were transferred into a microcentrifuge tube containing 50 μl of 1M Tris-HCl (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 μl of the infected cells in 150 μl 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 μl 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 μm filter unit. The phage supernatant was precipitated as described above and the phage pellet was re-suspended in 200 μl 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=A260×1010×100×22.14.
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 μg/ml of FC5DIE3IR and FC5 VHH (the negative control) were coated onto a microtiter plate. All positive clones specifically binding to FC5DIE3IR were sequenced and unique VHH sequences were selected for sub-cloning, large-scale expression, and purification. A total of 6 VHHs specific to FC5DIE3IR were isolated. Six VHH clones specific to FC5DIE3R (DI-A, DI-B, DI-C, DI-D, DI-E and DI-H) were identified by phage ELISA using FC5 parental FC5 VHH protein as negative control (
Expression and Purification of Soluble Monomeric VHHs. DNA constructs (see Table 7 for a description of the nucleotide sequence) encoding the isolated VHHs were synthesized commercially by GeneArt (Life Technologies, Carlsbad, Calif.) as described (Baral et al., 2013). 6×His- and c-Myc-tagged VHH 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, N.J.) as described in Baral et al., 2013 and Arbabi-Ghahroudi et al., 2009. The integrity and aggregation status of soluble VHH 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 al., 2009.
It was determined, by performing an protein-ELISA assay that all VHH antibodies showed specific binding to the FC5DIE3R protein with little or no cross-reactivity to FC5 VHH (data not shown).
In vitro Identification of Functional VHHs. To determine the functional effect of the isolated VHHs on hNav1.7 channels, Nav currents were evoked using the whole-cell patch-clamp technique in hNav1.7-HEK293 cells as described in Example I. Five VHHs (DI-A, DI-B, DI-C, DI-D, and DI-H) were tested using patch-clamp whole-cell. The VHH DI-E protein was not stable and consequently not tested. VHHs were tested at an initial concentration of 1 μM. At this concentration, DI-D reduced the amplitude of the Nav1.7 currents of 11.7%±5.8 (n=12), while DI-A, DI-B, DI-C, DI-H had no effect (DI-A, 2.73%±10.52, n=4; DI-B, 1.73%±1.96, n=6; DI-C, 0.46%±2.07, n=4; DI-H, 1.72%±1.32, n=3;
Next, it was evaluated if the VHH DI-D had an effect on the kinetics and voltage dependence properties of the hNav1.7 channels. To do this, the effect of DI-D on the current-voltage (I-V) relationship as well as the voltage-dependence of activation and steady-state fast inactivation of hNav1.7 channels in hNav1.7-HEK293 cells were examined (
In vivo validation of the VHH: rat Hargreaves model of hyperalgesia. To evaluate if the functional in vitro effect of the VHH DI-D translated in a functional in vivo effect against pain, DI-D was tested on CFA-induced thermal hyperalgesia in rats and compared it with the selective blocker for hNav1.7 channels TC-N1752 (Tocris, Bristol, United Kingdom). The VHH DI-D was tested also along with a negative control anti-GFP mAb 11E5. 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
To further evaluate the functionality of the VHH DI-D and target engagement, the VHH 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 (
VHHs from a FC5DIE3IR llama immune library. The FC5DIE3IR recombinant protein (100 μg, 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 μg 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, N.Y.) were coated overnight at 4° C. with 5 μg/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 μl 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, Tex.). Signal was detected by adding 100 μl/well TMB peroxidase substrate (Kirkegaard and Perry Laboratories, Gaithersburg, Md., USA). Reactions were stopped by adding 100 μl of 1M phosphoric acid and A450 was measured using a standard ELISA plate reader.
The VHH library was constructed as described previously (Baral et al., 2013). In brief, total RNA was isolated from approximately 1×107 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 μg 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 (IgG2 and IgG3) 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 VHH fragments were ligated with 40 μg (3:1 molar ratio, respectively) Sfi-digested pMED1 phagemid vector using LigaFast Rapid DNA ligation system and its protocol (Promega, Madison, Wis.), transformed into commercial electrocompetent TG1 E. coli cells (Stratagene, La Jolla, Calif.) as described previously (Baral et al., 2013) and a library size of 5×107 transformants was obtained. The VHH 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 VHH fragments. The library was grown for 3-4 h at 37° C., 250 rpm in 2×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 (5×1010 cells) was grown in for 1-2 hours at 37° C., 250 rpm in 2×YT/Amp-Glucose (2% w/v) medium (A600=0.4-0.5), infected with M13KO7 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×YT/Amp with 50 μg/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 VHH protein and a blocking buffer to remove/reduce the phage population binding to the parental FC5 VHH 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 μl of 0.4 mg/ml of FC5DIE3IR in PBS was added to one well of a NUNC MaxiSorp™ ELISA plate (eBioscience, San Diego, Calif.). Another well (blank) was coated with the same amount of FC5 VHH 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 VHH+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 μl of StartingBlock at room temperature (RT) for 1-2 h. After one hour, 100 μl of pre-adsorb phage (1012) was added to the blank well and continued the incubation for another hour. This step will further pre-adsorb undesired phage binders. Subsequently, 100 μl of pre-adsorbed phages were transferred from the blank well to the FC5DIE31R-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 2nd, 3rd and 4th rounds of panning, respectively). The bound phages were eluted with 0.1 M triethylamine, neutralized with 1M Tris-HCL, PH 7.4 and incubated with exponentially growing TG1 cells. After 30 min incubation at 37° C., the cells were superinfected with M13KO7 for additional 15 min and grown in 2×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 μg/well and washing was increased 7, 10 and 12× 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 μg/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 μg/ml of FC5DIE3IR and FC5 VHH (the negative control) were coated onto a microtiter plate. Four VHH clones specific to FC5DIE3IR (DI-4, DI-16, DI-28, DI-48) were identified by phage ELISA using FC5 parental VHH protein as negative control (
Expression and Purification of Soluble Monomeric VHHs. DNA constructs encoding the isolated VHHs (DI-4, D116, DI-28 and DI-48; Table 7) were synthesized and cloned into the pSJF2H expression vector commercially by GeneArt (Life Technologies, Carlsbad, Calif.) 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 His6 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 2×YT-Amp culture and grown at 37° C. with vigorous shaking. At OD600=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 re-suspended in 30 ml of cold TES buffer (0.2M Tris-CI pH 8.0, 20% sucrose, 0.5 mM 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 VHHs were dialyzed overnight against buffer (20 mM phosphate buffer (pH 7.4), 0.5M NaCl, 10 mM imidazole) at 4° C. then filtered through a 0.45 μm 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 ÄKTA FPLC purification system (GE Healthcare). The VHHs were eluted using gradient elution (buffer containing 500 mM imidazole); the fractions were pooled and dialyzed against PBS. VHH 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-11, DI-16, DI-28) (having a signal of at least 3× background) were identified and further characterized. Sequencing data showed that clones DI-11 and DI-16 have identical sequences and the difference in ELISA signal could be due to lower phage titer for clone DI-11. Additional rounds of screening (data not shown) identified a new VHH clone, named DI-48.
Surface Plasmon Resonance (SPR). To determine the binding affinity of the VHHs DI-4, DI-16, DI-28 and DI-48 with the recombinant protein FC5DIE3IR used to isolate the VHHs, SPR binding assays were performed. Before starting, pure monomeric fractions of the VHHs 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 VHH 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 (
CAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAG
CTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAACTACTGGATG
CACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGA
GAGATTAATCCTAGCAACGGTCGTGCTAACTACAATGAGAAGTTC
AAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCC
TACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTAT
TACTGTGCAAGATCCCCTTATGGTTACTACGACTACTGGGGCCAA
GGCACCACTCTCACAGTCTCCTCAGCCAAAACAACAGCCCCATCG
CTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCT
AGTCAGAGCCTTTTACACAGTAATGGAAACACCTATTTCCATTGG
TACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAA
GTTTCCAACCGATTTTTTGGGGTCCCAGACAGGTTCAGTGGCAGT
GGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCT
GAGGATCTGGGAGTTTATTTCTGCTCTCAAATTACACATGTTCCG
CTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGAT
CAGCAGCCTGGGGCTGAGCTTGTGAAGCCTGGGGCTTCAGTGAAG
CTGTCCTGCAAGGCTTCTGGCTACACTTTCACCAGCTACTGGATA
AACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGA
AATATTTATCCTGGTAATAGTAATACTAACTACAATGAGAAGTTC
AAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCC
TACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTAT
TATTGTGCAAGACGGTACTACTATGATTACGACGATGCTATGGAC
TACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACG
GTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAG
AGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTAT
GGCAATGGTTTTATGCACTGGTACCAGCAGAAACCAGGACAGCCA
CCCAAACTCCTCATCTATCGTGCATCCAACCTAGAATCTGGGATC
CCTGCCAGGGTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTC
ACCATTAATCCTGTGGAGGCTGATGATGTTGCAACCTATTACTGT
CAGCAAAGTAATGAGGATCCGTGGACGTTCGGTGGAGGCACCAAG
CTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTC
GNGFMHWYQQKPGQPPKWYRASNLESGIPARVSGSGSRTDFILTI
Homo sapiens
AAGGAGTCAGGACCTGGCCTGGTGGCACCCTCACAGAGCCTGTCC
ATCACATGCACTGTCTCTGGGTTCTCATTATCCAGATATAATGTA
CACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGA
ATGATATGGGGTGGTGGAAGCACAGACTATAATTCAGCTCTCAAA
TCCAGACTTAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTC
TTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATGTACTAC
TGTGCCAGAAATGGAGCTAACTGGGACTGGTTTGCTTACTGGGGC
CAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCA
TCTCCATCCTCCCTACCTGTGTCAGTTGGAGAGAAGGTTACTATG
AGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAG
AACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAA
CTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGAT
CGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATC
AGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAA
TATTATAGCTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAA
ATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCA
NYLAWYQQKPGQSPKWYWASTRESGVPDRFTGSGSGTDFILTISS
CAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAG
CTGTCCTGCAAGGCTTCTGGCTACACCTTCACCACCTACTGGATG
CACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGA
GAGATTAATCCTAGCAACGGTCGTGCTAACTACAATGAGAAGTTC
AAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCC
TACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTAT
TACTGTTTAAGATCACTAGGCTACTTTGACTACTGGGGCCAAGGC
ACCACTCTCACAGTCTCCTCAGCCAAAACAACAGCCCCATCGGTC
GTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAG
AGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTG
CAGAAGGCAGGCCAGTCTCCAAAGTTCCTGATCTACAAAGTTTCC
AACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCA
GGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGAT
CTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTACACG
TTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCA
SLVHSNGNTYLHWYLQKAGQSPKFLIYKVSNRFSGVPDRFSGSGS
GTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGG
TCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGAAGT
TATGCCATGTCTTGGGTTCGCCAGACTCCAGAAAAGGGGCTGGAG
TGGGTCGCATCCATTAGTAGTGGTGGTAGCACCTACTATCCAGAC
AGTGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCGGGAAC
ATCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCC
ATGTATTACTGTGCAAGAGGCTATGATGGTTACTACGAGAGGATA
TGGTACTATGCTATGGACTATTGGGGTCAAGGAACCTCAGTCACC
GTCTCCTCAGCCAAAACGACACCCCCATCTGTCTAT
YAMSWVRQTPEKGLEWVASISSGGSTYYPDSVKGRFTISRDNAGN
GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTA
GGAGACAGGGTCACTGTCTCCTGCAAGGCCAGTCAGAATGTGGGT
ACTATTGTAGCCTGGTATCAACAAAAACCAGGTCAATCTCCTAAA
GCACTGATTTACTCGGCATCCTACCGGTTCAGTGGAGTCCCTGAT
CGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATC
AGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAA
TATAACACCTATCCTCTCACGTTCGGCTCGGGGACAAAGTTGGAA
ATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCA
TIVAWYQQKPGQSPKALIYSASYRFSGVPDRFTGSGSGTDFTLTI
MPLLLLLPLLWAGALAQVQLQQPGAELVKPGASVKLSCKASGYTF
MRLPAQLLGLLMLVVVSGSSGDVLMTQTPLSLPVSLGDQASISCR
MPLLLLLPLLWAGALAQVQLKESGPGLVAPSQSLSITCTVSGFSL
MRLPAQLLGLLMLWVSGSSGDIVMSQSPSSLPVSVGEKVTMSCKS
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.
This application is a national stage application, filed under 35 U.S.C. § 371, of International Patent Application No. PCT/CA2019/050676, filed on May 17, 2019, which claims the benefit of U.S. Provisional Patent Application No. 62/673,210, filed May 18, 2018, which is incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2019/050676 | 5/17/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62673210 | May 2018 | US |
