JP6335152B2 - Antibodies against ion channels - Google Patents

Description

  The present disclosure relates to an anti-ion channel antibody directed to the E1 portion of an ion channel with functionally modifying properties and fragments of the antibody, pharmaceutical compositions comprising the antibody, treatment, eg, treatment / modulation of pain It relates to the use of antibodies and compositions comprising them, and to methods for making and preparing said antibodies.

  Ion channels are pore-forming proteins that help establish and control the cell membrane potential of all living cells by allowing the flow of ions down an electrochemical gradient of ions. They are present in the membrane that surrounds all biological cells. The human genome exhibits over 400 different numbers of ion channel genes that exhibit extensive diversity and play an important role in many cellular processes such as secretion, muscle contraction, and the generation and propagation of action potentials in heart and nerve tissues. Including.

  Ion channels are integral membrane proteins that can take the form of large molecular structures based on a collection of several proteins. Such “multi-subunit” assemblies typically comprise an arrangement of identical or homologous proteins that are tightly packed around a water-filled pore through the plane of the membrane or lipid bilayer. The pore-forming subunit (s), commonly referred to as alpha subunits, are auxiliary subunits, either membrane bound or cytoplasmic, that help control the activity and cell surface expression of ion channel proteins. Can meet. The X-ray structure of various ion channels has recently been elucidated (Doyle et al., Science 280: 69 (1998); Jiang et al., Nature 423: 33 (2003); Long et al., Science 309: 897 (2005)), pore structure. This configuration is largely conserved among ion channel family members. The opening and closing of ion channel pores, called gating processes, can be triggered by various cellular or biochemical processes.

  The largest family of ion channel proteins includes, for example, voltage-gated channels, including sodium, calcium, and potassium ion channels, transient receptor potential ion channels, hyperpolarization activated ion channels, inward rectifying ion channels, It is composed of a 2-pore domain potassium channel and a voltage-gated proton channel. The last is depolarized in a pH sensitive manner.

  The inwardly rectifying ion channel is composed of 15 official members and 1 informal member. The family can be further subdivided into 7 subfamilies based on homology.

  There are currently about 10 voltage-gated calcium channels that have been identified.

  Transient receptor potential ion channels are subdivided into six subfamilies based on homology: classical (TRPC), vanilloid receptor (TRPV), melastatin (TRPM), polycystin (TRPP) , Mucolipin (TRPML), and ankyrin transmembrane protein 1 (TRPA).

Hyperpolarization activated ion channels are sensitive to the cyclic nucleotides cAMP and cGMP, which changes the voltage sensitivity of the channel opening. These channels are permeable to monovalent cations K ⁺ and Na ⁺ . There are four members of this family, all of which form a tetramer of six transmembrane α subunits. Since these channels open under hyperpolarized conditions, they function as pacing channels in the heart, particularly in the sinoatrial node.

  Voltage-gated and ligand-gated ion channels are the most representative members of the ion channel protein family. The activity of voltage-gated ion channels (eg, calcium channels, sodium channels, and potassium channels) is controlled by changes in cell membrane potential, while ligand-gated ion channels (eg, GABA-A receptor, acetylcholine receptor) Is controlled by the binding of specific intracellular or extracellular ligands. The gating mechanism is very complex, involves various membranes, pore structures and cytoplasmic structures, and varies between ion channel classes.

  Voltage-gated ion channels, sometimes referred to as voltage-sensitive ion channels, are a class of transmembrane proteins that provide the basis for cell excitability in heart and nerve tissues. These channels are activated by either cell hyperpolarization or cell depolarization, resulting in ion fluxes that result in control of cell membrane potential. Voltage-gated sodium channels are generally involved in triggering action potentials, whereas voltage-gated potassium channels mediate cell membrane repolarization. Fine-tuned interactions between various voltage-gated ion channels are important in taking the form of cardiac and neural action potentials.

One class of voltage-gated sodium channels includes nine different isoforms (Nav1.1-1.9), and four different sodium channel specific accessory proteins have been described (SCN1b-SCN4b). The separate functional activities of these isoforms are described in various neuronal cell types (Llinas et al., J. Physiol. 305: 197-213 (1980); Kostyuk et al., Neuroscience 6: 2423- 2430 (1981); Bossu et al., Neurosci. Lett. 51: 241-246 (1984) 1981; Gilly et al., Nature 309: 448-450 (1984); French et al., Neurosci.Lett. 56: 289-294 (1985); Ikeda et al., J. Neurophysyl.55. Jones et al., J. Physiol.389: 605-627 (1987); Alonso & Linas, 1989; Gilly et al., J. Neurosci.9: 13. 2-1374 (1989)) and skeletal muscle (Gonoi et, J.Neurosci.5: 2559~2564 (1985); Weiss et al., Science 233: is described in 361 to 364 (1986)). Nav1.5 and Nav1.4 channels are the major sodium channel isoforms expressed in heart and muscle tissues, respectively, Na _v 1.1, 1.2, 1.3, 1.6, 1.7 , 1.8, and 1.9 are specifically expressed in the central and peripheral nervous systems. Using the natural toxin, tetrodotoxin (TTX), it was possible to establish a pharmacological classification of sodium channel isoforms based on their affinity for the toxin. Therefore, voltage-gated sodium channels were classified as TTX resistant (Na _v 1.5, 1.8, 1.9) and TTX sensitive.

Certain ion channels have been implicated in the regulation of pain (see, eg, PNAS Nov. 6, 2001, Vol. 98, No. 23, 13373-13378, and The Journal of Neuroscience 22, 2004 24 (38) 832-836). . The ion channel Na _v 1.7 is believed to be capable of modulating pain such as neuropathic pain and is therefore a particularly interesting therapeutic intervention target. Na _v 1.8 and Na _v 1.9 are also thought to have a role in the regulation of pain.

Na _v 1.7 is a voltage-activated tetrodotoxin sensitive sodium channel encoded by the gene SCN9A. Both gain-of-function and loss-of-function mutations of Na _v 1.7 result in overt pain-related abnormalities in humans.

  Originally, gain-of-function mutations in SCN9A were identified by linkage analysis as the cause of erythema (or primary lupus erythema) and paroxysmal extreme pain disorder (formerly familial rectal pain) . Erythema is a rare autosomal dominant disorder with seizures of burning pain along with fever and redness in the extremities. It is a very rare phenotype that an individual without neuropathy and otherwise cannot feel pain at all. Very recently, two studies reported by Cox et al. (2006) and Goldberg et al. (2007) describe phenotypes such as autosomal recessive traits that map to chromosome 2q24.3 (the region containing the gene SCN9A). doing. In both studies, detailed neurological examinations revealed that these people can distinguish between sharp / dull and hot / cold stimuli but have an overall lack of pain. All had lip and / or tongue wounds caused by biting themselves. All suffered frequent contusions and cuts, and most people experienced fractures or meningitis.

This data suggests that SCN9A channel disease resulting in loss of function of the ion channel Na _v 1.7 is associated with neuropathy or insensitivity to pain in the absence of cognitive, affective, or neuropathy There is good evidence and clinically confirms Na _v 1.7 as a pain-related target. Furthermore, from the KO study and animal pain models, Na _v 1.7 appears to play a major role in inflammatory pain.

FIG. 2a is a diagrammatic representation of an ion channel such as Na _v 1.7 containing four domains, A, B, C, and D (also referred to as domains I, II, III, and IV). Each domain contains six transmembrane protein helices S1, S2, S3, S4, S5, and S6. The exact amino acid number of each transmembrane protein depends on the database registry used, but UniProtKB / Swiss-Prot provides the following information for Na _v 1.7: In domain A, transmembrane protein S1 , S2, S3, S4, S5, and S6 are assigned to amino acids 122-145, 154-173, 187-205, 212-231, 248-271, and 379-404, respectively;
In domain B, transmembrane proteins S1, S2, S3, S4, S5, and S6 are assigned to amino acids 739-763, 775-798, 807-826, 833-852, 869-889, and 943-968, respectively. Be
In domain C, transmembrane proteins S1, S2, S3, S4, S5, and S6 are assigned to amino acids 1188-1211, 1225-1250, 1257-1278, 1283-1304, 1324-1351, and 1431-1457, respectively. Be
In domain D, the transmembrane proteins S1, S2, S3, S4, S5, and S6 are assigned to amino acids 1511 to 1534, 1546 to 1569, 1576 to 1599, 1610 to 1631, 1647 to 1669, and 1736 to 1760, respectively. It is done.
There are several natural variations of that sequence available in public databases, see for example UniProtKB / Swiss-Prot Q15858.

  In the present disclosure, S1, S2, S3, S4, S5, and S6 refer to the entities described above, including those given different amino acid assignments, or corresponding entities in alternative ion channels, and their natural Or non-natural varieties and corresponding entities in different isotypes.

  Each domain also includes extracellular hydrophilic loops E1, E2, and E3. The amino acid sequence of E1 in each domain begins after the transmembrane region S1 and ends with S2. E1 in each domain is different from E1 in other domains. The amino acid sequence of E2 in each domain begins after the transmembrane region S3 and ends with S4. E2 in each domain is different from E2 in other domains. The amino acid sequence of E3 in each domain begins after the transmembrane region S5 and ends with S6. E3 in each domain is also different from E3 in other domains.

Na _v and Ca _v ion channels, four domains A, wherein B, C, and D, but each containing six transmembrane protein helix, _{K v} ion channels, HCN ion channels, and the like TRP ion channels Other ion channels contain one domain. As for each domain in the Na _v and Ca _v ion channels, _{K v} ion channels, HCN ion channels, and TRP ion channels, as described above, six transmembrane protein helix S1, S2, S3, S4, S5 , And S6, and three extracellular hydrophilic loops E1, E2, and E3.

In the Na _v 1.7 ion channel, the extracellular loop (E loop) is the following amino acid residue of SEQ ID NO: 105 in FIG. 2c:

The extracellular loops in some domains of Na _v 1.7 are similar to the extracellular loops found in other ion channels.

Na _v 1.7 is expressed in the peripheral nervous system, ie nociceptive dorsal root ganglia (DRG), most notably in nociceptive small-diameter DRG neurons and almost in the brain Not presented. Na _v 1.7 distribution (eg sensory end) and physiology predispose it to play a major role in transmitting painful stimuli.

Expression of Na _v 1.7 in the peripheral nervous system makes it a very attractive target for the production of function-blocking antibodies, which are free of side effects or minimize side effects to acceptable levels. Shows an innovative approach to valuable treatment for pain.

  Neuropathic pain is a very common condition. In the United States, it is estimated that between 0.6% and 1.5% of the population, ie 1.8 to 4.5 million, are affected (Pullar and Palmer, 2003). Every year, at least 1.4 million people are diagnosed with three major causes of neuropathic pain: painful diabetic neuropathy (PDN), postherpetic neuropathy (PHN), or trigeminal neuralgia (TN). Other causes of neuropathic pain include spinal cord injury, multiple sclerosis, phantom limb pain, post-stroke pain, and HIV-related pain. If including patients with neuropathy-related chronic back pain, osteoarthritis, and cancer, the total number will be at least doubled. Nonsteroidal anti-inflammatory drugs (NSAIDs) are often used but are not very effective in the treatment of neuropathic pain. In addition, their long-term use can cause severe stomach damage. On the other hand, the use of opioids (morphine and derivatives) is limited to the most severe form of neuropathic pain, ie cancer-related neuropathy. This is because serious side effects such as nausea, vomiting, respiratory depression, constipation, and tolerance, as well as the potential for epilepsy and abuse are associated with long-term treatment. The latter have prevented the use of opioids in other neurological disorders (Delemijn, 1999; Namaka et al., 2004). Antiepileptic drugs (AEDs) are known to attenuate abnormal nerve hyperexcitability in the brain. Given that neuroexcitability plays an important role in neuropathic pain, it can be seen that AED has been directed to the treatment of chronic neuropathic pain (Renfrey, Downton, and Featherstone, 2003). Very recent important examples are gabapentin (Neurontin) and pregabalin (Lyrica, Frampton and Scott, 2004). However, even gabapentin, the gold standard for the treatment of neuropathic pain, only reduces pain by as much as 50% in about 40% of patients (Dworkin, 2002). Furthermore, in contrast to opioids, gabapentin has not been used to treat cancer-related neuropathic pain.

As noted above, the “loss of function” mutation of Na _v 1.7 in humans results in insensitivity to pain (Cox et al., 2006). Moreover, Na _v 1.7 “gain-of-function” mutations in humans lead to pain phenotype erythema and paroxysmal extreme pain disorders (Dib-Hajj, Yang, Waxman, 2008). In addition, peripherally acting small molecules that block Na _v 1.7 reverse hyperalgesia and allodynia in a rat model of inflammatory and neuropathic pain (McGowan et al., 2009). Thus, peripherally acting Na _v 1.7 blocking antibodies should be beneficial in the treatment of pain.

  To date, potent chemical inhibitors of ion channels have been identified, but generally they are characterized by poor selectivity for other ion channel isoforms. Considering the ubiquitous distribution in the living body, the use of these non-selective inhibitors is limited.

Antibodies are clearly desirable due to their elaborate specificity, but making functionally modified antibodies is, in part, quite simple, because ultimately clonal antibodies are required for therapeutic applications. Rather, some researchers in the field point out that polyclonal antibodies are required to effect alterations in ion channel function. Kionsky et al. (The Journal of Pharmacology and Experimental Therapeutics Vol. 319, pp. 192-198) states as follows:
“Neither rabbit, mouse, or fully human monoclonal antibodies raised against the pre-pore region of human TRPV1 were effective in blocking channel activation, so they We assume that it is impossible to lock the channel conformation through a high affinity binding agent (Since no rabbit, mouse or fully human monoclonal antibodies generated against the prepore region of human TRPV1 .. were effective in blocking channel activation we hypothesisethat it may not be possible to lock the channel conformation through high-affinity binders to small epitopes in this region). "

Sodium channels, in particular Na _v 1.7, Na _v 1.8, Na _v 1.9 appear to have been particularly difficult targets for generating functionally modified antibodies. However, the present inventors have now found that the activity of the ion channel can be altered using a functionally modified antibody, eg, a clonal population of antibodies. To date, antibodies against ion channels have been produced, but it seems that no E1-binding function-modified antibodies against ion channels involved in the regulation of pain have been disclosed.

  The inventors have now established that functionally modified antibodies can be produced against the E1 loop of ion channels involved in the regulation of pain. This is surprising because the E1 loop in each of the domains is a relatively short amino acid sequence.

Doyle et al., Science 280: 69 (1998) Jiang et al., Nature 423: 33 (2003). Long et al., Science 309: 897 (2005). Llinas et al. Physiol. 305: 197-213 (1980) Kostyuk et al., Neuroscience 6: 2423-2430 (1981) Bossu et al., Neurosci. Lett. 51: 241-246 (1984) 1981 Gilly et al., Nature 309: 448-450 (1984). French et al., Neurosci. Lett. 56: 289-294 (1985) Ikeda et al. Neurophysiol. 55: 527-539 (1986) Jones et al. Physiol. 389: 605-627 (1987) Alonso & Linnas, 1989; Gilly et al., J. MoI. Neurosci. 9: 1362-1374 (1989) Gonoi et al. Neurosci. 5: 2559-2564 (1985 Weiss et al., Science 233: 361-364 (1986). PNAS Nov. 6, 2001, Vol. 98, No. 23, 13373-13378 The Journal of Neuroscience 22, 2004 24 (38) 832-836 Kionsky et al., The Journal of Pharmacology and Experimental Therapeutics 319: 1, 192-198

  Accordingly, the present invention provides an anti-E1 ion channel antibody or binding fragment thereof, wherein the ion channel is functioning in the regulation of pain, and the antibody or fragment binds the ion channel to it and then functions. Modified.

FIG. 5 shows the functional effect of certain monoclonal antibodies on human Na _v 1.7 current in HEK cells. It is a diagrammatic representation of Na _v 1.7. Na _v 1.7 Domain A (SEQ ID NO: 101), Domain B (SEQ ID NO: 102), Domain C (SEQ ID NO: 103), and shows the amino acid sequence of the domain D (SEQ ID NO: 104). FIG. 3 is a diagram showing the complete amino acid sequence of Na _v 1.7 (SEQ ID NO: 105). FIG. 11 shows that clonal 983 anti-Na _v 1.7 antibody reduces the frequency of electrically induced DRG spikes in vitro. FIG. 11 shows that anti-Na _v 1.7 monoclonal antibody 983 reduces the frequency of electrically induced DRG spikes in vitro. FIG. 5 shows that anti-Na _v 1.7 monoclonal antibody 1080 in vitro reduces the frequency of electrically induced DRG spikes. (A) Automatic patch clamp analysis of recombinant human Nav1.7 channel expressed in HEK cells. The 983 monoclonal antibody produces a dose-dependent inhibition of the Nav1.7 current. (B) Automatic patch clamp analysis of recombinant human Nav1.7 channel expressed in HEK cells. The 1080 monoclonal antibody produces a dose-dependent inhibition of the Nav1.7 current. FIG. 6 shows automated patch clamp analysis of recombinant rat Nav1.7 channel expressed in HEK cells. The 983 monoclonal antibody produces a dose-dependent inhibition of the Nav1.7 current. The 1080 monoclonal antibody produces about 26% inhibition of the Nav1.7 current at 25 μg / ml. It is a figure which shows the dynamics of human Nav1.7 inhibition by a 983 monoclonal antibody. FIG. 10 shows ELISA data for specific binding of antibody 983 to Nav1.7 peptide. FIG. 10 shows ELISA data for specific binding of antibody 1080 to Nav1.7 peptide. It shows the amino acid sequence of a particular _anti-Na v 1.7 antibody. It shows the amino acid sequence of a particular _anti-Na v 1.7 antibody. It shows the amino acid sequence of a particular _anti-Na v 1.7 antibody. It shows the amino acid sequence of a particular _anti-Na v 1.7 antibody.

  In one aspect, the disclosure provides an ion channel E1 loop binding entity that functionally modifies the activity of an ion channel associated with modulation of pain, comprising an antibody, antibody fragment, protein or proteinaceous scaffold, Examples include nucleic acids or nucleotides, small molecules such as synthetic molecules, in particular antibodies, antibody fragments, proteins or proteinaceous scaffolds, or nucleic acids or nucleotides.

An ion channel believed to participate and / or associated with the modulation of _{pain, Na v 1.3, Na v 1.6} , Na v 1.7, Na v 1.8, Na v 1.9, Ca v 3 _{.1, Ca v 3.2, Ca v} 3.3, Ca v 2.1, Ca v 2.2, Ca v 2.3, K v 2.1, K v 2.2, K v 7. x, HCN1, HCN2, TRPV1, TRPA1, ASIC1, TRPM8, TRPV3, and TRP4, but are not limited thereto.

In one embodiment, the ion _channels, such as Na v _1.7, Na v _1.7, a sodium channel of Na v 1.8, or _Na v 1.9,. The peptide used for immunization may comprise at least part of the extracellular sequence of the ion channel, the extracellular sequence being the E1 loop, and derived from the A domain, B domain, C domain, or D domain of the ion channel There may be. In a preferred embodiment, the peptide comprises at least a portion of the E1 extracellular region from the A domain, B domain, C domain, or D domain of the ion channel. In a further preferred embodiment, the peptide comprises at least part of the E1 extracellular region from the A or B domain of the ion channel. Preferably, the peptide comprises at least a portion of the BE1 extracellular region.

  In one embodiment of the invention, the ion channel is not Nav1.7. In one embodiment, the invention provides an anti-E1 ion channel antibody or binding fragment thereof that binds to the E1 extracellular loop of the ion channel, wherein the ion channel functions in the regulation of pain, A fragment functionally modifies the ion channel after binding it to it, and the ion channel is not Nav1.7.

In one embodiment, the ion channel is a potassium ion channel, K _v 2.1, K _v 2.2, or K _v 7. x.

In one embodiment, ion channels, calcium ion channels, for _{example, Ca v 3.1, Ca v 3.2} , Ca v 3.3, Ca v 2.1, Ca v 2.2, or _Ca v 2. 3.

  In one embodiment, the ion channel is the hyperpolarization activated channel HCN1 or HCN2.

  In one embodiment, the ion channel is a non-gated ion channel, eg, TRPV1, TRPA1, ASIC1, TRPM8, TRPV3, or TRP4.

  In one embodiment, an E1-binding anti-ion channel antibody for use in treatment, eg, modulation of pain, particularly pain amelioration is provided.

  In one embodiment, an E1-binding anti-ion channel antibody for use in modulation, eg, pain amelioration, is provided.

In one embodiment, there is provided an E1-binding anti-Na _v 1.7 antibody for use in modulation, eg, remission of pain, particularly the pain subgroups described herein.

In one embodiment, there is provided an E1-binding anti-Na _v 1.8 antibody for use in modulation, eg, remission of pain, particularly the pain subgroups described herein.

In one embodiment, an E1-binding anti-Na _v 1.9 antibody for use in modulation, eg, remission of pain, particularly the pain subgroups described herein, is provided.

  In one embodiment, there is provided an E1-binding anti-HCN1 antibody for use in modulation, eg, remission of pain, particularly the pain subgroups described herein.

  In one embodiment, there is provided an E1-binding anti-HCN2 antibody for use in modulation, eg, remission of pain, particularly the pain subgroups described herein.

  In one embodiment, there is provided an E1-binding anti-TRPA1 antibody for use in modulation, eg, remission of pain, particularly the pain subgroups described herein.

  In one embodiment, there is provided an E1-binding anti-TRPV1 antibody for use in modulation, eg, remission of pain, particularly the pain subgroups described herein.

  In one embodiment, an E1-binding anti-TRPV3 antibody for use in modulation, eg, remission of pain, particularly the pain subgroups described herein, is provided.

  In one embodiment, there is provided an E1-binding anti-TRPM8 antibody for use in modulation, eg, remission of pain, particularly the pain subgroups described herein.

  In one embodiment, there is provided an E1-binding anti-TRP4 antibody for use in modulation, eg, remission of pain, particularly the pain subgroups described herein.

  In one embodiment, there is provided an E1-binding anti-ASIC1 antibody for use in modulation, eg, remission of pain, particularly the pain subgroups described herein.

In one embodiment, modulating, for example, pain, E1 binding anti Ca v _3.1 antibody for use in ameliorating subgroup of pain as specifically described herein are provided.

In one embodiment, modulating, for example, pain, E1 binding anti Ca v _3.2 antibody for use in ameliorating subgroup of pain as specifically described herein are provided.

In one embodiment, modulating, for example, pain, E1 binding anti Ca v _3.3 antibody for use in ameliorating subgroup of pain as specifically described herein are provided.

In one embodiment, modulating, for example, pain, E1 binding anti Ca v _2.1 antibody for use in ameliorating subgroup of pain as specifically described herein are provided.

In one embodiment, modulating, for example, pain, E1 binding anti Ca v _2.2 antibody for use in ameliorating subgroup of pain as specifically described herein are provided.

In one embodiment, modulating, for example, pain, E1 binding anti Ca v _2.3 antibody for use in ameliorating subgroup of pain as specifically described herein are provided.

  A functionally modified antibody as used herein reduces the activity of an ion channel, eg, at least 5%, such as 20% in at least one in vitro or in vivo assay, eg, 10% or 15%. Is intended to refer to an antibody or fragment (such as a binding fragment) to be altered. Suitable in vitro assays include patch clamp assays or other assays as described herein. In one embodiment, the functionally modified antibody has a current amplitude of 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70% or more by a patch clamp assay. Decrease percentage.

  An antibody that gives a functional modification to an ion channel and a functional modified antibody are terms that are used interchangeably herein.

  In one embodiment, the functional modification is sufficient to, for example, block, close or inhibit the pores of the ion channel. This functional modification may be effected by any mechanism, such as physically blocking the pore, eg blocking the pore, causing a conformational change in the ion channel, or ion Inducing the channel to assume a non-functional state (resting state or inactivated state) and / or maintaining the ion channel in a non-functional state (allosteric modulation).

  In one embodiment, the functional modification is sufficient to reduce the cell surface level of the ion channel protein. This functional modification may be effected by any mechanism that causes antibody-induced internalization or endocytosis of ion channels leading to a reduction in the number of functional ion channel proteins on the cell surface. Or it may include but is not limited to increased cycling.

  The mechanism proposed above for functional modification of ion channels is an example, and is not limited in terms of the manner in which functionally modified antibodies can produce functional modification effects in ion channels.

By examining the differences in the sequence of the Na _v 1.7 extracellular domain versus the extracellular domains of other family members, it becomes possible to identify specific areas of interest, for example those sequences. Can be used for the production of antibodies. In Na _v 1.7, domain A amino acids 146 to 153, domain B amino acids 764 to 774, domain C amino acids 1213 to 1224 and 1216-1224, and domain D amino acids 1535 to 1545 are particularly different / difference regions. Thus, it may be particularly suitable for making antibodies.

  In one embodiment, the antibody or fragment binds in the E1 loop to the extracellular (or extracellular accessible) region of domain A of the ion channel.

  In one embodiment, the antibody or fragment binds in the El loop to the extracellular (or extracellular accessible) region of domain B in the ion channel.

  In one embodiment, the antibody or fragment binds in the E1 loop to the extracellular (or extracellular accessible) region of domain C in the ion channel.

  In one embodiment, the antibody or fragment binds in the E1 loop to the extracellular (or extracellular accessible) region of domain D in the ion channel.

  In one embodiment, the antibody or fragment according to the invention is converted to another entity that functionally modifies the ion channel in question, for example the E3 region of the ion channel, eg domain A, B, C, or D, such as domain A. It may be used in combination with an antibody or fragment that binds to a portion of the E3 region.

In one embodiment, an E1-binding anti-Na _v 1.7 antibody is provided.

An anti-Na _v 1.7 antibody is an antibody that specifically binds to Na _v 1.7. An E1-binding anti-Na _v 1.7 antibody is an antibody that specifically binds to Na _v 1.7 in the E1 region.

Specific binding means that an antibody is selective for an associated ion channel, such as Na _v 1.7, and distinguishes it from other ion channels and proteins, such as other ion channels in the same family. It refers to the fact that it can. Selective antibodies are those that can be used, for example, to affinity purify related ion channels such as Na _v 1.7 from other ion channels.

In one embodiment, the anti-Na _v 1.7 antibody is specific for mammalian Na _v 1.7, eg, human Na _v 1.7.

In one embodiment, E1-binding anti-ion channel antibodies, such as anti-Na _v 1.7 antibodies, cross-react with related human ion channels and corresponding rat ion channels.

  In one embodiment, the functionally modified antibody or fragment is a clonal population of antibodies, eg, a monoclonal population. It has been suggested in the literature that polyclonal antibodies are required to obtain functional modification of ion channels. Thus, it is particularly surprising that the present disclosure provides monoclonal antibodies that functionally modify the relevant ion channels.

  Monoclonal as used herein shall refer to an antibody or fragment derived from a single cell, eg, by using hybridoma technology.

  A clonal population shall refer to a population of antibodies or fragments having the same properties and characteristics, including the same amino acid sequence and specificity.

  In one embodiment, a clonal population of two or more, e.g., 3 or 4 anti-ion channel antibodies, is used in admixture, the mixture comprising at least one, e.g., 2, 3, or according to the invention Contains 4 antibodies.

  In one embodiment, an antibody according to the present disclosure is a whole antibody.

  In one embodiment, the antibody or fragment thereof is multivalent and / or bispecific. As used herein, multivalent refers to a plurality of binding sites (eg, at least two binding sites) in an antibody or fragment entity. A multivalent entity in the context of the present specification has at least two binding sites with the same specificity. A multivalent antibody having a binding site that binds to the same epitope is not considered bispecific unless the entity includes a third binding site that has a different specificity than the first and second binding sites. Shall. Thus, bispecificity as used in the present disclosure requires two or more binding sites in an antibody or fragment that are different, ie, bind to distinct target antigens.

  In entities having multiple binding sites, where each binding site binds to a different epitope on the same or different target antigen, the antibody or fragment entity is considered bispecific within the meaning of this disclosure. Shall.

  As used herein, a binding site shall refer to the area of an antibody or fragment that specifically binds a target antigen. Heavy chain and light chain variable domain pairings shall be considered as examples of binding sites herein.

  In one embodiment, the antibody fragment according to the present disclosure is monovalent. In other words, it has only one binding site.

  In one embodiment, the antibody is a fragment, eg, a single domain antibody, single chain Fv, Fab, Fab ', or a complete heavy and light chain pairing.

  The antibodies of the present disclosure or fragments thereof used in the present invention can be derived from any species, but are preferably derived from monoclonal antibodies, human antibodies, or humanized fragments. Antibody fragments for use in the present invention can be derived from any class (eg, IgG, IgE, IgM, IgD, or IgA) or subclass of immunoglobulin molecules, such as mouse, rat, shark, rabbit, pig, hamster, It may be obtained from any species including camel, llama, goat, or human.

  Residues in the antibody variable domain are usually numbered by a system devised by Kabat et al. This system is shown in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereinafter “Kabat et al. (Supra)”). This numbering system is used herein unless otherwise indicated.

  Kabat residue designations do not always correspond directly to the linear numbering of amino acid residues. The actual linear amino acid sequence is the exact Kabat corresponding to the shortening or insertion of the basic variable domain structure (whether it is a framework or complementarity determining region (CDR)) structural component. May contain fewer or additional amino acids in the numbering. The correct Kabat numbering of residues can be determined for a given antibody by alignment of residues of homology in the sequence of that antibody with the “standard” Kabat numbering sequence.

  The CDRs of the heavy chain variable domain are located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2), and residues 95-102 (CDR-H3) according to the Kabat numbering system. Kabat numbering shall be used herein.

  An alternative numbering system not used herein is Chothia (Chothia, C. and Lesk, AM J. Mol. Biol., 196, 901-917 (1987)), CDR-H1. Is looping from residue 26 to residue 32. The combination of Chothia and Kabat numbering for “CDR-H1” would include, for example, residues 26-35.

  The CDRs of the light chain variable domain are located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2), and residues 89-97 (CDR-L3) according to the Kabat numbering system.

CDRs for various rabbit antibodies are provided below:

  In one embodiment, the disclosure herein extends to an antibody comprising one, two, three, four, five, or six CDR sequences disclosed herein.

  In one embodiment, the present disclosure extends to an antibody comprising a single variable domain or a pair of variable domains derived from the sequence (s) herein.

  In one embodiment, the variable domain of the heavy chain comprises at least one of the CDRs having the sequence shown in the table listed above, eg, when the CDR is in its “native position”. The natural positions of CDRs such as H1, H2, H3, L1, L2, or L3 are shown in the table above, for example, the natural position for the CDR of SEQ ID NO: 4 is H1, The natural position for the CDR of 5 is H2, the natural position for the CDR of SEQ ID NO: 6 is H3, and so forth. Similar interpretations also apply to light chain sequences.

  In one example, an antibody of the invention comprises a heavy chain, wherein at least two of the heavy chain variable domains CDR-H1, CDR-H2, and CDR-H3 are selected from the sequences shown in the table above, For example, the CDRs are in their natural position and optionally in their natural pairing. Natural pairing as used herein shall refer to pairing of CDRs from the same antibody (ie, from the one table above). Examples of natural pairing for CA167_00983 are SEQ ID NOs: 1 and 2, 1 and 3, and 2 and 3.

In one embodiment, the antibody according to the invention comprises a heavy chain, the variable domain of which is shown below:
SEQ ID NO: 4 CDR-H1,
SEQ ID NO: 5 CDR-H2 and SEQ ID NO: 6 CDR-H3,
Or SEQ ID NO: 10 CDR-H1,
SEQ ID NO: 11 CDR-H2 and SEQ ID NO: 12 CDR-H3,
Or SEQ ID NO: 16 CDR-H1,
SEQ ID NO: 17 CDR-H2 and SEQ ID NO: 18 CDR-H3,
Or
SEQ ID NO: 22 CDR-H1,
SEQ ID NO: 23 CDR-H2 and SEQ ID NO: 24 CDR-H3,
Or SEQ ID NO: 28 CDR-H1,
SEQ ID NO: 29 CDR-H2 and SEQ ID NO: 30 CDR-H3,
Or SEQ ID NO: 34 CDR-H1,
SEQ ID NO: 35 CDR-H2 and SEQ ID NO: 36 CDR-H3,
Or SEQ ID NO: 40 CDR-H1,
SEQ ID NO: 41 CDR-H2 and SEQ ID NO: 42 CDR-H3,
Or SEQ ID NO: 46 CDR-H1,
SEQ ID NO: 47 CDR-H2 and SEQ ID NO: 48 CDR-H3,
Or SEQ ID NO: 52 CDR-H1,
SEQ ID NO: 53 CDR-H2 and SEQ ID NO: 54 CDR-H3,
Or SEQ ID NO: 58 CDR-H1,
SEQ ID NO: 59 CDR-H2 and SEQ ID NO: 60 CDR-H3,
Or a sequence having at least 60%, 70%, 80% identity or similarity, such as at least 90%, 95%, or 98%.

  In one embodiment, an antibody according to the invention comprises a heavy chain, wherein the variable domain of the heavy chain is a variable domain or variable domain as disclosed herein, eg, as listed herein. Contains components.

  As used herein, a variable domain component shall refer to CDRs and combinations thereof, particularly CDRs and combinations thereof as specifically disclosed herein.

  In another embodiment, an antibody of the invention comprises a heavy chain, and the variable domain of the heavy chain is at least 90%, 95%, or 98% identical to the heavy chain variable region disclosed herein, or Includes sequences having at least 60%, 70%, 80% identity or similarity, such as similarity.

“Identity” as used herein indicates that an amino acid residue is identical between the sequences at any particular position in the aligned sequences. “Similarity” as used herein indicates that at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. For example, leucine may be used in place of isoleucine or valine. Other amino acids that can often be substituted for each other include, but are not limited to:
-Phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains);
-Lysine, arginine, and histidine (amino acids with basic side chains);
-Aspartate and glutamate (amino acids with acidic side chains)
Asparagine and glutamine (amino acids with amide side chains); and cysteine and methionine (amino acids with sulfur-containing side chains).
The degree of identity and similarity can be easily calculated (Computational Molecular Biology, edited by Lesk, AM, Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Project, Genome Project. Hen, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M. and Griffin, H. G. Ed., Humana, 19th. Heinje, G., Ac ademic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., M Stockton Press, New York, 1991).

The present invention also provides an E1-binding anti-ion channel antibody or fragment, wherein the ion channel has a role / function in the regulation of pain in vivo, in particular, selectively inhibits the function of the ion channel. Antibodies directed against the ion channels described in the document, for example anti-Na _v 1.7 antibodies, wherein the antibody or fragment is the sequence shown herein for the light chain CDRs (see the tables and lists provided). A light chain comprising at least one CDR with a reference), for example when the CDR is in its “native position”.

  In one embodiment, an antibody of the invention comprises a light chain, wherein at least two of CDR-L1, CDR-L2, and CDR-L3 in the variable domain of the light chain are the sequences set forth herein for light chain CDRs. For example, the CDRs are in their natural position, optionally in their natural pairing. Natural pairing as used herein means that a CDR from one antibody is paired in the variable domain with a CDR from the same chain of the same antibody, particularly the same antibody (ie, from the one table above). It shall refer to the fact that it forms / co-located.

  Needless to say, all permutations are included to avoid misunderstandings.

In one example, an antibody of the invention comprises a light chain, the variable domain of which is the sequence shown below:
SEQ ID NO: 1 CDR-L1,
SEQ ID NO: 2 CDR-L2 and SEQ ID NO: 3 CDR-L3,
Or SEQ ID NO: 7 CDR-L1,
SEQ ID NO: 8 CDR-L2 and SEQ ID NO: 9 CDR-L3,
Or SEQ ID NO: 13 CDR-L1,
SEQ ID NO: 14 CDR-L2 and SEQ ID NO: 15 CDR-L3,
Or
SEQ ID NO: 19 CDR-L1,
SEQ ID NO: 20 CDR-L2 and SEQ ID NO: 21 CDR-L3,
Or SEQ ID NO: 25 CDR-L1,
SEQ ID NO: 26 CDR-L2 and SEQ ID NO: 27 CDR-L3,
Or SEQ ID NO: 31 CDR-L1,
SEQ ID NO: 32 CDR-L2 and SEQ ID NO: 33 CDR-L3,
Or SEQ ID NO: 37 CDR-L1,
SEQ ID NO: 38 CDR-L2 and SEQ ID NO: 39 CDR-L3,
Or SEQ ID NO: 43 CDR-L1,
SEQ ID NO: 44 CDR-L2 and SEQ ID NO: 45 CDR-L3,
Or SEQ ID NO: 49 CDR-L1,
SEQ ID NO: 50 CDR-L2 and SEQ ID NO: 51 CDR-L3,
Or SEQ ID NO: 55 CDR-L1,
SEQ ID NO: 56 CDR-L2 and SEQ ID NO: 57 CDR-L3,
It includes sequences having at least 60%, 70%, 80% identity or similarity, such as at least 90%, 95%, or 98%.

  In one embodiment, the invention includes a light chain, and the light chain variable domain includes a variable domain or variable domain component as disclosed herein, eg, from any heavy chain described. .

  In another embodiment, an antibody of the invention comprises a light chain and the variable domain of the light chain is at least 90%, 95%, or 98% identical to the heavy chain variable region disclosed herein, or Includes sequences having at least 60%, 70%, 80% identity or similarity, such as similarity.

  In one embodiment, a pair of variable domains is provided, eg, a heavy chain variable domain and a light chain variable domain. In one aspect, a cognate pair, a heavy chain variable domain and light chain variable domain pair is provided.

  The antibody molecule of the present invention comprises a complementary light chain or a complementary heavy chain, respectively.

  In one embodiment, the heavy and light chains are naturally paired, in other words, from the same antibody, eg, as shown in a single table herein.

  In one embodiment, the heavy and light chains have non-natural pairing.

  One antibody provided by the present invention is referred to herein as antibody 983 shown in FIG.

  In a further aspect, the present invention also provides a nucleotide sequence encoding an antibody or fragment thereof according to the present disclosure.

For example, a CDR grafted (or humanized) anti-ion channel antibody (according to the invention) directed to an ion channel as described herein, in particular, functionally modifying said ion channel Such anti-Na _v 1.7 antibodies are also provided by the present invention. In one embodiment, one or more of the CDRs in the CDR-grafted antibody molecule has been obtained from rabbit antibody 983. As used herein, the term “CDR grafted antibody molecule” refers to heavy and / or light chain heavy chain variable region frameworks and / or light chain variable region frames of acceptor antibodies (eg, human antibodies). One or more CDRs (optionally including one or more modified CDRs) from a donor antibody grafted to the work (eg, a rat or rabbit antibody as described herein) ). For a review, see Vaughan et al., Nature Biotechnology, 16, 535-539, 1998.

  When CDRs are grafted, any suitable acceptor variable region framework sequence may be used in view of the class / type of donor antibody from which the CDR is derived, including rat, rabbit, mouse, primate And human framework regions. Preferably, a CDR-grafted antibody of the invention has a variable domain comprising a human acceptor framework region and one or more of the CDRs from a donor antibody as referred to herein. Accordingly, CDR grafted antibodies are provided wherein the variable domain comprises a human acceptor framework region and a non-human, preferably rat, mouse, or rabbit donor CDR.

  Examples of human frameworks that can be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (Kabat et al., Supra). For example, KOL and NEWM can be used for heavy chains, REI can be used for light chains, and EU, LAY, and POM can be used for both heavy and light chains. Alternatively, human germline sequences may be used; these are http: // vbase. mrc-cpe. cam. ac. Available at uk /. In a further alternative, a database of affinity matured human V region sequences may be used as a framework.

In the CDR-grafted antibody of the present invention, the acceptor heavy chain and the light chain are not necessarily derived from the same antibody, and may include a complex chain having a framework region derived from a different chain, if necessary.
In the CDR-grafted antibody of the present invention, the framework region does not have to have exactly the same sequence as that of the acceptor antibody. For example, a normal residue may be changed to a residue that is more frequently present for that acceptor chain class or type. Alternatively, selected residues in the acceptor framework region may be altered so that they correspond to residues found at the same position in the donor antibody (Reichmann et al., 1998, Nature, 332, 323 324). Such changes should be kept to the minimum necessary to restore donor antibody affinity. A protocol for selecting residues in the acceptor framework region that may need to be changed is shown in WO 91/09967.

  A donor residue is a residue from a donor antibody, ie, an antibody from which the CDR was originally derived, which in one embodiment of the invention may be derived from a rat, mouse, or rabbit antibody, as needed. Accordingly, it can be incorporated into the final antibody or fragment.

  In one embodiment, the antibody (or fragment, such as a Fab or Fab 'fragment) is a monoclonal, fully human, humanized, or chimeric antibody fragment. In one embodiment, the antibody, Fab or Fab 'fragment is a fully human or humanized antibody.

Thus, an antibody for use in the present invention may comprise a complete antibody molecule having a full-length heavy and light chain, or a fragment thereof, Fab, modified Fab, Fab ′, F (ab ′) ₂ , Fv, single Domain antibodies (VH, VL, VHH, IgNAR V domains, etc.), scFv, bivalent, trivalent or tetravalent antibodies, bis-scFv, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and Any of the above epitope-binding fragments may be, but are not limited to (eg, Holliger and Hudson, 2005, Nature Biotech. 23 (9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews- Online 2 (3), 209-217). Methods for making and producing these antibody fragments are well known in the art (see, eg, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments used in the present invention include Fab fragments and Fab ′ fragments described in International Patent Application Publications WO2005 / 003169, WO2005 / 003170, and WO2005 / 003171. Multivalent antibodies may contain multiple specificities or may be monospecific (see, eg, WO92 / 22853 and WO05 / 113605).

  In one example, the antibodies used in the present invention may be derived from camelids such as camels or llamas. Camelids have a functional class of antibodies that lack light chains, referred to as heavy chain antibodies (Hamers et al., 1993, Nature, 363, 446-448; Muyldermans et al., 2001, Trends. Biochem. Sci. 26, 230. ~ 235). The antigen binding sites of these heavy chain antibodies are limited to only the three hypervariable loops (H1-H3) provided by the N-terminal variable domain (VHH). The initial crystal structure of VHH revealed that the H1 and H2 loops are not restricted to the known canonical structure classes defined for normal antibodies (Decanniere et al., 2000, J. Mol. Biol, 300, 83-91). The H3 loop of VHH is on average longer than those of normal antibodies (Nguyen et al., 2001, Adv. Immunol., 79, 261-296). The majority of dromedary heavy chain antibodies prefer binding to the active site of the enzyme from which they are produced (Lawereys et al., 1998, EMBO J, 17, 3512-3520). In some cases, the H3 loop protrudes from the remaining paratope and has been shown to insert into the active site of chicken egg white lysozyme (Desmyter et al., 1996, Nat. Struct. Biol. 3, 803-811, and De Genst). Et al., 2006, PNAS, 103, 12, 4586-4591, and WO97049805).

It has been suggested that these loops can be displayed in other scaffolds and CDR libraries can be made in those scaffolds (see, eg, WO03050531 and WO97049805).
In one example, the antibody used in the present invention may be derived from cartilaginous fish such as sharks. Cartilage fish (sharks, goose rays, rays, and chimeras) have an atypical immunoglobulin isotype known as IgNAR. IgNAR is a heavy chain homodimer that is not associated with a light chain. Each heavy chain has one variable domain and five constant domains. An IgNAR V domain (or V-NAR domain) has several non-canonical cysteines that allow classification into two closely related subtypes, I and II. The type II V region has an additional cysteine in CDR1 and CDR3, which has been proposed to form a domain-restricted disulfide bond similar to that observed in camelid VHH domains. In addition, CDR3 may have a more elongated conformation and protrude from the antibody framework, similar to camelid VHH. Indeed, like the VHH domain described above, certain IgNAR CDR3 residues have also been demonstrated to be capable of binding in the chicken egg white lysozyme active site (Stanfield et al., 2004, Science, 305, 1770-1773). .

  Examples of methods for generating VHH domains and IgNAR V domains are described, for example, in Lauwereys et al., 1998, EMBO J. et al. 1998, 17 (13), 3512-20; Liu et al., 2007, BMC Biotechnol. 7, 78; Saerens et al., 2004, J. MoI. Biol. Chem. 279 (5), 51965-72.

  The antibodies used in the present invention include any appropriate class, for example, IgA, IgD, IgE, IgG, or IgM, or whole antibodies of subclasses such as IgG1, IgG2, IgG3, or IgG4, and functionally active fragments thereof or Derivatives include, but are not limited to, monoclonal antibodies, clonal antibodies, humanized antibodies, fully human antibodies, or chimeric antibodies.

  In one embodiment, the constant region used for an antibody according to the present disclosure or a particular fragment thereof is a hybrid constant region or a mutant constant region. A hybrid constant region includes parts or domains from two or more distinct constant regions, eg, two or more distinct human constant regions.

  Examples of hybrid constant regions include those disclosed in US Patent Application Publication No. 2007/0041972, in which at least the CH1 and hinge regions are derived from one or more IgG2 antibodies, and the CH2 and CH3 regions Are derived from one or more IgG4 CH2 and CH3 regions. Eculizumab (Alexion Pharmaceuticals) is a humanized anti-human C5 monoclonal antibody for paroxysmal nocturnal hemoglobinuria containing a hybrid constant region. It has a hybrid chain of IgG2 derived CH1 and hinge and IgG4 derived CH2 and CH3 domains. It does not bind to FcγR and does not activate complement. It also has low immunogenicity (low titer of anti-eculizumab antibody detected in only 3 (3%) of 196 patients).

  WO 2008/090958 discloses certain hybrid constant regions comprising CH1 from IgG1, hinge, and CH2, and chains of IgG3 derived CH3 domains. The hybrid has higher CDC activity than that of IgG1 or IgG3 antibody, and has protein A binding activity equivalent to IgG1.

  Additional hybrid constant regions can be found in Tao et al. (SL Morrison group) J. Mol. Exp. Med 173 1025-1028, 1991. This paper includes multiple IgG domain exchanges from all classes, but the important hybrids are g1g4 and g4g1, each linked in the CH2 domain. In contrast to IgG1, IgG (1-1-1 / 4-4) cannot activate complement at all. However, IgG (4-4-4 / 1-1) showed significant activity compared to IgG4, but was slightly impaired compared to IgG1. An important difference appears to be the hinge, and many papers later demonstrate that the hinge regulates but does not mediate complement activation.

  Tao et al. (SL Morrison group) Exp. Med 178 661-667, 1993 discloses the structural features of human IgG that determine isotype-specific differences in complement activation. Ser331 (CH2) in IgG4 prevents C1q binding and complement activation. Mutagenesis of Ser331 to Pro in IgG4 and IgG (1-1-1 / 4-4) allows binding and activation, but at a lower level than that of IgG1. Interestingly, P331S in IgG1 allows binding but does not allow activation.

  Zucker et al., Canc Res 58 3905-3908 1998 uses chimeric human-mouse IgG antibodies with recombinant constant region exons to demonstrate that multiple domains contribute to in vivo half-life. In particular, this paper examines the half-life of IgG (1-1-1 / 4-4) hybrids and the like. In SCID mice, IgG (1-1-1 / 4-4) has a significantly longer half-life than IgG1, although significantly longer than IgG4. IgG (4-4-4 / 1-1) has the longest half-life.

  Examples of mutated constant regions include those used for Abatacept, which is a fusion of human CTLA-4 and IgG1 hinge-Fc. The hinge was changing from CPPC to SPPS. The latter is O-gly. Mutant constant regions do not mediate ADCC or CDC and have low immunogenicity (3% incidence).

The hinge is thought to have a possible role in complement activation. The functional hinge deduced from crystallographic studies ranges from 216 to 237 of IgG1,

of

, Consisting of each. In one embodiment, an antibody or fragment according to the present disclosure comprises a functional hinge.

  Mutations / modifications to the constant region can result, for example, in increased stability, for example, US Patent Application Publication No. 2004/0191265 discloses IgG1 hinge mutagenesis, IgG stability was increased by introducing one or more amino acid modifications in the hinge region at positions 233-239 or 249 of human IgG1. This reduced degradation when heated to 55 ° C. for 1 week.

  Alternatively, the upstream hinge portion (human IgG1 residues 226-243 and corresponding residues in immunoglobulins from other IgG subtypes and / or other species) and / or adjacent CH1 and / or CH2 sequences (human IgG1 residues) Point-to-point to labile amino acids (eg histidine or threonine) or reactive amino acids (eg lysine or glutamic acid) in group 249, and corresponding residues in immunoglobulins from other IgG subtypes and / or other species Modifications may be brought about by causing mutations.

  Monoclonal antibodies include hybridoma technology (Kohler & Milstein, Nature, 1975, 256, 495-497), trioma technology, human B cell hybridoma technology (Kozbor et al., Immunology Today, 1983, 4, 72), and EBV hybridoma technology (Cole et al., It may be prepared by any method known in the art, such as “Monoclonal Antibodies and Cancer Therapy,” pages 77-96, Alan R. Liss, Inc., 1985).

  The antibodies used in the present invention are also described in, for example, Et al., Proc. Natl. Acad. Sci. USA, 1996, 93 (15), 7843-7848, WO92 / 02551, WO2004 / 051268, and WO2004 / 106377, resulting from a single lymphocyte selected for production of a particular antibody It may also be made using a single lymphocyte antibody method by cloning and expressing the immunoglobulin variable region cDNA.

  A humanized antibody is an antibody molecule derived from a non-human species having one or more complementarity determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (eg, a US patent). No. 5,585,089).

  Antibodies used in the present invention can also be generated using various phage display methods known in the art, including those described by Brinkman et al. Immunol. Methods, 1995, 182, 41-50; Ames et al., J. Biol. Immunol. Methods, 1995, 184, 177-186; Kettleborough et al., Eur. J. et al. Immunol. 1994, 24, 952-958; Persic et al., Gene, 1997 187, 9-18; and Burton et al., Advances in Immunology, 1994, 57, 191-280; WO 90/02809; WO 91/10737; WO 92/01047; WO93 / 11236; WO95 / 15982; and WO95 / 20401; and US Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717. 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; No. 5,780,225; No. 5,658,727; No. 5,733,743 And those disclosed by Patent 5,969,108. Transgenic mice or other organisms including other mammals may also be used to make humanized antibodies.

  Fully human antibodies have both heavy and light chain variable regions and constant regions (if present) that are all from human origin, or not necessarily from the same antibody, but in substantial sequence with human origin. Are identical. Examples of fully human antibodies include, for example, antibodies produced by the phage display method described above, and, for example, EP0546073B1, US Pat. No. 5,545,806, US Pat. No. 5,569,825, US Pat. 625,126, U.S. Pat.No. 5,633,425, U.S. Pat.No. 5,661,016, U.S. Pat.No. 5,770,429, EP 0438474 B1, and EP 0463151 B1, Mention may be made of antibodies produced by mice in which mouse immunoglobulin variable region genes and / or constant region genes have been replaced by their human counterparts.

  The antibodies or fragments used in the present invention can be obtained from any whole antibody, in particular whole monoclonal antibody, using any suitable enzymatic cleavage and / or digestion technique, for example by treatment with pepsin.

  Alternatively, or in addition, antibody starting materials may be prepared by using recombinant DNA techniques involving the manipulation and reexpression of DNA encoding antibody variable and / or constant regions. Standard molecular biology techniques may be used to modify, add, or remove amino acids or domains as appropriate. Any changes to the variable region or constant region are still included in the terms “variable” region and “constant” region as used herein.

  As discussed, antibody fragments “starting material” may be obtained from any species including, for example, mouse, rat, rabbit, hamster, shark, camel, llama, goat, or human. Portions of antibody fragments may be obtained from more than one species, for example, antibody fragments may be chimeric. In one example, the constant region is from one species and the variable region is from another species. The antibody fragment starting material may also be modified. In another example, the variable region of an antibody fragment may be generated using recombinant DNA manipulation techniques. Such engineered versions include those generated from natural antibody variable regions by insertions, deletions or changes in or in the amino acid sequences of the natural antibodies. Specific examples of this type are engineered comprising at least one CDR, and optionally one or more framework amino acids from one antibody, and the remainder of the variable region domain from a second antibody. Variable region domains. Methods for making and producing these antibody fragments are well known in the art (eg, Boss et al., US Pat. No. 4,816,397; Cabilly et al., US Pat. No. 6,331,415). Shrader et al., WO 92/02551; Ward et al., 1989, Nature, 341, 544; Orlandi et al., 1989, Proc. Natl. Acad. Sci. USA, 86, 3833; Riechmann et al., 1988, Nature, 322, 323; Bird et al., 1988, Science, 242, 423; Queen et al., US Pat. No. 5,585,089; Adair, WO 91/09967; Mountain and Adair, 1992, Biotechnol. Genet. Eng. Rev. 10, 1-142; Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

  Thus, in one embodiment, the constant region domain can be a human IgA, IgD, IgE, IgG, or IgM domain. In particular, human IgG constant region domains, particularly IgG1 and IgG3 isotype human IgG constant region domains may be used where the antibody molecule is intended for therapeutic use and antibody effector function is required. Alternatively, IgG2 and IgG4 isotypes may be used when the antibody molecule is intended for therapeutic purposes and antibody effector function is not required. It will be appreciated that sequence variants of these constant region domains may also be used. For example, an IgG4 molecule in which the serine at position 241 is changed to proline as described in Angal et al., Molecular Immunology, 1993, 30 (1), 105-108 may be used. It will also be appreciated by those skilled in the art that antibodies can undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell system and culture conditions used to express the antibody. Such modifications can include variations in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization, and asparagine deamidation. A common modification is the loss of a carboxy terminal basic residue (such as lysine or arginine) due to the action of a carboxypeptidase (as described in Harris, RJ. Journal of Chromatography 705: 129-134, 1995).

  In one embodiment, the light chain of the antibody or fragment comprises either a kappa or lambda CL domain.

  The term “antibody” or fragment as used herein can also include a binding agent comprising one or more CDRs incorporated into a biocompatible framework structure. In one example, the biocompatible framework structure can display a conformationally stable one or more amino acid sequences (eg, CDRs, variable regions, etc.) that bind to antigens in localized surface regions. Polypeptides or portions thereof that are sufficient to form a structural support, or framework, or scaffold. Such a structure can be a natural polypeptide or polypeptide “fold” (structural motif), or one or more of amino acid additions, deletions, or substitutions relative to the natural polypeptide or fold. May have modifications. These scaffolds can be derived from polypeptides of any species (or more than one species) such as humans, other mammals, other vertebrates, invertebrates, plants, bacteria, or viruses.

  Typically, the biocompatible framework structure is based on a protein scaffold or backbone other than the immunoglobulin domain. For example, those based on fibronectin, ankyrin, lipocalin, neocarzinostatin, cytochrome b, CP1 zinc finger, PST1, coiled coil, LACI-D1, Z domain, and tendramistat may be used. (See, eg, Nygren and Uhlen, 1997, Current Opinion in Structural Biology, 7, 463-469).

  In one embodiment, the total charge of the antibody or fragment is neutral.

  In one embodiment, the total charge of the antibody or fragment is negative.

  In one embodiment, the total charge of the antibody or fragment is positive.

  In one embodiment, the CDRs may be grafted or engineered into alternative types of scaffolds, such as fibronectin or actin-binding repeats.

  In one embodiment, the antibody or fragment comprises an effector molecule such as a toxin including a biotoxin such as a polymer, toxicant or chemical inhibitor conjugated thereto.

Biotoxins and toxicants are natural regulators such as blockers of cell signaling. When conjugated to an antibody or fragment according to the invention, they can be used to enhance the functional effect on the ion channel while maintaining the selectivity provided by the antibody. For example, the tarantula spider venom peptide ProTxII has been shown to selectively inhibit Na _v 1.7, see, eg, Mol Pharmacol 74: 1476-1484, 2008. In one embodiment, the toxin is a botulinum toxin, such as botulinum toxin A. Other toxins include tetrodotoxin and saxitoxin.

  In one embodiment, the antibody or fragment is conjugated to an aptamer. Aptamers are single-stranded oligonucleotide sequences that can take and bind secondary and tertiary structures to modulate protein activity. Conjugation to such structured antibodies results in target-specific modulation of ion channels (direct effect).

In one embodiment, the antibody or fragment is conjugated to a chemical inhibitor, such as a synthetic chemical inhibitor of the ion channel. Examples of chemical inhibitors include the following compounds of formula (I):

Where n is 0 or 1;
m is 0 or 1;
p is 0 or 1;
Y is CH, N, or NO;
X is oxygen or sulfur;
W is oxygen, H ₂ or F ₂ ;
A is N or C (R ² );
G is N or C (R ³ );
D is N or C (R ⁴ );
Provided that not more than one of A, G and D is nitrogen, but at least one of Y, A, G and D is nitrogen or NO;
R1 is hydrogen or a _C 1 -C ₄ alkyl;
R ² , R ³ , and R ⁴ are independently hydrogen, halogen, C ₁ -C ₄ alkyl, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl, aryl, heteroaryl, OH, OC ₁ -C _{4 ^{alkyl, CO 2 R 1, -CN,}} -NO 2, -NR 5 R 6, -CF 3, an -OSO ₂ CF _3, or ^{R 2} and ^{R 3,} or ^{R 3} and ^{R 4} are each Together, another 6 containing A and G, or G and D, each containing between 0 and 2 nitrogen atoms and substituted with 1 to 2 of the following substituents: membered to form an aromatic ring or a heteroaromatic ring: independent hydrogen, _halogen, C 1 -C _{4 alkyl,} C 2 -C _{4 alkenyl,} C 2 -C ₄ alkynyl, aryl, heteroaryl, OH, OC ₁ -C _{4 alkyl,} CO ² R _1, -CN ^{_{-NO 2, -NR 5 R 6,}} -CF 3, -OSO 2 CF 3;
R ⁵ and R ⁶ are independently hydrogen, C ₁ -C ₄ alkyl, C (O) R ⁷ , C (O) NHR ⁸ , C (O) OR ⁹ , SO ₂ R ¹⁰ , or Taken together can be (CH ₂ ) _j Q (CH ₂ ) _k where Q is O, S, NR ¹¹ , or a bond; j is 2-7; k is 0-2 R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , and R ¹¹ are independently C ₁ -C ₄ alkyl, aryl, or heteroaryl, or an enantiomer thereof, and their pharmaceuticals Is a strong ligand for the nicotinic acetylcholine receptor).
See EP0996622.

  Bioorg Med Chem (2003) 11: 2099-113. RA Hill, S Rudra, B Peng, DS Roane, JK Bonds, Yg, A Adloo, T Lu disclose certain hydroxyl-substituted sulfonylureas as inhibitors.

  4,4-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) has been used as an anion transport inhibitor.

The following are also chemical inhibitors of Na _v 1.7:

The compound named 2 in the table above, N-[(R) -1- (R) -7-chloro-1-isopropyl-2-oxo-2,3,4,5-tetrahydro-1H-benzo [b ] Azepin-3-ylcarbamoyl) -2- (2-fluorophenyl) -ethyl] -4-fluoro-2-trifluoromethyl-benzamide was prepared according to McGowan et al., Annesthesia and Analgesia 109, 3, September 2009. (Peripheral acting Na _v 1.7 sodium channel blocker reverses hyperalgesia and allodynia in a rat model of inflammatory and neuropathic pain (A Peripherally Acting Na _v 1.7 Sodium Channel) Blocker Reverses Hyperalgesia and Allody ia on Rat Models of Inflammatory and Neuropathic Pain) "a is) are discussed in the article by.

Turnawa et al. (2007) ("Blockers of voltage-gate sodium for fortient sequen ss ent essen ss ent ss ent sens ss ent ss ent ss ent sens ss ent ss ent sens ss ent sen ss ent ss ent sen ss s s s s)? 2:57) reviewed recent medicinal chemistry of sodium channel blockers. Some older drugs such as lidocaine, mexiletine, carbamazepine, phenytoin, lamotrigine, and newly developed drugs such as lacosamide, oxycarbazepine, clobenetine, ralfinamide are effective in treating various types of chronic pain in animal models Sodium channel blockers that have been proven to be effective, some of which are also used clinically. Some other examples of chemical regulators of voltage-gated sodium channels are listed below:

Figure (15a). Compound with a combination of aromatic and heteroaromatic rings patented by Merck

Figure (16). Biaryl carboxamide compound patented by Atkinson

Figure (17). Compound having a combination of aromatic and heteroaromatic rings patented by Ehring

Figure (18b). Compound with combined aromatic and heteroaromatic rings patented by Vertex

Figure (19). Other references describing compound voltage-gated sodium channel chemical modulators with combined aromatic and heteroaromatic rings, patented by Ionix: Anger et al. (2001) J. MoI. Med. Chem. 44 (2): 115; Hoyt et al. (2007), Bioorg. Med. Chem. Lett. 17: 6172; Yang et al. (2004), J. MoI. Med. Chem. 47: 1547; Benes et al. (1999) J. MoI. Med. Chem. 42: 2582
US Pat. No. 7,456,187 discloses certain potassium channel inhibitors of the following formula:

In which R ₁ is aryl, heteroaryl, cycloalkyl, or alkyl;
R ₂ is H, alkyl, nitro, —CO ₂ R ₇ , CONR ₄ R ₅ , or halo;
R ₃ is H, NR ₄ R ₅ , NC (O) R ₈ , halo, trifluoromethyl, alkyl, nitrile, or alkoxy;
R ₄ and R ₅ may be the same or different and may be H, alkyl, aryl, heteroaryl, or cycloalkyl; or R ₄ and R _{5 taken} together are saturated, unsaturated, Or may form a partially saturated 4-7 membered ring provided that said ring optionally comprises one or more additional heteroatoms selected from N, O, or S;
X is O, S, or NR ₆ ;
R ₆ is H or alkyl;
R ₇ is hydrogen, methyl, or ethyl;
R ₈ is methyl or ethyl;
L is (CH ₂ ) _n , where n is 1, 2, or 3; and Y is aryl, heterocyclic, alkyl, alkenyl, or cycloalkyl, or a pharmaceutically acceptable salt thereof Salt.

Bioorganic and Medical Chemistry Letters, Volume 19, 11th Edition, June 1, 2009, pages 3063-3066, disclose specific inhibitors of the following formula:

More specifically:

  In one embodiment, the entity conjugated to the antibody or fragment changes the overall charge of the molecule.

  The term effector molecule as used herein refers to, for example, antineoplastic agents, drugs, toxins, bioactive proteins such as enzymes, other antibodies or antibody fragments, synthetic or natural polymers, nucleic acids and fragments thereof. Reporter groups such as, for example, DNA, RNA and fragments thereof, radionuclides, in particular radioiodides, radioisotopes, chelating metals, nanoparticles, and compounds that can be detected by fluorescent compounds or NMR or ESR spectroscopy Including.

  Examples of effector molecules can include cytotoxic agents, or cytotoxic agents including any agent that is detrimental to cells (eg, kills cells). Examples include combretastatin, dolastatin, epothilone, staurosporine, maytansinoid, spongistatin, lysoxine, halichondrin, loridine, hemiasterin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide , Tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, and puromycin, As well as their analogs or homologues.

  Effector molecules also include antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (eg, mechloretamine, thioepachlorambucil, melphalan, carmustine) (BSNU), and lomustine (CCNU), cyclotosfamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (eg, daunorubicin (formerly daunomycin) ), And doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mitramycin, anthramycin (AMC), calicheamic Emissions, or duocarmycins), and anti-mitotic agents (e.g., but may include vincristine and vinblastine), but are not limited to.

Other effector molecules include chelating radionuclides such as ¹¹¹ In and ⁹⁰ Y, Lu ¹⁷⁷ , bismuth ²¹³ , californium ²⁵² , iridium ¹⁹² , and tungsten ¹⁸⁸ / rhenium ¹⁸⁸ ; or, without limitation, alkylphosphocholine, topoisomerase I inhibition Mention may be made of drugs such as agents, taxoids, and suramin.

  Other effector molecules include proteins, peptides, and enzymes. Enzymes of interest include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, transferases. Proteins, polypeptides, and peptides of interest include, but are not limited to, toxins such as immunoglobulins, abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin.

Other effector molecules can include, for example, detectable substances useful for diagnosis. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radionuclides, positron emitting metals (for positron emission tomography), and non-radioactive paramagnetic metal ions. . See generally US Pat. No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostics. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin, and biotin; suitable fluorescent materials include Umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin , And aequorin; and suitable radionuclides include ¹²⁵ I, ¹³¹ I, ¹¹¹ In, and ⁹⁹ Tc.

  In another example, the effector molecule increases the half-life of the antibody in vivo and / or decreases the immunogenicity of the antibody and / or delivers the antibody to the immune system across the epithelial barrier. Can be enhanced. Examples of suitable effector molecules of this type include polymers, albumin, albumin binding proteins, or albumin binding compounds such as those described in WO05 / 117984.

  Where the effector molecule is a polymer, it may generally be a synthetic polymer or a natural polymer, for example, an optionally substituted linear or branched polyalkylene, polyalkenylene, or poly It may be a polymer of oxyalkylene or a branched or unbranched polysaccharide, for example a homopolysaccharide or a heteropolysaccharide.

  Specific optional substituents that may be present on the above synthetic polymers include one or more hydroxy groups, methyl groups, or methoxy groups.

  Specific examples of synthetic polymers include optionally substituted linear or branched poly (propylene glycol) poly (vinyl alcohol) or derivatives thereof, particularly optionally substituted poly (ethylene glycol) Ethylene glycol) or a derivative thereof.

  Suitable polymers include poly (ethylene glycol), or in particular polyalkylene polymers such as methoxy poly (ethylene glycol) or derivatives thereof, particularly those having a molecular weight in the range of about 15000 Da to about 40000 Da.

  The size of the polymer can vary as needed, but is generally in the average molecular weight range from 5000 Da to 40000 Da, such as from 500 Da to 50000 Da, for example from 20000 Da to 40000 Da. The polymer size may be selected based on, among other things, the intended use of the product, eg, the ability to localize to a particular tissue, such as a tumor, or to increase the circulation half-life (for review, see Chapman, 2002). , Advanced Drug Delivery Reviews, 54, 531-545). Thus, for example, if the product is intended to exit the circulation and penetrate the tissue, eg, for use in the treatment of tumors, use a low molecular weight polymer, eg, a polymer having a molecular weight of about 5000 Da. Can be advantageous. For applications where the product remains in the circulation, it may be advantageous to use higher molecular weight polymers, for example polymers having a molecular weight in the range of 20000 Da to 40000 Da.

  In one embodiment, the PEG used is a releasable PEG supplied by, for example, Enzon Pharmaceuticals.

  In one example, the antibody used in the present invention is attached to a poly (ethylene glycol) (PEG) moiety. In one particular example, the antibody is an antibody fragment and the PEG molecule is any available amino acid side chain or terminal amino acid functional group located on the antibody fragment, such as any free amino group, imino group, thiol group. , A hydroxyl group, or a carboxyl group. Such amino acids may be naturally present within the antibody fragment or may be engineered into fragments using recombinant DNA methods (eg, US Pat. No. 5,219,996; 5,667,425; see WO98 / 25971). In one example, an antibody molecule of the invention is a modified Fab fragment, where the modification is the addition of one or more amino acids to the C-terminus of its heavy chain that allows attachment of an effector molecule. Suitably, the additional amino acid forms a modified hinge region comprising one or more cysteine residues to which the effector molecule can be attached. Multiple sites can be used to attach two or more PEG molecules.

  In one embodiment, the Fab or Fab 'is PEGylated with one or two PEG molecules.

  In one embodiment, the PEG molecule is linked to cysteine 171 in the light chain, see for example WO 2008/038024, incorporated herein by reference.

  In one embodiment, the Fab or Fab 'is PEGylated through a surface accessible cysteine.

  Suitably, the PEG molecule is covalently linked through a thiol group of at least one cysteine residue located in the fusion protein. Each polymer molecule attached to the fusion protein can be covalently bound to a sulfur atom of a cysteine residue located within the protein. The covalent bond is generally a disulfide bond or, in particular, a sulfur-carbon bond. If a thiol group is used as the point of attachment, a suitably activated PEG molecule, for example, a thiol selective derivative such as maleimide and a cysteine derivative can be used. Activated PEG molecules can be used as starting materials in the preparation of polymer fusion proteins containing such molecules. The activated PEG molecule can be any polymer containing a thiol reactive group such as an α-halocarboxylic acid or ester, eg, iodoacetamide, imide, eg, maleimide, vinyl sulfone, or disulfide. Such starting materials are commercially available (eg, from Nektar (formerly Shearwater Polymers Inc.), Huntsville, AL, USA) or from commercially available starting materials using conventional chemical procedures. Can be prepared. Specific PEG molecules include 20K methoxy-PEG-amine (available from Nektar (formerly Shearwater); Rapp Polymere; and SunBio), and M-PEG-SPA (available from Nektar (formerly Shearwater)). It is done.

  Effector molecules such as PEG molecules can be linked to aldehyde sugars, or more commonly, any available amino acid side chain or terminal amino acid functional group located on the antibody fragment, such as any free amino group, imino group, thiol It can be attached to the antibody by several different methods, including through groups, hydroxyl groups, or carboxyl groups. The site of attachment of the effector molecule can be random or site specific.

  Random attachment is often achieved through an amino acid such as lysine, which results in an effector molecule such as a PEG molecule attached to several sites throughout the antibody fragment depending on the position of lysine. . This has been successful in some cases, but the exact position and number of effector molecules, such as PEG molecules attached, cannot be controlled, which is, for example, too little attached. Too much activity can result in loss of activity and / or affinity, for example if they interfere with antigen binding sites (Chapman 2002 Advanced Drug Delivery Reviews, 54, 531-545). As a result, controlled site-specific attachment of effector molecules such as PEG molecules is usually the preferred method.

  Site specific attachment of effector molecules such as PEG molecules is most commonly achieved by attachment to cysteine residues, since such residues are relatively rare in antibody fragments. Antibody hinges are a commonly used region for site-specific attachment because they contain cysteine residues and are remote from other regions of the fusion protein that are likely to be involved in antigen binding. Suitable hinges may exist naturally within the fragment or may be made using recombinant DNA technology (eg, US Pat. No. 5,677,425; WO 98/25971; Leon et al., 2001). Cytokine, 16, 106-119; Chapman et al., 1999 Nature Biotechnology, 17, 780-783). Alternatively, or in addition, site-specific cysteines may also be engineered into antibody fragments to generate, for example, surface exposed cysteine (s) for effector molecule attachment (US Pat. No. 5,219, 996).

  Thus, in one embodiment, the PEG molecule is attached to a surface exposed cysteine.

  Surface-exposed cysteine (free cysteine) as used herein is accessible for conjugating an effector molecule such as a PEG molecule to the protein when the protein assumes a “natural” folded conformation. We shall refer to a certain cysteine. An example of how to manipulate this type of free cysteine is also provided in US Pat. No. 7,521,541.

  Specific natural polymers include lactose, amylose, dextran, glycogen, or derivatives thereof.

  As used herein, a “derivative” is intended to include a reactive derivative, eg, a thiol-selective reactive group such as maleimide. The reactive group can be linked to the polymer directly or through a linker segment. It will be appreciated that the residues of such groups in some cases form part of the product as a linking group between the fusion protein and the polymer.

  The present invention also provides an isolated DNA encoding an antibody described herein, or a fragment thereof of heavy or light chain.

  In a further aspect, a vector comprising the DNA is provided.

  The general methods by which vectors can be constructed, transfection methods, and culture methods are well known to those skilled in the art. In this connection, “Current Protocols in Molecular Biology”, 1999, F.M. M.M. Reference is made to Maniatis Manual produced by Ausubel (eds.), Wiley Interscience, New York and Cold Spring Harbor Publishing.

  In a further aspect, a host cell comprising the vector and / or DNA is provided.

  Any suitable host cell / vector system can be used for expression of the DNA sequence encoding the fusion protein molecule of the invention. Bacterial systems, such as E. coli systems, and other microbial systems may be used, or eukaryotic, eg, mammalian host cell expression systems may also be used. Suitable mammalian host cells include CHO cells, myeloma cells, or hybridoma cells.

  The present invention also provides a method for producing an antibody or fragment thereof according to the present invention, wherein a host cell comprising the vector (and / or DNA) of the present invention is treated with a protein from DNA encoding the antibody or fragment thereof. A method is provided comprising culturing under conditions suitable to effect expression and isolating it.

  For the production of products containing both heavy and light chains, the cell line is divided into two vectors, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. It may be transfected. Alternatively, a single vector may be used, the vector comprising sequences encoding the light and heavy chain polypeptides.

In one aspect, there is provided a method of making an antibody using a peptide whose sequence is derived from an ion channel involved in the regulation of pain, eg, the E1 extracellular region of Na _v 1.7. Such peptides are used both in immunization protocols to produce polyclonal antibodies and / or screening or panning protocols to select specific anti-ion channel antibodies, eg, anti-Na _v 1.7 antibodies. It is done. One embodiment uses peptides corresponding to separate sequences of the aforementioned extracellular regions that have the greatest differences from other ion channels and their isoforms.

Based on the amino acid sequence numbering of the Na _v 1.7 sequence deposited in the Swiss Prot database as the human protein SCN9A (accession number Q15858), these sequences are as follows:
Domain A amino acids 146-153,
Domain B amino acids 764-774,
Domain C amino acids 1216-1224 and domain D amino acids 1535-1545 are regions with particular differences / differences and are therefore particularly suitable for generating antibodies.

  The selected peptide may be a normal linear peptide or a cyclic peptide and may comprise at least 4 consecutive amino acids from the above sequence. In either case, the peptide is designed to contain a single functional group that is used for subsequent conjugation of the carrier protein or reporter group to the polymeric carrier. The functional group may be a side chain thiol of a cysteine residue, a side chain carboxyl of aspartic acid or glutamic acid, or a primary amine of an N-terminal amino group or a lysine side chain residue.

It said amino acid residue having a functional group, may correspond to the naturally occurring Na v _1.7 sequences, may be an addition to the natural Na v _1.7 sequence. The position of this residue in the Na _v 1.7 peptide sequence may be at either end of the sequence or at any internal position. In one embodiment, the peptide is, for example, a linear peptide comprising any of the following sequences, and domain A, B, C, or D of Na _v 1.7 from which the peptide is derived is shown in parentheses: Has been. The cysteine underlined in the peptide is a non-natural cysteine residue in the ion channel. Cysteine residues in the following peptides can be used to attach carrier proteins.

  These sequences may be capped at either the N-terminus or C-terminus, for example, with an Nα acetyl group or an amide group, respectively.

Other Nav1.7 sequences include the following:

In one embodiment of the invention, the Na _v 1.7 peptide used to make the anti-E1 ion channel antibody has an amino acid sequence selected from the group consisting of SEQ ID NOs: 107-122. In a further embodiment, the Na _v 1.7 peptide used to make the anti-E1 ion channel antibody is selected from the group consisting of SEQ ID NOs: 107, 108, 110-112, 114-116, and 119-127. It has an amino acid sequence.

  Immunogens from the E1 loop of other ion channels can be prepared similarly, so that, for example, each immunogen is, for example, at least 4 consecutive amino acid residues, shown in bold below , And additional residues at either the N-terminus and / or C-terminus in the sequence order as shown. Again, the selected peptide may be capped with a functional group such as N-acetyl at the N-terminus and / or with a functional group such as amide at the C-terminus. The peptide may be synthesized as a linear peptide or may be synthesized as a cyclic peptide. In either case, the peptide is designed to contain a single unique functional group for covalent attachment to a polymeric carrier such as a heterologous protein.

  If the peptide does not contain aspartate, glutamate, or lysine residues and the N-terminus is capped, the unique functional group can be derivatized directly for attachment to the carrier by amide chemistry. It can be a C-terminal carboxylic acid.

  If the peptide does not contain a lysine residue, the unique functional group can be an N-terminal amino group that can be derivatized to introduce additional reactive groups such as maleimide.

  If the peptide contains a single cysteine residue, its side chain thiol can be a unique functional group that can be attached to the carrier by maleimide chemistry.

  Intrinsic functional groups may be incorporated by additional residues at either terminus (either natural or non-natural amino acids), eg, cysteine, to allow specific binding.

Examples of sequences derived from specific ion channels are listed below, and the domain A, B, C, or D from which the peptides are derived is represented before that sequence for each peptide:

Thus, in one embodiment, the Na _v 1.3 peptide used to make the anti-E1 ion channel antibody has an amino acid sequence selected from the group consisting of SEQ ID NOs: 131-134.

In one embodiment of the invention, the Na _v 1.6 peptide used to make the anti-E1 ion channel antibody has an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-138.

In one embodiment of the invention, the Na _v 1.8 peptide used to make the anti-E1 ion channel antibody has an amino acid sequence selected from the group consisting of SEQ ID NOs: 139-145.

In one embodiment of the invention, the Na _v 1.9 peptide used to make the anti-E1 ion channel antibody has an amino acid sequence selected from the group consisting of SEQ ID NOs: 146-149.

  In one embodiment of the invention, the HCN1 or HCN2 peptide used to make the anti-E1 ion channel antibody has the amino acid sequence of SEQ ID NO: 150 or 151.

  In one aspect, a method for making an antibody using a cyclic peptide is provided.

  A cyclic peptide as used herein is a sequence of amino acids linked by a bond such as a disulfide bond, thereby forming a loop or circle having no discernable starting and / or ending point. It is a peptide. Cyclic peptides can be formed from the corresponding linear peptide by various means such as, but not limited to: ligation of the C-terminal carboxyl group to the N-terminal α-amino group to form a peptide bond; or The side chain carboxyl group (of the aspartic acid or glutamic acid residue) may be ligated to the side chain amino group or N-terminal α-amino group of lysine, or the C-terminal carboxyl group may be ligated to the side chain amino group of lysine. Formation of disulfide bonds between side chain thiols of two cysteine residues separated from each other by at least three residues in a linear sequence. It may be desirable to form “ring completion bonds” in overlapping areas in a linear array. As used herein, a region of overlap shall refer to where there are two or more amino acid repeats present in the sequence. Thus, an overlapping sequence as used herein is where there is some commonality in the sequence, for example at least 2 amino acids, such as 3 or 4 amino acids, in two separate positions. It is assumed that they are located in the same order in the array in FIG. These overlapping regions can be aligned and ligated such that the amino acid at one position replaces the corresponding amino acid at the second position to form a cyclized peptide.

  Thus, in one embodiment, the peptide is cyclized by forming an amide bond. In one embodiment, the peptide is cyclized by forming a disulfide bond.

  In one embodiment, the sequences are ligated with overlapping regions in a linear array.

  Cyclic peptides can be synthesized using any suitable method known in the art. In one embodiment, the cyclic peptide uses protecting groups to prevent amino acid side chain reactions (Barlos, K .; Gatos, D .; Kutsogianni, S .; Papaphotiou, G .; Poulos, C .; Tsegenidis, T Int. J. Pept. Protein Res. 1991, 38, 6 pp. 562-568) followed by cyclization and removal of the protecting group (Kessler H et al., 1989, Computer Aided Drug Design, 461-). 484; Dekker M et al., 1990, J. Peptide Research, 35, 287-300; Gurrath M. et al., 1992, Eur. J. Biochem., 210, 911-921; N. et al., 1981, Biopolymers, 20, 1785-1791; Brady SF et al., 1983, Peptides, Structure and Function, Proceedings of the American Peptide H. V, H. 127-130 pages, Pierce Chemical Company, Rockford, Illinois; He JX et al., 1994, Lett. Peptide Sci., 1, 25-30).

  Surprisingly, the functionally modified antibodies are, for example, only 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 It can be made with very short cyclic peptide sequences containing 18, 18 or 19 amino acids. This is obtained by the high stiffness provided by cyclizing the peptide.

In one embodiment, a cyclized peptide comprising a fragment of at least 4 consecutive amino acids from Na _v 1.7 is selected from, for example, domain A, B, C, or D from which the peptide is derived. And the extracellular loop E1, E2, or E3 is shown in parentheses:

In one embodiment, a cyclic peptide comprising at least 4 consecutive amino acid fragments from Na _v 1.7 and, for example, one additional cysteine residue for attachment to a carrier is selected from: The domain A, B, C or D from which the peptide is derived and the extracellular loop E1, E2 or E3 are represented in parentheses:

Thus, in one embodiment of the invention, the Na _v 1.7 cyclic peptide used to make anti-E1 ion channel antibodies has an amino acid sequence selected from the group consisting of SEQ ID NOs: 123-130.

Each peptide requires a covalent conjugation to a carrier protein in order to prepare an immunogen for the purpose of producing an anti-ion channel antibody in the host animal. Carrier proteins are selected based on their “foreign” nature to the host species; thus, for rabbit or rodent immunization, examples of suitable carrier proteins include keyhole limpet hemocyanin (KLH), ovo Albumin (OVA) and bovine serum albumin (BSA). Each of the peptides, whether linear or cyclic, can be conjugated through a cysteine thiol to each one of the proteins, with the latter lysine side chain amino group being covalently linked by a maleimide functional group. Each of these results in the following:
KLH-maleimide, ovalbumin-maleimide, or BSA-maleimide

The present disclosure clearly contemplates each of the peptides described herein separately conjugated to each of the carriers listed above, i.e. 45 different molecules for immunizing a host. Specifically provided, for example,

Or

It is. Thus, any of the peptides having an amino acid sequence selected from SEQ ID NOs: 107-151 can be conjugated with each of the carrier proteins listed above.

  As described above, the carrier protein can be conjugated through a unique functional group such as a cysteine residue. However, any alternative natural or non-natural residue may be used in place of the cysteine residue to conjugate the peptide to the carrier protein. An example of a non-natural residue that can be used in place of cysteine is a homocysteine residue, which is a homologue of cysteine that further contains an additional methylene group in the side chain. Thus, any peptide having an amino acid sequence selected from SEQ ID NOs: 107-151 containing a cysteine residue is suitable as an alternative suitable natural or non-natural for conjugation to a carrier protein such as a homocysteine residue Residues may be modified to replace cysteine residues.

  The present disclosure also extends to the novel peptides disclosed herein and compositions comprising the same.

  In general, between 0.001 mg and 1 mg of each peptide-carrier protein is required for each immunization dose per host animal.

Alternative immunogens suitable for producing functionally modified antibodies include the following: related full-length human ion channels such as Na _v 1.7, individual subdomains and their cleavage, including truncated forms of subdomains Type; chimeric molecules with regions of ion channels fused to regions of other transmembrane proteins to aid expression or present extracellular loops to the immune system, and constrain the regions of ion channels to the desired conformation For ion channel mutant type.

  These immunogens may be expressed in mammalian cells for direct cell immunization or purification of proteins for immunization.

  These immunogens may be expressed in E. coli or cell-free expression systems for purification of proteins for immunization.

  The purified protein may be incorporated into lipid vesicles or micelles for immunization.

  These ion channel versions may also be made as lipoparticles for immunization.

  In addition, any of the above immunogens can be used as a screening tool for identifying functionally modified antibodies.

  Thus, in one embodiment, several different peptides, for example at least one ion channel E1 peptide-carrier protein conjugate or separately conjugated to the same carrier protein, where at least one is E1. Or by immunization as a mixture conjugated to the same carrier protein is provided.

  In one embodiment, the method comprises 1, 2, 3, 4, or 5 immunizations.

  In one embodiment, the method comprises at least 2 immunizations, such as 2 or 3 times, with each conjugated peptide (s).

  In one embodiment, the second immunization uses a different conjugate where the peptide (s) are in common but the carrier protein is different from the carrier protein used for the first immunization.

  Thus, in one embodiment, the third immunization is the same as that of the first and second immunizations of the peptide (s), but the carrier protein is the first and / or second immunizations. Use a different conjugate that is different from that used in Undesirable antibody specificity for the carrier protein can thus be minimized.

  Suitable carrier protein combinations for sequential immunization include KLH and ovalbumin and BSA in any order.

  Changing the carrier can be advantageous in optimizing the response to the peptide.

  Each immunization also generally involves the administration of an adjuvant to stimulate the immune response. Suitable adjuvants include Freund's complete or incomplete adjuvant and adjuvants including alum, QS21, MPL, and / or CPG.

  The method may further comprise separating the antibody or antibody producing cell from the host.

  In one embodiment, the host is a rodent such as a mouse or rat, a camel, a llama, a pig, a dog, a primate, a shark, or a rabbit, particularly a rabbit.

  The aforementioned peptides may also be conjugated to a reporter group. The reporter group can be any group that allows for detection or isolation of, for example, biotin, fluorescent groups, or enzyme tags, or anti-ion channel antibodies. Reporter group-peptide conjugates can be used to screen the resulting polyclonal sera and monoclonal antibodies. For example, a screening ELISA can include streptavidin-coated microwells and captured biotinylated peptides. Titration of immune serum against this results in binding of the peptide-specific antibody to the surface, which can then be revealed by a fourth anti-species (such as anti-rabbit) IgG-peroxidase layer.

  Reporter group-peptide conjugates can also be used to isolate specific ion channel antibodies, such as by library-based techniques such as phage panning.

  The method may also include further purification steps, eg, to provide polyclonal or monoclonal antibodies.

  The method may also include the step of generating a recombinant clonal antibody derived from said immunization.

  Before a cloned antibody can be prepared recombinantly, it may be necessary to clone and / or sequence portions such as variable regions, or the entire antibody.

  The present disclosure also extends to antibody producing cells and antibodies obtainable or obtained from the methods herein.

  The present disclosure also extends to antibodies or suitable antibody fragments obtainable or obtained using the methods herein.

  The disclosure also includes pharmaceutical compositions comprising the antibodies or fragments herein.

  The pharmaceutical composition suitably comprises a therapeutically effective amount of an antibody of the invention. The term “therapeutically effective amount” as used herein is used to treat, ameliorate, or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or prophylactic effect. Refers to the amount of therapeutic agent required. For any antibody, the therapeutically effective dose should be estimated initially either in cell culture assays or in animal models, usually rodents, rabbits, dogs, pigs, or primates. Can do. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

  The exact therapeutically effective amount for a human subject is the severity of the condition, the subject's general health, the subject's age, weight, and sex, diet, administration time and frequency, combination (s), Depends on response sensitivity and tolerance / response to treatment. This amount can be determined by routine experimentation and is within the judgment of the clinician. Generally, a therapeutically effective amount is from 0.01 mg / kg to 50 mg / kg, such as from 0.1 mg / kg to 20 mg / kg. The pharmaceutical composition may conveniently be provided in the form of a unit dose containing a predetermined amount of the active substance of the present invention per dose.

  The composition may be administered to the patient individually or in combination with other agents, drugs, or hormones (eg, simultaneously, sequentially, or separately).

  The dose to which the antibody or fragment of the invention is administered depends on the nature of the condition being treated, the extent of the disease and / or the symptoms present, and whether the antibody or fragment is to be used prophylactically or Depends on what is to be used to treat the condition.

  The frequency of dosing will depend on the half-life of the antibody molecule and the duration of its effect. If the antibody molecule has a short half-life (eg, 2-10 hours), it may be necessary to dose one or more times per day. Alternatively, if the antibody molecule has a long half-life (eg 2 to 15 days), dosing once a day, once a week, or once a month or once every two months Can only be needed.

  A pharmaceutically acceptable carrier should not itself induce the production of antibodies that are detrimental to the individual receiving the composition, and should not be toxic. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactive virus particles.

  Pharmaceutically acceptable salts, such as mineral salts such as hydrochloride, hydrobromide, phosphate, and sulfate, or acetate, propionate, malonate, and benzoate Organic acid salts can be used.

  Pharmaceutically acceptable carriers in therapeutic compositions may additionally include liquids such as water, saline, glycerol, and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents, or pH buffering substances may be present in such compositions. Such carriers can be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, and suspensions for oral ingestion by the pharmaceutical composition.

  Suitable forms for administration include forms suitable for parenteral administration, such as by injection or infusion, for example by bolus injection or continuous infusion. If the product is for injection or infusion, it can take the form of a suspension, solution, or emulsion in an oily or aqueous medium, and is a suspension, preservative, stabilizer, and / or Or you may contain formulation agents, such as a dispersing agent. Alternatively, the antibody molecule may be in a dry form for reconstitution prior to use with a suitable sterile liquid.

  Once formulated, the compositions of the invention can be administered directly to the subject. The subject to be treated can be an animal. However, in one or more embodiments, the composition is adapted for administration to a human subject.

  Suitably, in a formulation according to the present disclosure, the pH of the final formulation is not similar to the isoelectric point value of the antibody or fragment, eg, 8-9 or above if the pH of the formulation is 7. pI may be appropriate. While not intending to be bound by theory, this can ultimately provide a final formulation with improved stability, eg, the antibody or fragment remains dissolved.

  The pharmaceutical compositions of the present invention may be administered by any number of routes, including oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracerebroventricular, transdermal. Transcutaneous (see, eg, WO 98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal, or rectal routes, but is not limited thereto. A hypodermic injector may also be used to administer the pharmaceutical composition of the present invention. Typically, the therapeutic composition may be prepared as an injection, either as a solution or a suspension. Solids suitable for solution or suspension in a liquid medium prior to injection may also be prepared.

  Direct delivery of the composition is generally accomplished by subcutaneous, intraperitoneal, intravenous, or intramuscular injection, or injection delivered to the interstitial space of the tissue. The composition can also be administered to a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule.

  It will be appreciated that the active ingredient in the composition is a protein molecule. It is therefore prone to degradation in the gastrointestinal tract. Thus, if the composition is to be administered by route using the gastrointestinal tract, the composition protects the antibody from degradation, but releases the antibody once it has been absorbed from the gastrointestinal tract. It may be necessary to include an active agent.

  A thorough discussion of pharmaceutically acceptable carriers is available at Remington's Pharmaceutical Sciences (Mack Publishing Company, NJ 1991).

  In one embodiment, the formulation is provided as a formulation for topical administration, including inhalation.

  Suitable inhalable preparations include inhalable powders, metered aerosols with nebulizing gas, or inhalable solutions without nebulizing gas. An inhalable powder according to the present disclosure comprising an active substance may consist solely of the aforementioned active substance or a mixture of the aforementioned active substance with physiologically acceptable excipients.

  These inhalable powders include monosaccharides (eg glucose or arabinose), disaccharides (eg lactose, saccharose, maltose), oligosaccharides and polysaccharides (eg dextran), polyalcohols (eg sorbitol, mannitol, Xylitol), salts (eg, sodium chloride, calcium carbonate), or mixtures thereof with each other. Suitably monosaccharides or disaccharides are used, in particular lactose or glucose in the form of hydrates, but not limited thereto.

  Particles for lung deposition require a particle size of less than 10 microns, such as 1 to 9 microns, for example from 0.1 μm to 5 μm, in particular from 1 μm to 5 μm. The particle size of the active ingredient (such as an antibody or fragment) is most important.

  Nebulizing gases that can be used to prepare inhalable aerosols are known in the art. Suitable atomizing gases include hydrocarbons such as n-propane, n-butane, or isobutane, and halohydrocarbons such as chlorinated and / or fluorinated derivatives of methane, ethane, propane, butane, cyclopropane, or cyclobutane. It is selected from the inside. The atomizing gas described above may be used alone or as a mixture thereof.

  Particularly suitable atomizing gases are halogenated alkane derivatives selected from among TG11, TG12, TG134a, and TG227. Of the aforementioned halogenated hydrocarbons, TG134a (1,1,1,2-tetrafluoroethane) and TG227 (1,1,1,2,3,3,3-heptafluoropropane) and mixtures thereof are particularly Is suitable.

  Nebulized gas-containing inhalable aerosols may also include other components such as co-solvents, stabilizers, surfactants (surfactants), antioxidants, lubricants, and means for adjusting the pH. All of these components are known in the art.

  The nebulized gas-containing inhalable aerosol according to the invention may contain up to 5% by weight of active substance. The aerosol according to the present invention is, for example, 0.002 to 5 wt%, 0.01 to 3 wt%, 0.015 to 2 wt%, 0.1 to 2 wt%, 0.5 to 2 wt%, or 0 0.5 to 1% by weight of active ingredient.

  Alternatively, topical administration to the lung can also be achieved, for example, by a nebulizer, eg, a Pali LC-Jet connected to a compressor (eg, a Pari Master® compressor manufactured by Pari Respiratory Equipment, Inc., Richmond, Va) connected to a compressor. It may be by administration of a solution or suspension formulation using a device such as a Plus® nebulizer.

  The antibodies or fragments of the invention can be delivered dispersed in a solvent, for example, in the form of a solution or suspension. It can be suspended in a suitable physiological solution, such as saline, or other pharmaceutically acceptable solvent or buffer solution. Buffer solutions known in the art are 0.05 mg to 0.15 mg disodium edetate, 8.0 mg to 9.0 mg per ml water to reach a pH of about 4.0 to 5.0. Of NaCl, 0.15 mg to 0.25 mg polysorbate, 0.25 mg to 0.30 mg anhydrous citric acid, and 0.45 mg to 0.55 mg sodium citrate. As the suspension, for example, a freeze-dried antibody can be used.

  A therapeutic suspension or solution formulation may also include one or more excipients. Excipients are well known in the art and include buffering agents (eg, citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohol , Ascorbic acid, phospholipids, proteins (eg, serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. Solutions or suspensions can be encapsulated in liposomes or biodegradable microspheres. The formulations are generally provided in a substantially sterile form using sterile manufacturing methods.

  This includes production by filtration of the buffer / solution used in the formulation, aseptic suspension of the antibody in sterile buffer / solution, and dispensing the formulation into a sterile container, by methods well known to those skilled in the art. Can include sterilization.

  Nebulizable formulations according to the present disclosure may be provided, for example, as a single dose unit (eg, a sealed plastic container or vial) packaged in foil. Each vial contains a unit dose in a volume, for example 2 ml of solvent / solution buffer.

  The fusion protein molecules of the present disclosure are considered suitable for delivery by spray.

  The antibodies and fragments of the present disclosure are suitable for treating pain, eg, neuropathic pain including painful diabetic neuropathy (PDN), postherpetic neuropathy (PHN), or trigeminal neuralgia (TN) obtain. Other causes of neuropathic pain include spinal cord injury, multiple sclerosis, phantom limb pain, post-stroke pain, and HIV-related pain. Conditions such as chronic back pain, osteoarthritis, and cancer can also result in the development of neuropathy related pain and therefore may be suitable for treatment with antibodies or fragments according to the present disclosure.

  In one embodiment, the antibody or fragment according to the invention treats pain including somatic pain, visceral pain, neuropathic pain, nociceptive pain, acute pain, chronic pain, breakthrough pain, and / or inflammatory pain. Or it is suitable for prevention.

  In one embodiment, an antibody or fragment according to the invention is suitable for the treatment or prevention of one or more of the following types of pain: allodynia, painful sensory loss, angina pain, breakthrough pain, complex Local pain syndrome type I, complex local pain syndrome type II, hyperalgesia, hyperpathia, idiopathic pain, malignant pain, illusion, phantom limb pain, psychogenic pain.

  In one embodiment, an antibody or fragment according to the present disclosure is useful for the treatment of asthma, airway hyperresponsiveness in asthma, eg, chronic cough and / or chronic obstructive airway in asthma.

  In one embodiment, an antibody or fragment according to the present disclosure is useful for the treatment of inflammation, osteoarthritis, rheumatoid arthritis, and / or pain associated with any of them.

  In one embodiment, an antibody or fragment according to the present disclosure is useful for the treatment of pain associated with acute injury, eg, wounds such as lacerations, cuts, burns, bullet injury, and / or shrapnel injury.

  As discussed, antibodies or fragments according to the present disclosure treat pain such as acute and chronic pain, neuropathic pain, nociceptive pain, visceral pain, back pain, and pain associated with disease and degeneration. Likely to be useful.

  Pain can result from one or more causes, including peripheral neuropathy, central neuropathy, carpal tunnel syndrome, tarsal tunnel syndrome, ulnar nerve strangulation, compression nerve root disorder, lumbar spinal canal Nerve compression or strangulation syndrome such as stenosis, sciatic nerve compression, spinal cord compression, intercostal neuralgia, compression radiculopathy, and nerve root back pain, spinal root destruction, back pain, neuritis, autoimmune disease, postoperative pain, toothache , Direct trauma, inflammation, HIV infection, smallpox infection, herpes infection, toxic exposure, invasive cancer, chemotherapy, radiation therapy, hormonal treatment, foreign body, burn, congenital defect, phantom limb pain, rheumatoid arthritis, degenerative joint , Fracture pain, gout pain, fibromyalgia, multiple sclerosis, pain associated with diarrhea, irritable bowel syndrome, migraine, encephalitis, diabetes, chronic alcoholism, hypothyroidism, uremia, and vitamin deficiency There are But it is not limited to, et al. Accordingly, an antibody or fragment according to the present disclosure may be useful for the treatment or amelioration of one or more symptoms of the above indications.

  In one embodiment, an antibody or fragment according to the present disclosure is used as a standard in an assay to screen for ion channel inhibitors.

  Comprising in the context of this specification shall mean including.

  In theory, suitable embodiments of the invention can be combined.

  Embodiments have been described to include specific features / elements. The present disclosure also extends to separate embodiments consisting of, or consisting essentially of, the above features / elements.

  The invention is further described, by way of example only, in the following examples, with reference to the accompanying figures:

Generation / Selection of Na _v 1.7 Therapeutic Antibodies Peptides are available from Peptide Protein Research Ltd. Fareham, U.S.A. K. Linear peptides were synthesized by Fmoc solid phase peptide chemistry according to the method of Atherton and Sheppard (1989) Solid Phase peptide synthesis: a practical approach Oxford, England: IRL Press. For N-terminal to C-terminal cyclic peptides, see Computer Aided Drug Design, Methods and Applications, J. et al. Perun and C.I. L. Probst ed., Pages 461-484, Marcel Dekker, New-York; 1990, Int. J. et al. Pept. Protein Res. , 35, 287-300; Et al., 1992, Eur. J. et al. Biochem. , 210, 911-921; Izumiya N .; Et al., 1981, Biopolymers, 20, 1785-1791; Brady S. et al. F. 1983, Peptides, Structure and Function, Proceedings of the Eighth American Peptide Symposium, V. et al. J. et al. Hruby and D.H. H. Rick, pages 127-130, Pierce Chemical Company, Rockford, Illinois; X. Et al., 1994, Lett. Peptide Sci. , 1, 25-30, Barlos et al., Int. J. et al. Pept. Protein Res. It was synthesized as a side chain protected peptide by the method of 1991 and cyclization was performed in the liquid phase, followed by deprotection of the side chain by the method of Kessler H et al., 1989.

Rabbits were immunized with a combination of human Na _v 1.7 peptides conjugated to either KLH, OVA, or BSA (Table 1). After 5 subcutaneous immunizations (KLH, OVA, BSA, KLH, OVA), animals were sacrificed and PBMC, spleen, and bone marrow were collected. Serum was tested in an ELISA for binding to human biotinylated peptides.

  The table shows the rabbit number to be immunized, the peptide combination used for immunization, and the peptide sequence. B11 is a peptide derived from loop E1 in domain B. C11 is a peptide derived from loop E1 in domain C. D11 is a peptide derived from loop E1 in domain D.

  SLAM was performed substantially using the method described in Tickle et al., 2009 (JALA, Vol. 14, No. 5, pages 303-307). Briefly, SLAM cultures were set up using rabbit splenocytes or PBMC and supernatants were first screened for the ability to bind to biotinylated peptides in a bead-based assay in FMAT. This was a homogeneous assay that revealed binding using a goat anti-rabbit Fc-Cy5 conjugate (Jackson ImmunoResearch) using a biotinylated human peptide conjugated to streptavidin beads (Bangs Laboratories). Subsequently, those that were positive from this screening were subjected to a negative screening to identify non-specific antibodies. This identified streptavidin beads without peptide or streptavidin beads with an irrelevant peptide, revealed binding to goat anti-rabbit Fc-Cy5 conjugate (Jackson immunoResearch), and identified peptide-specific conjugates .

  From 10 SLAM experiments, several B11-specific, C11-specific, and D11-specific antibody-containing wells were identified using the above screen.

  Isolation of single B cells from several of these wells by fluorescence focus method and subsequent cloning of variable region genes resulted in heavy and light chain variable region gene pairs after reverse transcription (RT) -PCR. I got it well. These V region genes were cloned as rabbit IgG1 full-length antibodies and reexpressed in HEK293 transient expression system.

Sequence analysis of the cloned V region revealed the presence of several unique families that are specific for anti-human B11 (see Table 2 below). The DNA and amino acid sequences of these antibodies are shown in the figure. The antibody was expressed in a transient CHO system and then purified to allow further characterization in vitro and in vivo.

Figures 4-7 show the sequences for the anti-Na _v 1.7 antibody. The immunized rabbits from which the antibodies are derived and their peptide specificity are detailed.

Procedure for h Na _v 1.7 recording for antibody testing Solution and antibody manipulation The extracellular solution contained (mM): 130 NaCl, 4 KCl, 1.5 CaCl ₂ , 1 MgCl ₂ , 30 glucose, 10 HEPES (pH 7.4 in Tris-Base, and 300-305 mmol osmotic pressure). The intracellular solution contains (mM): 5 NaCl, 115 CsF, 20 CsCl, 110 HEPES, 10 EGTA free acid (pH 7.2 with CsOH, and 290-295 mmol osmotic pressure) to make it fresh Or frozen. Extracellular and intracellular solutions were filtered before use. The antibodies were diluted and prepared fresh (within 15 minutes) directly in extracellular solution prior to transfer to 96-well polypropylene compound plates (Sarsted, # 83.1835.500). For experiments with selective peptides, equal concentrations of antibody and peptide were preincubated at 4 ° C. for at least 30 minutes prior to patch clamp experiments.

Cell Preparation HEK293 cells stably expressing human Na _v 1.7 channel (type IX voltage-gated sodium channel α subunit) were purchased from Upstate (Upstate, Millipore, catalog # CYL3011). 10% FBS (Lonza, # DE14-802F), 1% penicillin + streptomycin (Lonza, DE17-603E), 1% non-essential amino acids (Lonza, BE13-114E), and 400 μg / ml G418 (GIBCO, # 10131-027) Cells in a T-75 (BD BioCoat ™ Collagen I Cellware, Becton Dickinson Labware, Bedford, MA, # 356485) coated with collagen type I using a glutamine-containing standard medium DMEM-F12 Was cultured. Cells are used for 2 or 3 days prior to use with PatchXpress® 7000A (Axon instrument, a new member of MDS Analytical Technologies) at a density of 15,000 cells / cm 2 or 8,000 cells / cm 2, respectively. Plated. Cell confluence did not exceed 90%. On the day of the experiment, cells were harvested using Accumax (Sigma, A7089). Briefly, the cells are washed twice in PBS without calcium and magnesium (Lonza, # BE12-516F), a 1: 4 dilution of Accumax solution is added, and 1.5-2 at 37 ° C. Incubated for minutes. Accumax digestion was terminated by the addition of 15 mM HEPES and L-glutamine containing DMEM-F12 (Lonza, # BE12-719F) (recovery medium) containing 10% FBS. Subsequently, the cells are centrifuged in a 50 ml Falcon tube at 1,000 rpm for 5 minutes and the pellet is resuspended in 10 ml of recovery medium. Cells were counted (Coulter Z2), suspended at approximately 100,000 cells / ml and transferred to a 15 ml screw-cap tube for a minimum of 90 minutes at room temperature. The cells were then centrifuged at 1,000 rpm for 60 seconds. The pellet was gently resuspended in 1,000 μl of extracellular solution and centrifuged a second time at 1,000 rpm for 30 seconds. The pellet was resuspended in 150 μL extracellular solution and immediately tested with PatchXpress®.

PatchXpress® Procedure The AVIVA Biosciences SealChip16 ™ electrode array (purchased from Axon Instruments, Union City, Calif.) Is manually placed in the holder of the PatchXpress® system and cell apply is automatically prepared. It was. Intracellular solution was injected into the bottom of each chamber, and extracellular solution was perfused through the 16-nozzle washer into the top of the chamber. During this time, the positive pressure from the inside of the cell (+10 mmHg) was maintained with a pressure control device in order to keep the cell debris from being in the hole. The cells were ground by an integrated Cavro pipetting robot before 4 μl (containing 10K-30K cells) was added to each well.

PatchXpress® h Na _v 1.7 Assay After 10 seconds, the pressure was switched from +4 mmHg to −30 mmHg and the suspended cells were drawn into each of the 16 holes (electrodes). Seal formation was achieved by repeating a negative pressure ramp from -1 mm Hg to -35 mm Hg every 36 seconds at a rate of 1.6 mm Hg / second until a gigaohm seal was obtained and verified for 20 seconds. Whole cell access was achieved by breaking the membrane patch over the hole using a ramp increase in negative pressure from -40 mmHg to -150 mmHg at a rate of 7.5 mmHg / sec with a pipette potential of -80 mV. After whole cell recording, the cells were washed with extracellular solution for 66 seconds to remove excess cells in the wells. The cells were dialyzed for 5 minutes, during which time the access resistance was monitored. Cells were held at −80 mV between potential protocols from the time of the whole cell trial run to the end of the experiment. A time course protocol was applied to assess antibody potency to sodium currents induced by a -80 mV to 0 mV depolarization step at 10 second intervals for 20 milliseconds. Whole cell compensation was automatically made before each trial began, and the electrical access resistance (Ra) was corrected by 65%. Linear leak subtraction was performed online using the P / N leak subtraction protocol (N = 4) while maintaining -80 mV.
After the stabilization period (up to 10 minutes), a negative control solution (extracellular solution) was applied for 5 minutes, followed by two doses of antibody. The interval between two additions of the same concentration of compound to the wells was approximately 11 seconds. Antibody solution (45 μL) was added online (30 μL / sec) at the desired concentration with continuous aspiration. Current was monitored continuously during the 18 minute exposure to the antibody.

Data analysis Cells were not analyzed in the following cases:
(1) The membrane resistance was initially less than 200 M ohms,
(2) Current amplitude <200 pA,
(3) Access resistance is 20 M ohms or less, and (4) There is no substantial stabilization current after negative control addition.
Current amplitude was measured using DataXpress2 software (Axon instruments), rundown current correction by linear or exponential fitting method, last 10-15 data points after wash time, and last after negative control addition. Measurements related to 10-15 data points were performed.
The current was normalized by the average current correction amplitude prior to antibody addition. Current inhibition was estimated by the residual response after 18 minutes of antibody application. The data is shown in Table 3 below.
Table 3:

FIG.
FIG. 1 shows the functional effect of selected antibodies (25 μg / ml) on human Nav1.7 current expressed in HEK cells in the presence or absence of specific peptides. Nav1.7 current is recorded by automatic patch clamp using a repetitive stimulation protocol and data is presented as normalized Nav1.7 current after the last stimulation. Selected antibodies were incubated for 30 minutes at 4 ° C. in the presence of specific peptides (25 μg / ml) and then transferred to the PatchXpress system for Nav1.7 current recording. The presence of the peptide systematically reverses the inhibitory effect of the antibody, thus indicating that the inhibition of the Nav1.7 current is mediated by specific interaction of the antibody with the Nav1.7 extracellular loop. Yes.

FIG. 3D (a)
Automated patch clamp analysis of recombinant human Nav1.7 channel expressed in HEK cells. The 983 monoclonal antibody produces a dose-dependent inhibition of the Nav1.7 current. Data points represent the normalized peak current amplitude (endpoint) after application of the repetitive potential step protocol in the presence of antibody.

FIG. 3D (b)
Automated patch clamp analysis of recombinant human Nav1.7 channel expressed in HEK cells. The 1080 monoclonal antibody produces a dose-dependent inhibition of the Nav1.7 current. Data points represent the normalized peak current amplitude (endpoint) after application of the repetitive potential step protocol in the presence of antibody.

FIG.
Automated patch clamp analysis of recombinant rat Nav1.7 channel expressed in HEK cells. The 983 monoclonal antibody produces a dose-dependent inhibition of the Nav1.7 current. The 1080 monoclonal antibody produces about 26% inhibition of the Nav1.7 current at 25 μg / ml. Data points represent the normalized peak current amplitude (endpoint) after application of the repetitive potential step protocol in the presence of antibody.

3F
Kinetics of human Nav1.7 inhibition by the 983 monoclonal antibody. HEK cells expressing recombinant human Nav1.7 channel are stimulated for about 20 minutes at 0.1 Hz using a voltage step protocol. Data points represent the normalized peak current amplitude (rundown corrected) for the Nav1.7 channel recorded every 10 seconds. Nav1.7 current is reduced in the presence of antibody (25 μg / ml), but only if repeated activation of the channel at 0.1 Hz is maintained. Stimulation of the Nav1.7 channel only at the end of the protocol (and after antibody incubation) does not result in inhibition of the Nav1.7 current. The data suggests that specific inhibition by the 983 monoclonal antibody requires repetitive activation (channel cycling) of the Nav1.7 channel protein.

Dorsal Root Ganglion In Vitro Test Primary Culture Preparation Dorsal root ganglia were isolated from 1-2 wild type rat pups between 1 and 3 days of life. The ganglia were washed in PBS after incision and immediately placed in a DMEM (Lonza, # BE12-604F) solution containing 2 mg / ml collagenase (Sigma-Aldrich, # C2674) for enzymatic digestion. Incubated at 37 ° C for approximately 45 minutes. Removing the collagenase solution, 10% fetal calf serum (Lonza, # DE14802F), 0.5 mM L-glutamine (Lonza, # BE17-605E), 1% penicillin / streptomycin (Lonza, # BE17-603E), and Replaced with DMEM supplemented with 20 ng / ml nerve growth factor (NGF, Invitrogen). The ganglia were then mechanically crushed, centrifuged at 1000 g for 5 minutes, and resuspended in the same medium. Count the separated cells and suspension at 100,000-120,000 cells / ml on coverslips pre-coated with 50 μg / ml poly-D-lysine (Sigma) and 30 μg / ml laminin (Invitrogen) Dilute to solution and incubate at 37 ° C., 5% CO ₂ until ready for use.

Primary culture electrophysiology After in vitro culture (DIV) within 2 days of preparation, isolated DRGs were harvested for use. Cells were visualized with an Olympus BX50WI upright microscope with an Ikegami ICD-42B CCD camera. Using 5 kHz digital sampling, electrophysiological recordings are obtained and filtered at 3 dB, 3 kHz frequency in an Axopatch 1D (Molecular Devices) amplifier and converted to a digital signal using Digidata 1322A analog-to-digital converter (Molecular Devices). It was. All recordings were obtained using pClamp 10 software (Molecular Devices) and subsequently analyzed in Clampfit 10 (Molecular Devices). The recording electrode was withdrawn from the borosilicate glass pipette with a Sutter p-97 horizontal pipette puller to a final resistance of 4.5-6 MΩ and filled with (mM) internal solution containing: 140 K-methanesulfonate, 5 NaCl, 1 CaCl2, 2 MgCl2, 11 EGTA, 10 HEPES, 2 Mg-ATP, and 1 Li-GTP; pH was adjusted to 7.2 with Tris-base, and osmolality was adjusted to 310 mOsm with sucrose. The bath solution contained (mM): 130 NaCl, 25 glucose, 10 HEPES, 4 KCl, 2 CaCl ₂ , 1 MgCl ₂ , 1.25 NaPO ₄ ; pH adjusted to 7.35 with NaOH, osmolality The concentration was adjusted to 310 mOsm with sucrose. The liquid junction potential is calculated to be 14.2 mV and all reported potentials have been corrected to compensate.
After releasing the positive pressure in the potential clamp mode and forming a seal (> 1 GΩ) by manual suction, the capacitive current was compensated and the command potential was set to -70 mV. The cell membrane was broken and the cells were allowed to dialyze the intracellular solution for 5 minutes. After dialysis, whole cell parameters were recorded. Cells were discarded if the whole cell capacitance was> 35 pF or a stable access resistance of less than 3 times the electrode resistance could not be achieved. The amplifier was switched to current clamp mode and the resting membrane potential was recorded. The cells were then injected with a series of 1.5 second duration increasing amplitude depolarizing current steps intended to induce action potential (AP) or a stretch of AP. Cells that failed to induce more than one AP were discarded during a single step after depolarization to a maximum of -35 mV.
The cells were then treated with either control solution or antibody solution by high speed bath perfusion directly over the cells to be recorded for 90 seconds to fully fill the recording chamber, and the recording chamber When the was fully filled, both perfusion and aspiration were stopped. The previous series of depolarizing current steps was repeated over a period of 40 minutes at 2 minute intervals, typically with a 1.5 second delay between individual steps, allowing membrane repolarization. Occasionally, a constant current was injected when the resting membrane potential (RMP) was adjusted to maintain a constant RMP of -65 mV during the course of the experiment. During the course of the experiment, cells in which the RMP deflected more than 20% in either the positive or negative direction or the holding current changed more than 100 pA were discarded. Individual holding and injection currents for each step, as well as any electrophysiological parameters that changed during the course of the experiment, were noted individually for each cell.

Data analysis The action potential (AP) was manually counted for each depolarization step and the total number of evoked APs was summed for each time point. In Microsoft Excel 2003, the number of APs at each time point was normalized against the number of induced APs at time = 0 and plotted as a function of time using Graphpad Prism 5.0 software. Each plotted data point represents the average value of all recorded cells under the specified experimental conditions, and the error bar represents the calculated standard error.
FIG. 3A: Current clamp traces of evoked action potentials from representative DRG neurons before treatment (time = 0) and after treatment (time = 30 minutes).
Time = potential compared to control treated with vehicle or control antibody after antibody administration at 2 minutes.
FIG. 3B: Antibody 983 (25 μg / ml) significantly reduced the number of evoked action potentials after administration of antibody at time = 2 minutes compared to controls treated with vehicle or control antibody.

FIG. 3C: Electrophysiology (current clamp recording) study on action potential induction in cultured rat dorsal root ganglion (DRG) neurons. The 1080 monoclonal antibody at a dose of 25 μg / ml reduces the frequency of electrically induced spikes in DRG neurons. Data points represent the normalized spike frequency compared to the initial frequency observed at time 0 prior to antibody application.

Peptide binding ELISA
Nunc 96 well plates were coated overnight at 4 ° C. in 100 ul / well of 5 ug / ml streptavidin (Jackson 016-000-114) in carbonate coating buffer. Plates were washed 4 times in PBS / Tween and 200 ul / well blocking solution (1% BSA in PBS) was added for 1 hour at room temperature. Plates were washed 4 times in PBS / Tween and 100 ul / well of 5 ug / ml biotinylated peptide was added for 1 hour at room temperature. Plates were washed 4 times in PBS / Tween and 100 ul / well antibody was added for 1 hour at room temperature (starting at 10 ug / ml along the plate and diluted in a 3.16 fold series in blocking solution. To go). Plates were washed 4 times in PBS / Tween and 100 ul / ml goat anti-rabbit Fc HRP (Jackson 111-036-046) was added for 1 hour at room temperature. Plates were washed 4 times in PBS / Tween and 100 ul / well of TMB (3,3 ′, 5,5′-tetramethylbenzidine) solution was added. 50 ul / well NaF was added to stop the reaction and the absorbance was read at 630 nm.
FIG. 3G shows ELISA data for binding of antibody 983 to various cyclic Nav ion channel peptides Table 4.
FIG. 3H shows ELISA data for binding of antibody 1080 to various cyclic Nav ion channel peptides Table 4.
Specific binding in both cases was observed only for the B11.7 peptide and no binding to equivalent loops from other Nav ion channels was observed.

Claims (6)

An anti-E1 ion channel monoclonal antibody or binding fragment thereof that binds to the E1 extracellular loop of an ion channel, comprising:
The ion channel functions in the regulation of pain, and the antibody or fragment functionally modifies after binding the ion channel to the ion channel;
The ion channel is a sodium ion channel selected from the group consisting of Na _v 1.3, 1.6, 1.8, and 1.9;
The antibody or the binding fragment thereof, which binds to the E1 loop in domain B or domain C of the ion channel. The anti-E1 ion channel monoclonal antibody or binding fragment thereof according to claim 1, wherein the antibody or fragment thereof binds to a Na _v 1.3 peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 131 to 134. The anti-E1 ion channel monoclonal antibody or a binding fragment thereof according to claim 1, wherein the antibody or a fragment thereof binds to a Na _v 1.6 peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 135 to 138. The anti-E1 ion channel monoclonal antibody or binding fragment thereof according to claim 1, wherein the antibody or fragment thereof binds to a Na _v 1.8 peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 139 to 145. The anti-E1 ion channel monoclonal antibody or binding fragment thereof according to claim 1, wherein the antibody or fragment thereof binds to a Na _v 1.9 peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 146 to 149. 6. The anti-E1 ion channel monoclonal antibody according to any one of claims 1 to 5, or an antibody thereof, wherein the antibody inhibits the function of the relevant ion channel to which it is specific by at least 5 percent in an in vitro patch clamp assay. Binding fragment.