Anti-Kv1.3 antibodies, and methods of production and use thereof

Anti-Kv1.3 antibodies (mAbs), particularly mAbs that specifically bind to Kv1.3 with high affinity and/or inhibit Kv1.3 function, are disclosed. The amino acid sequences of the CDRs of the light chains and the heavy chains, as well as consensus sequences for these CDRs, of these anti-Kv1.3 mAbs are provided. Additionally, canonical structures for CDRs in the VH and VL regions of anti-Kv1.3 antibodies are provided. The disclosure also provides nucleic acid molecules encoding the anti-Kv1.3 mAbs, expression vectors, host cells, methods for making the anti-Kv1.3 mAbs, and methods for expressing the anti-Kv1.3 mAbs. Finally, methods of using the anti-Kv1.3 mAbs as therapeutics, such as for preventing or treating an autoimmune disorder, are disclosed.

FIELD OF THE INVENTION

The present disclosure relates generally to antibodies, their production and use. Specifically, the present disclosure pertains to antibodies which specifically bind to the human Kv1.3 protein, modulate the activity of the human Kv1.3 protein, methods of producing such antibodies, and diagnostic, therapeutic and clinical methods of using such antibodies.

BACKGROUND

Peptide toxins and their derivatives are being developed as therapeutic agents that inhibit Kv1.3 function for the treatment of autoimmune disease. For example, the peptide toxin ShK derived from the sea anemoneStichodactyla helianthusbinds to the outer vestibule of the Kv1.3 tetramer with high affinity and occludes ion conductance through the pore (Beeton et al. (2003), J. Biol. Chem., 278, 9928-37)).

Therapeutic antibodies that modulate the function of Kv1.3 represent an alternative class of biologics that could be developed to treat a variety of TEM-mediated auto immune disease. A number of anti-Kv1.3 antibodies that recognize both intracellular epitopes and extracellular epitopes are commercially available (e.g., Alomone Labs, Jerusalem, Israel: Anti-Kv1.3 (Extracellular), Cat# APC101; Anti-Kv1.3 (Intracellular), Cat# APC002), but are not functionally active and do not modulate Kv1.3 activity. However, rabbits immunized with a peptide consisting of 14 amino acids located at the external end of the human Kv1.3 pore region produced polyclonal antibodies capable of functionally inhibiting Kv1.3 activity (Yang et al. (2012), PLoS One 7, e36379), indicating that functional anti-Kv1.3 immunoglobulins are viable.

Nevertheless, there remains a need for the identification and development of high-affinity monoclonal antibodies (mAbs) that recognize Kv1.3 and, in particular, the extracellular loop regions that are expected to be critical in exerting a modulating effect on Kv1.3 activity.

SUMMARY

The present invention depends, in part, upon the development of improved immunogenic preparations of human Kv1.3 protein, which have permitted the production of anti-Kv1.3 monoclonal antibodies which are directed to the extracellular domains of the tetrameric Kv1.3 ion channel and which have superior affinity and specificity for Kv1.3. These antibodies have both therapeutic and diagnostic utility.

Thus, in one aspect, the invention provides anti-Kv1.3 monoclonal antibodies (mAbs), particularly mAbs that modulate Kv1.3 functional activity. In particular, the invention provides the amino acid sequences of the CDRs of the light chains and the heavy chains, as well as consensus sequences for these CDRs, and enables the production of a variety of antibodies and other immunoglobulin-based molecules comprising these CDRs. Additionally, the invention provides predicted canonical structures for the CDRs in the light and heavy chain variable domains, and thereby enables the production of additional antibodies and other immunoglobulin-based molecules which specifically bind to Kv1.3

In another aspect, the invention provides nucleic acid molecules encoding the anti-Kv1.3 mAbs and other immunoglobulin-based molecules, expression vectors comprising such nucleic acids, host cells comprising such nucleic acids or vectors, methods for making the anti-Kv1.3 mAbs and other immunoglobulin-based molecules, and methods for expressing the anti-Kv1.3 mAbs and other immunoglobulin-based molecules. Finally, methods of using the anti-Kv1.3 mAbs and other immunoglobulin-based molecules as therapeutic drugs or diagnostic reagents are provided.

In another aspect, the invention provides an antibody that specifically binds to a human Kv1.3 protein comprising: an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the variable region of said light chain comprises: (i) a CDR1 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-69; 249-267; 381-386; (ii) a CDR2 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 70-88; 268-280; 387-391; and/or (iii) a CDR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-112; 281-297; 392-398.

In another aspect, the invention provides an antibody that specifically binds to a human Kv1.3 protein comprising: an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the variable region of said heavy chain comprises: (i) a CDR1 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 165-177; 317-334; 409-415; (ii) a CDR2 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 178-202; 335-352; 416-422; and/or (iii) a CDR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 203-229; 353-370; 423-429.

In another aspect, the invention provides an antibody that specifically binds to a human Kv1.3 protein comprising: an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the variable region of said light chain comprises: (i) a CDR1 region comprising an amino acid sequence selected from the group consisting of VL CDR1 Motifs 1-6; (ii) a CDR2 region comprising an amino acid sequence selected from the group consisting of VL CDR2 Motifs 1-6; and/or (iii) a CDR3 region comprising an amino acid sequence selected from the group consisting of VL CDR3 Motifs 1-6.

In another aspect, the invention provides an antibody that specifically binds to a human Kv1.3 protein comprising: an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the variable region of said heavy chain comprises: (i) a CDR1 region comprising an amino acid sequence selected from the group consisting of VH CDR1 Motifs 1-6; (ii) a CDR2 region comprising an amino acid sequence selected from the group consisting of VH CDR2 Motifs 1-6; and/or (iii) a CDR3 region comprising an amino acid sequence selected from the group consisting of VH CDR3 Motif 1-6.

In some embodiments, the invention provides an antibody preparation comprising an antibody as described herein. In some embodiments, the invention provides an antibody preparation wherein said preparation is a monoclonal antibody preparation. In some embodiments, the invention provides an antibody preparation wherein said preparation is a mixture of at least two monoclonal antibody preparations.

In another aspect, the invention provides an isolated nucleic acid molecule encoding a heavy chain or light chain of any one of the antibodies as described herein. In some embodiments, the invention provides an isolated nucleic acid molecule wherein said nucleic acid molecule is selected from the group consisting of a cloning vector, an expression vector, a heterologous recombination vector and a viral integration vector. In some embodiments, the invention provides a cell transformed with the nucleic acid. In some embodiments, said cell is a mammalian cell. In some embodiments, said cell is a rodent cell. In some embodiments, said cell is a Chinese Hamster Ovary (CHO) cell. In some embodiments, said cell is a human cell.

In another aspect, the invention provides a method of isolating a cell expressing a Kv1.3 protein comprising: (a) obtaining a population of cells; (b) contacting the population of cells with a multiplicity of antibodies as described herein; and (c) separating cells in the population that specifically bind the antibodies from cells in the population that do not specifically bind the antibodies. In some embodiments, the cells are separated by fluorescence activated cell sorting. In some embodiments, the cells are separated using an immobilized secondary antibody by fluorescence activated cell sorting.

In another aspect, the invention provides a method for preventing or treating an autoimmune disorder in humans a subject comprising administering to the subject a therapeutically effective amount of the antibody preparation described herein. In some embodiments, the antibody preparation inhibits Kv1.3 potassium channels, thereby preventing or treating the autoimmune disorder. In some embodiments, autoreactive effector memory T cells of the subject are depleted, thereby preventing or treating the autoimmune disorder. In some embodiments, the autoimmune disorder is selected from the group of: Multiple sclerosis; Myasthenia gravis; Autoimmune neuropathies; Guillain-Barre Syndrome; Autoimmune uveitis; Crohn's Disease; Ulcerative colitis; Primary biliary cirrhosis; Autoimmune hepatitis; Autoimmune thrombocytopenia; Type-1 diabetes mellitus; Addison's Disease; Grave's Disease; Hashimoto's thyroiditis; Autoimmune orchitis; Behcet's Disease; Rheumatoid arthritis; Bone resorption associated with periodontal disease; Systemic lupus erythematosus; Scleroderma Polymyositis, dermatomyositisis; Pemphigus vulgaris; Spondyloarthropathies; Ankylosing spondylitis; and Sjogren's syndrome.

In another aspect, the invention provides a method for preventing or treating graft versus vs host disease in a subject said method comprising administering to the subject a therapeutically effective amount of an antibody preparation described herein. In some embodiments, the antibody preparation inhibits Kv1.3 potassium channels, thereby preventing or treating graft versus host disease. In some embodiments, autoreactive effector memory T cells of the subject are depleted, thereby preventing or treating graft versus host disease.

These and other aspects and embodiments of the invention are illustrated and described below. Other compositions, methods and features will become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional compositions and methods and features are within the scope of the present invention.

DETAILED DESCRIPTION

The present disclosure relates to isolated antibodies (Abs), particularly Abs that bind specifically to human Kv1.3 with high affinity and Abs that modulate Kv1.3 functional activity. In certain embodiments, the anti-Kv1.3 Abs are derived from particular heavy and light chain sequences and/or comprise particular structural features, such as CDR regions, comprising particular amino acid sequences. This disclosure provides isolated anti-Kv1.3 Abs, methods of making such anti-Kv1.3 Abs, immunoconjugates and bispecific molecules comprising such anti-Kv1.3 Abs, and methods of expressing such anti-Kv1.3 Abs. This disclosure also relates to methods of using the anti-Kv1.3 Abs as therapeutic treatment for auto-immune diseases or as diagnostic reagents.

Definitions

All scientific and technical terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent or later-developed techniques which would be apparent to one of skill in the art. In addition, in order to more clearly and concisely describe the subject matter which is the invention, the following definitions are provided for certain terms which are used in the specification and appended claims.

The term “antibody” or abbreviation “Ab,” as used herein, includes whole antibodies and any antigen binding fragment (i.e., “antigen-binding portion”) or single chains thereof, with or without native glycosylation. A complete “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds or an antigen binding portion thereof. Each heavy chain includes a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2, and CH3. Each light chain includes a light chain variable region (VL) and a light chain constant region with one domain, CL. The VHand VLregions can be further subdivided into complementarity determining regions (CDR) and framework regions (FR). The VHand VLregions each include three CDRs, designated CDR1, CDR2 and CDR3, that interact with an antigen (e.g., Kv1.3).

The term “antigen-binding portion” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., Kv1.3). Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include a Fab fragment, F(ab′)2fragment, Fab′ fragment, Fd fragment, Fv fragment, scFv fragment, dAb fragment, and an isolated CDR.

The term “monoclonal antibody” or “monoclonal antibody preparation,” as used herein, refers to a preparation of antibody molecules consisting essentially of antibodies having a single heavy chain amino acid sequence and a single light chain amino acid sequence (but which may have heterogeneous glycosylation).

The term “humanized antibody,” as used herein, includes antibodies having constant region and variable region framework regions (FRs) but not CDRs derived from human germline immunoglobulin sequences.

The term “recombinant antibody,” as used herein, includes all antibodies prepared, expressed, created, or isolated by recombinant means. In certain embodiments, recombinant antibodies are isolated from a host cell transformed to express the antibody (e.g., from a transfectoma). In other embodiments, recombinant antibodies are isolated from a recombinant, combinatorial antibody library, such as a phage display library. Recombinant antibodies may also be prepared, expressed, created, or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.

The term “isotype,” as used herein, refers to the heavy chain class (e.g., IgA, IgD, IgE, IgG, and IgM for human antibodies) or light chain class (e.g., kappa or lambda in humans) encoded by the constant region genes. The term “subtype” refers to subclasses within the subtype (e.g., IgA1, IgA2, IgG1, IgG2, IgG3, IgG4in humans).

The phrase “an antibody specific for” a specified antigen is used interchangeably herein with the phrase “an antibody which specifically binds to” a specified antigen. As used herein, the term “Ka” refers to the association rate and the term “Kd” to the dissociation rate of a particular antibody-antigen complex. The term “KD” refers to the dissociation constant, which is obtained from the ratio of Kdto Kaand expressed as a molar concentration (M). According to some embodiments, an antibody that “specifically binds to human Kv1.3” is intended to refer to an antibody that binds to human Kv1.3 with a KDof 5×10−8M or less, more preferably 1×10−8M or less.

The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued U.S. patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

The invention provides a variety of new antibodies with high affinity against the human Kv1.3 protein, particularly epitopes on the extracellular loops and which modulate the activity of Kv1.3 function. The antibodies may comprise the complete VH and VL regions disclosed herein, or may comprise only the CDR sequences disclosed herein in combination with known human or other mammalian (e.g., human) or avian (e.g., chicken) framework regions. In addition, based upon specific CDR sequences disclosed herein, sequence motifs for consensus CDR sequences are provided, and antibodies that comprise CDR sequences defined by these motifs, in combination with known human or other mammalian or avian framework regions, are also provided. Furthermore, possible canonical structures for each CDR of the VL and VH regions are assigned, and antibodies that comprise CDRs belonging to the disclosed structural motifs, in combination with known human or other mammalian or avian framework regions, are also provided.

The CDR sequences of the invention (including both the CDRs disclosed inFIGS.11-22and the CDRs defined by the sequence motifs disclosed herein) can be combined with other immunoglobulin sequences according to methods well known in the art to produce immunoglobulin molecules with antigen-binding specificity determined by the CDRs of the invention.

In some embodiments, the CDRs of the invention are combined with framework region (FR) and constant domain (CH or CL) sequences from other antibodies. For example, although some of the CDRs disclosed herein are derived from chicken B cells and have chicken FR and constant domain sequences, they can be recombined with human or other mammalian or avian FR and constant domain sequences to produce humanized or other recombinant antibodies. Similarly, CDRs disclosed herein that are derived from llamas can be recombined with human or other mammalian constant domain sequences to produce humanized or other recombinant antibodies. The production of such recombinant antibodies is well known to those of skill in the art and requires only routine experimentation.

The type of constant regions included in such recombinant antibodies can be chosen according to their intended use. For example, if the antibodies are intended for therapeutic use to target Kv1.3-expressing cells for destruction, heavy chain constant domains (i.e., Fc regions) of IgG subtypes can be used. If the antibodies are intended only as reagents for labeling cells (e.g., for fluorescence-activated cell sorting (FACS)), a complete antibody, antigen binding fragment (Fab), single-chain variable fragment (scFV), single domain antibody (sdAb) or even non-antibody immunoglobulin molecule (e.g., an MHC receptor extracellular domain) can be used with the CDRs of the invention.

The CDRs of the invention can be selected independently such that the CDR1, CDR2 and CDR3 sequences of a given variable light (VL) chain or variable heavy (VH) chain can be chosen from different original VL and VH chains, from different VL and VH CDR motifs, or from a combination of the disclosed CDRs and motifs. However, sequences for light chain CDRs should be selected from the disclosed VL CDRs or VL CDR motifs, and sequences for heavy chain CDRs should be selected from the disclosed VH CDRs or VH CDR motifs. Similarly, the sequences for CDR1 regions should be selected from the disclosed CDR1 or CDR1 motif sequences, the sequences for CDR2 regions should be selected from the disclosed CDR2 or CDR2 motif sequences, and the sequences for CDR3 regions should be selected from the disclosed CDR3 or CDR3 motif sequences, for VL or VH chains as appropriate.

In certain aspects, the invention provides a Kv1.3 binding antibody or antigen binding portion thereof with the binding specificity of any one of the antibodies described inFIGS.11-22.

In certain aspects, the invention provides a Kv1.3 binding antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises a VH chain that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH chain of an antibody fromFIG.13,17, or21and a VL chain that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL chain of an antibody fromFIG.11,15, or19.

In certain aspects, the invention provides a Kv1.3 binding antibody or antigen binding portion thereof wherein the antibody or antigen binding portion thereof comprises the VH chain of an antibody fromFIG.13,17, or21and the VL chain of an antibody fromFIG.11,15, or19.

In certain embodiments, the antibody or antigen binding portion thereof is fully humanized and recombinantly produced. In certain embodiments, the antibody or antigen binding portion thereof is not naturally occurring. In certain embodiments, the antibody comprises the pairing of VH and VL chains as isolated from an animal immunized with Kv1.3. In certain embodiments, the antibody comprises the pairing of VH and VL chains as isolated from an animal immunized with Kv1.3, wherein the Fc portion of the antibody is not the isotype or portion of the pair of VH and VL chains as isolated from the immunized animal. In certain embodiments, the antibody comprises a pairing of VH and VL chains, wherein the VH or VL individually could be isolated from the immunized animal. In some embodiments, the antibody comprises VH chain or CDRs of a VH chain of one clonal cell line, and VL or CDRs of another clonal cell line. In certain embodiments, the antibody comprises the pairing of VH and VL chains as isolated from an animal immunized with Kv1.3 modified by substituting one or more amino acids.

In certain embodiments, the antibody or antigen binding portion thereof comprises a VH which comprises the CDR1, CDR2, and CDR3 of an antibody fromFIG.13,17, or21. In certain embodiments, the antibody or antigen binding portion thereof comprises a VL which comprises the CDR1, CDR2, and CDR3 of an antibody fromFIG.11,15, or19.

In certain embodiments, the antibody or antigen binding portion thereof comprises a VH which comprises the CDR1, CDR2, and CDR3 of an antibody fromFIG.13,17, or21and further comprises the complementary VL which comprises the CDR1, CDR2, CDR3 of an antibody fromFIG.11,15, or19.

In certain aspects, the invention provides a pharmaceutical composition comprising any one of the antibodies of the invention or antigen binding portion thereof or any combination thereof. In certain aspects, the invention provides pharmaceutical compositions including any one of the antibodies of the invention or antigen binding portion thereof and a pharmaceutically acceptable carrier.

Comparing the sequences of the antibodies and their affinity and inhibition of Kv1.3, a skilled artisan can readily determine sequence identity, compare sequence length and determine the percent sequence identity and/or changes, including percent sequence identity and/or changes in the VH and VL sequences, including percent sequence identity and/or changes in the CDRs, as well as the specific positions and types of substitutions which can be tolerated while affinity and inhibition of Kv1.3 is maintained.

Methods of Using Anti-Kv1.3 Antibodies

The anti-Kv1.3 antibodies of the invention can be used in standard methods of immunoaffinity purification, immunohistochemistry and immunotherapy, but with specific application to cells and tissue expressing the Kv1.3 protein.

For example, the anti-Kv1.3 antibodies of the invention can be used to isolate cells expressing Kv1.3 from a mixed population of cells including only a fraction of cells that express Kv1.3. For example, individual cells can be subjected to techniques such as FACs using fluorescently-labeled anti-Kv1.3 antibodies or immunoaffinity purification using immobilized anti-Kv1.3 antibodies.

Similarly, immobilized anti-Kv1.3 antibodies can be used for purification of Kv1.3 protein from lysates derived from Kv1.3 expressing cells. Kv1.3 protein can be purified while remaining associated with cell membrane fragments or following dissociation from biological membranes after, for example, treatment with a variety of detergents. Additionally, Kv1.3 can be purified in such a manner while in association with small molecules or biologics (e.g., peptides, mAbs, etc.) that specifically bind Kv1.3. Such purified Kv1.3 preparations will have utility in techniques used for determining structural information regarding Kv1.3 both with and without bound molecules (e.g., crystallography, cryoEM). Furthermore, such preparations will have utility in screening a variety of libraries (e.g., small molecule, mAb) for molecules that specifically interact with Kv1.3.

Alternatively, immunohistochemistry may be performed using the anti-Kv1.3 antibodies of the invention to identify cells or tissues expressing Kv1.3 and/or to quantify Kv1.3 expression in such cells.

In addition, the anti-Kv1.3 antibodies of the invention can be used therapeutically to target Kv1.3-expressing cells, particularly TEMcells, and/or to inhibit the function of Kv1.3 in such cells. Additionally, the anti-Kv1.3 antibodies of the invention that bind to Kv1.3 and that may or may not inhibit Kv1.3 activity may deplete target Kv1.3 expressing cells, particularly TEMcells, via cytotoxic Fc mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Furthermore, anti-Kv1.3 antibodies of the invention conjugated to moieties that inhibit Kv1.3 function can also be used therapeutically. Antibody-drug conjugates of the anti-Kv1.3 antibodies of the invention can also be used to deliver therapeutic drugs to Kv1.3-expressing cells.

In autoimmune diseases, specific autoreactive T cells can undergo differentiation into chronically activated memory T cells that contribute to pathogenesis by migrating to inflamed tissues and secreting cytokines. Although not bound by any mechanism of action, in some embodiments, blocking Kv1.3 activity may alter the phenotype of T cells in response to an antigen and convert T cells to a suppressive state that is beneficial in treating autoimmune disease. In chronic autoimmune diseases, there is clonal expansion of T effector memory (TEM) cells. It has been shown that in cells with a Kv1.3 loss of function mutation, T central memory (TCM) cells fail to differentiate into T effector memory (TEM) cells, and TEM cells even revert back into TCM cells. (Hu et al. (2012), J. of Biological Chemistry, 287(2), 1261-68). Thus, in some embodiments, the antibody or antigen binding portion thereof inhibits Kv1.3 function to treat autoimmune immune disorders. Accordingly, the Kv1.3 binding antibodies or antigen binding portion thereof of the invention can be used in methods for preventing or treating an autoimmune disorder in a subject. The method comprises administering to the subject a therapeutically effective amount of an antibody or antigen binding portion thereof as described herein. In some embodiments, the antibody or antigen binding portion thereof inhibits Kv1.3 potassium channels. In some embodiments, the autoreactive effector memory T cells of the subject are depleted or reduced. In some embodiments, the subject is suspected of or evaluated for having an autoimmune disease. In some embodiments, the subject is a human.

Graft versus host disease (GvHD) can occur following the transplantation of tissue from a donor to a recipient. GvHD is caused by recipient reactive T-cells from the donor organ, which recognize and destroy MHC-mismatched host tissues. Accordingly, the Kv1.3 binding antibodies or antigen binding portion thereof of the invention can be used in methods for preventing or treating GvHD in a subject. The method comprises administering to the subject a therapeutically effective amount of an antibody or antigen binding portion thereof as described herein. In some embodiments, the antibody or antigen binding portion thereof inhibits Kv1.3 potassium channels. In some embodiments, the autoreactive effector memory T cells of the subject are depleted or reduced. In some embodiments, the subject has received an organ transplant from a non-genetically identical donor. In some embodiments, the subject is a human.

In some embodiments, the Kv1.3 binding antibodies or antigen binding portion thereof of the invention can be used in methods for inhibiting Kv1.3 potassium channels in a subject. The method comprises administering to the subject the antibody described herein in an amount that is effective in inhibiting Kv1.3 potassium channels. In some embodiments, the subject is a human.

Nucleic Acid Molecules Encoding Anti-Kv1.3 Antibodies

The invention also provides nucleic acid molecules encoding the anti-Kv1.3 antibodies of the invention. Such nucleic acids can be designed using standard tables for the universal genetic code to choose codons that will encode the desired amino acid sequence, or specialized codon tables can be used that reflect codon biases characteristic of different organisms. Thus, for example, to optimize expression of the anti-Kv1.3 antibodies of the invention in CHO cells, a nucleic acid encoding the desired antibody can be designed using a codon table optimized for CHO cells.

The nucleic acids encoding the anti-Kv1.3 antibodies of the invention can be included in a wide variety of vectors known in the art, including cloning vectors (e.g., bacterial or mammalian cloning vectors), transformation vectors (e.g., homologous recombination, viral integration or autonomously replicating vectors) and expression vectors (e.g., high copy number, inducible or constitutive mammalian expression vectors).

Cells Expressing Anti-Kv1.3 Antibodies

Also provided are host cells expressing heterologous sequences encoding the anti-Kv1.3 antibodies of the invention. Such host cells can be useful for commercial production of the anti-Kv1.3 antibodies of the invention, and can be produced by transforming appropriate host cells with expression vectors described above.

In some embodiments the invention provides mammalian cells, including CHO cells, expressing the anti-Kv1.3 antibodies of the invention. However, those of skill in the art can express the antibodies in a variety of host cells, including bacterial, yeast, insect and mammalian systems. See, e.g., Verma et al. (1998),J. Immunol. Methods216(1-2):165-81, incorporated by reference in its entirety herein.

Pharmaceutical Compositions

In certain aspects, the invention provides a pharmaceutical composition comprising an antibody described herein wherein the composition is used for therapeutic purposes such as but not limited to treatments and/or prevention of autoimmune diseases or GvHD. In certain aspects, the invention provides a pharmaceutical composition comprising an antibody described herein in combination with any other suitable antibody or composition for treating and/or preventing autoimmune diseases or GvHD. In certain embodiments, the pharmaceutical compositions comprise one or more nucleic acids that encode the antibodies described herein. In certain embodiments, these nucleic acids can be expressed by any suitable vector for expression of antibodies.

Various methods to make pharmaceutical compositions are known in the art and are contemplated by the invention. In some embodiments, the compositions include excipient suitable for a biologic molecule such as the antibodies described herein. In some embodiments, the antibodies could be produced in specific cell lines and conditions so as to control glycosylation of the antibody.

In certain aspects, the invention provides that the antibodies or antigen binding portion thereof described herein can be formulated as a composition (e.g., a pharmaceutical composition). Suitable compositions can comprise the antibody or antigen binding portion thereof dissolved or dispersed in a pharmaceutically acceptable carrier (e.g., an aqueous medium). The compositions can be sterile and can be administered by intravenous, e.g., as a bolus or by continuous infusion. The administration can be by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral tropical, or by inhalation. The antibody or antigen binding portion thereof can also be formulated as a composition appropriate for topical administration to the skin or mucosa. Such compositions can take the form of liquids, ointments, creams, gels and pastes. Standard formulation techniques can be used in preparing suitable compositions.

The antibodies or antigen binding portion thereof described herein can be administered to subjects with autoimmune disease and/or with graft versus host disease and used to kill T cells by virtue of the antibodies or antigen binding portion thereof binding to the surface of T cells expressing Kv1.3 cells.

Suitable dose ranges can depend on the antibody or antigen binding portion thereof and on the nature of the formulation and route of administration. Optimum doses can be determined by one skilled in the art without undue experimentation.

EXAMPLES

Preparation of Kv1.3 Immunogen and B-Cell and Phage Screening Reagents

The gene encoding human Kv1.3 protein (FIG.1A; SEQ ID NO:1) was optimized for expression inTetrahymena thermophila. The optimized Kv1.3 gene was further modified by the incorporation of nucleotides encoding dual affinity FLAG (DYKDDDDK) and 10× His tags at the Kv1.3 C-terminus (FIG.1B: SEQ ID NO:2). The optimized Kv1.3 gene was synthesized and cloned into an expression cassette placing control of Kv1.3 gene expression under an inducibleTetrahymena thermophilametallothionein promoter (FIG.2). The entire expression cassette containing the Kv1.3 gene was subsequently cloned into aTetrahymena thermophilahigh copy number ribosomal DNA expression vector, pTRAS1 (FIG.2; U.S. Pat. No. 8,664,374). MatingTetrahymenacells were transformed with Kv1.3 containing pTRAS1 and viable transformants were selected in media containing a selective agent for transformants. Cells expressing Kv1.3 following induction of gene expression were selected for preparation of Kv1.3 immunogen. Cultures (>1 L) of transformantTetrahymenacells were grown and induced to express Kv1.3, harvested, and lysed by microfluidization. Membrane fractions were collected by centrifugation and then frozen for subsequent purification.

Kv1.3 was extracted fromTetrahymenamembranes in buffer containing Fos-Choline detergent and subsequently purified by NiNTA chromatography. Purified Kv1.3 was reconstituted into liposomes consisting of 10 mg/ml phosphatidylcholine to produce Kv1.3 proteoliposomes.FIG.3shows SDS-PAGE and Western analysis of Kv1.3 proteoliposome immunogen samples.

Antibody Generation

Chicken Derived Antibodies:

Kv1.3 proteoliposomes were used to immunize chickens, and to increase the immune response through boosting. Following a period of increased specific anti-Kv1.3 antibody titer in the sera, animals were sacrificed and splenocytes harvested. Table 1 shows anti-Kv1.3 antibody titer results. Specificity was determined by comparing ELISA signals in wells coated with either Kv1.3 or a non-related ion channel, Nav1.8. Additionally,FIG.4shows specific anti-Kv1.3 antibody titer in sera by FACS analysis using Kv1.3 coated magnetic beads. No specific signal was observed by FACS using Kv1.3 magnetic beads from sera derived from animals immunized with a non-related ion channel, i.e., Nav1.8. Splenic B cells producing anti-Kv1.3 antibody were identified by fluorescence using a GEM assay (U.S. Pat. Nos. 8,030,095 and 8,415,173) incorporating either Kv1.3 proteoliposomes or Kv1.3 attached to magnetic beads (FIGS.5Aand B).

Variable light and heavy chain genes from individual B cells identified by GEM assay were amplified by PCR and cloned into a mammalian expression vector as fusions to human IgG1 Fc to generate genes encoding bivalent scFv-Fc antibodies (FIG.6). Antibodies were expressed in HEK293 cells and supernatants assayed by ELISA to confirm specific binding to Kv1.3 compared to a non-related ion channel (Nav1.8). Table 2 below shows ELISA results of cloned scFv-Fc antibodies identified in GEM assays using Kv1.3 proteoliposome. Table 3 below shows ELISA results of cloned scFv-Fc antibodies identified in GEM assays using Kv1.3 magnetic beads.

TABLE 2ELISA analysis of anti-Kv1.3 antibodies derived from chickens.19724p1.A119724p1.A1119724p1.A519724p1.A919724p1.B119724p1.B11Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8501.2310.0721.2090.071.080.0541.240.0741.1190.0681.1340.0742501.4960.0521.4820.051.1690.0561.4860.0531.3750.0641.2350.05712501.2160.0751.2770.0591.1950.0661.4040.0671.3820.0650.1090.06762501.1010.0551.1560.0671.2240.0610.940.0641.3080.0570.950.0519724p1.C1219724p1.C419724p1.D1119724p1.D219724p1.D819724p1.E2Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8501.3370.061.0960.061.1810.0590.860.0621.0530.0651.2780.0612500.1030.0531.0290.071.1160.0631.0040.0471.2070.0481.430.04712501.5260.0831.2030.0561.0780.0720.9040.061.0660.0621.5330.05362501.0750.0511.1050.0510.8690.0570.6380.0740.6930.0581.2830.06219724p1.E319724p1.F319724p1.F619724p1.F719724p1.F819724p1.F9Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8501.0290.0611.1990.0771.3850.0721.3570.081.8021.4351.3990.0652501.3490.0531.2680.0541.3210.0661.3720.0641.4871.0071.3590.0512501.220.0511.1940.0531.5120.0671.3580.0611.1130.4361.3920.06262500.830.0680.9450.0751.2390.0681.3530.0680.5230.1831.3170.07219724p1.G619724p1.H1219724p1.H219724p1.H419724p1.H719724p1.B5 (neg)Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8501.640.0591.2910.0771.3280.0641.3440.0621.3580.0710.0780.0532501.540.0561.4240.091.4660.0851.2530.0661.2670.0650.0820.06712501.3070.0511.1670.0811.3970.0831.1220.0771.1490.080.0730.0862501.1550.0621.2130.0831.2020.0860.8110.0941.0090.0830.0880.08419724p1.E6 (neg)MockKv1.3Nav1.8Kv1.3Nav1.8500.0770.060.0710.042500.0740.0740.0710.05312500.0770.0770.0660.06762500.0840.0840.090.081

TABLE 3ELISA analysis of anti-Kv1.3 antibodies derived from chickens.19724p2.A219724p2.A319724p2.A519724p2.A719724p2.B519724p2.C4Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8500.6210.0390.9720.0580.6930.0620.7950.0420.5770.070.8760.0522500.4590.0510.9450.0530.7470.0590.7810.0510.3610.0520.8290.05112500.2190.0550.9310.0510.5840.0550.5680.0470.1490.0510.8280.04462500.0830.0540.6710.0530.290.0550.2780.0560.0610.0530.4120.06519724p2.D119724p2.D219724p2.D919724p2.E619724p2.F719724p2.G9Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8500.9510.0550.770.0560.8130.0560.8780.0710.7010.0610.9550.0592500.8530.0530.5010.0540.9740.0630.9210.0630.630.0610.9940.07312500.9140.0460.2540.0510.7530.0650.9340.0630.5010.0540.9190.07462500.6710.0530.1320.0560.5570.060.590.0630.3030.0660.6380.0719724p2.H1019724p2.H1219724p2.H419724p2.H6Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8Kv1.3Nav1.8500.8530.0490.5420.0460.5980.0570.9120.0652500.8820.0610.4770.0490.5060.0630.9660.05912500.8930.0640.3630.0620.2850.050.990.05462500.1730.0510.1460.0520.1360.0640.770.052

Llamas were immunized with DNA encoding human Kv1.3 and boosted with Kv1.3 proteoliposomes. Once specific anti-Kv1.3 antibody titers were detected phage libraries were constructed and phage panned using Kv1.3 magnetic beads. Positive clones were sequenced and purified scFv-Fc antibodies were confirmed to specifically bind to Kv1.3 by ELISA.

Analysis of Anti-Kv1.3 Antibodies

Anti-Kv1.3 antibody clones were tested (i) for their ability to bind native Kv1.3 on a human T-cell line, and/or (ii) their ability to modulate the functionality of Kv1.3 activity (Tables 4 and 7).

The human leukemic T cell line Jurkat expresses Kv1.3 on the cell surface (Gasiorowska et al. (2012),Cell Mol Biol Lett.17:559-570). The ability of each of the GEM discovered antibodies to bind Jurkat cells was analyzed by FACS. Table 4 shows that six clones (19724p1.A11, 19724p1.D8, 19724p1.H7, 19724p1.E6, 19724p2.A3, 19724p2.G9) bound Jurkat cells with signals significantly higher (average signal approximately 3,370) than background levels associated with other antibodies (average signal approximately 193). Additionally three clones (19724p2.D2, 19724p2.D9 and 19724p2.E6) show signals that are slightly higher than background levels (average signal approximately 314). Of the antibodies that are FACS positive for Jurkat binding, one clone (19724p1.E6) does not show binding to Kv1.3 by ELISA. This result may indicate that this antibody recognizes a conformational dependent Kv1.3 epitope that is maintained in the Kv1.3 proteoliposomes used for the initial GEM selection and in native Kv1.3 channels present on the Jurkat cell surface, but that is lost during the ELISA procedure, presumably when Kv1.3 is bound to the wells of the ELISA plate.

Anti-Kv1.3 antibodies were tested for inhibition of human Kv1.3 channels transiently expressed in L929 human fibroblast cells. Cells were plated on cover-slips coated with poly-L-lysine 24 hours post-transfection for whole-cell patch clamp using an EPC-10 HEKA amplifier. Control currents were recorded in normal Ringers solution containing: 160 mM NaCl, 4.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES (adjusted to pH 7.4 and 290-310 mOsm). Patch pipettes were pulled from soda lime glass (micro-hematocrit tubes, Kimble Chase, Rochester, N.Y.) to a resistance of 2-3 MΩ and filled with an internal pipette solution containing: 45 mM KF, 2 mM MgCl2, 10 mM HEPES, 10 mM EGTA (pH 7.2, 290-310 mOsm). Currents were recorded using depolarizing pulses to 40 mV applied every 30 seconds for 200 milliseconds. Antibodies were freshly diluted in normal Ringers solution immediately prior to bath perfusion. Cell capacitance, a direct measure of cell surface area, and access resistance were continuously monitored during recordings to ensure minimal current rundown.FIG.7shows that ten antibody clones (chicken antibodies p1A11, p1D8, p1H7, P1E6, p2A3, p2G9, p1A1, p1F8, p1H4 and llama antibody L1A3) functionally inhibit Kv1.3 activity. Dose-response analysis shows that the most potent antibodies have IC50values of 6 nM (p1E6), 46 nM (p2G9) and 109 nM (L1A3) (FIG.8). Additionally, antibodies have shown selective inhibition of Kv1.3 over other Kv family members (FIG.9) and inhibition of Kv1.3 in activated rhesus monkey T cells indicating that they will demonstrate similar activity against Kv1.3 in human T cells (FIG.10).

Antibody Sequence Analyses

Antibodies Derived from Chickens:

B-cell clones producing potentially useful anti-Kv1.3 antibodies were subjected to DNA sequencing and the corresponding amino acid sequences of light and heavy chain variable domains were deduced. Sequences are disclosed for forty antibodies derived from the GEM screen described above.

Variable Light Chain Sequences

Alignments of all of the VL sequences described above are shown inFIGS.11A-11B. The figure indicates the approximate locations of the three CDR regions (bold, underscore) and the SEQ ID NO corresponding to each sequence.

Unique VL CDR Sequences.

Alignments of the unique CDR sequences of the VLs ofFIGS.11A-11Bare shown inFIG.12. Of the 40 VL sequences, there are 27 unique CDR1 sequences, 19 unique CDR2 sequences and 24 unique CDR3 sequences, as shown inFIG.12.

Based on the sequences disclosed inFIG.12, as well as structure/function characteristics of the naturally occurring amino acids, consensus sequences for the VL CDRs can be determined.

One consensus sequence is VL CDR1 Motif 1:

Noting in particular that the VL CDR1 sequences of SEQ ID NOs: 3, 21, 22, 23, 24, 37, 38, 39 and 42 are derived from antibodies that inhibit Kv1.3 function distinct from the others inFIGS.11A-11B, an alternative consensus sequence is VL CDR1 Motif 2:

Noting in particular that the VL CDR1 sequences of SEQ ID NO: 21, 22, 23, 24, 39 and 42 are derived from antibodies that are FACS positive for Jurkat cell binding distinct from the others inFIGS.11A-11B, an alternative consensus sequence is VL CDR1 Motif 3:

Noting in particular that the VL CDR1 sequence of SEQ ID NO: 42 is derived from an antibody that may recognize a conformational Kv1.3 epitope distinct from the others inFIG.11A-11B, an alternative consensus sequence is VL CDR1 Motif 4:

For the VL CDR2, one consensus sequence is VL CDR2 Motif 1:

Noting in particular that the VL CDR2 sequences of SEQ ID NOs: 3, 21, 22, 23, 24, 37, 38, 39 and 42 are derived from antibodies that inhibit Kv1.3 function distinct from the others inFIGS.11A-11B, an alternative consensus sequence is VL CDR2 Motif 2:

Noting in particular that the VL CDR2 sequences of SEQ ID NO: 21, 22, 23, 24, 39 and 42 are derived from antibodies that are FACS positive for Jurkat cell binding distinct from the others inFIGS.11A-11B, an alternative consensus sequence is VL CDR2 Motif 3:

Noting in particular that the VL CDR2 sequence of SEQ ID NO: 42 is derived from an antibody that may recognize a conformational Kv1.3 epitope distinct from the others inFIGS.11A-11B, an alternative consensus sequence is VL CDR2 Motif 4:

For the VL CDR3, one consensus sequence is VL CDR3 Motif 1:

Noting in particular that the VL CDR3 sequences of SEQ ID NOs: 3, 21, 22, 23, 24, 37, 38, 39 and 42 are derived from antibodies that inhibit Kv1.3 function distinct from the others inFIGS.8A-8B, an alternative consensus sequence is VL CDR3 Motif 2:

Noting in particular that the VL CDR3 sequences of SEQ ID NO: 21, 22, 23, 24, 39 and 42 are derived from antibodies that are FACS positive for Jurkat cell binding distinct from the others inFIGS.11A-11B, an alternative consensus sequence is VL CDR3 Motif 3:

Noting in particular that the VL CDR3 sequence of SEQ ID NO: 42 is derived from an antibody that may recognize a conformational Kv1.3 epitope distinct from the others inFIGS.11A-11B, an alternative consensus sequence is VL CDR3 Motif 4:

Variable Heavy Chain Sequences

Alignments of all of the VH sequences described above are shown inFIGS.13A-13C. The figures indicate the approximate locations of the three CDR regions (bold, underscore) and the SEQ ID NO corresponding to each sequence.

Unique VH CDR Sequences.

Alignments of the unique CDR sequences of the VHs ofFIGS.13A-13Care shown inFIG.14. Of the 40 VH sequences, there are 13 unique CDR1 sequences, 25 unique CDR2 sequences and 27 unique CDR3 sequences, as shown inFIG.14.

Based on the sequences disclosed inFIG.14, as well as structure/function characteristics of the naturally occurring amino acids, consensus sequences for the VH CDRs can be determined.

For the VH CDR1, one consensus sequence is VH CDR1 Motif 1:

Noting in particular that the VH CDR1 sequence of SEQ ID NOs: 125, 129, 130, 131, 132, 133, 141, 159 and 160 are derived from antibodies that inhibit Kv1.3 function distinct from the others inFIGS.13A-13C, an alternative consensus sequence is VH CDR1 Motif 2:

Noting in particular that the VH CDR1 sequences of SEQ ID NOs: 129, 130, 131, 132, 133 and 141 are derived from antibodies that are FACS positive for Jurkat cell binding distinct from the others inFIGS.13A-13C, an alternative consensus sequence is VH CDR1 Motif 3:

Noting in particular that the VH CDR1 sequence of SEQ ID NO: 129 is derived from an antibody that may recognize a conformational Kv1.3 epitope distinct from the others inFIGS.10-10C, an alternative consensus sequence is VH CDR1 Motif 4:

For the VH CDR2, one consensus sequence is VH CDR2 Motif 1:

Noting in particular that the VH CDR2 sequence of SEQ ID NOs: 125, 129, 130, 131, 132, 133, 141, 159 and 160 are derived from antibodies that inhibit Kv1.3 function distinct from the others inFIGS.13A-13C, an alternative consensus sequence is VH CDR2 Motif 2:

Noting in particular that the VH CDR2 sequences of SEQ ID NOs: 129, 130, 131, 132, 133 and 141 are derived from antibodies that are FACS positive for Jurkat cell binding distinct from the others inFIGS.13A-13C, an alternative consensus sequence is VH CDR2 Motif 3:

Noting in particular that the VH CDR2 sequence of SEQ ID NO: 129 is derived from an antibody that may recognize a conformational Kv1.3 epitope distinct from the others inFIGS.13A-13C, an alternative consensus sequence is VH CDR2 Motif 4:

For the VH CDR3, one consensus sequence is VH CDR3 Motif 1:

Noting in particular that the VH CDR3 sequence of SEQ ID NOs: 125, 129, 130, 131, 132, 133, 141, 159 and 160 are derived from antibodies that inhibit Kv1.3 function distinct from the others inFIGS.13A-13C, an alternative consensus sequence is VH CDR3 Motif 2:

Noting in particular that the VH CDR3 sequences of SEQ ID NOs: 129, 130, 131, 132, 133 and 141 are derived from antibodies that are FACS positive for Jurkat cell binding distinct from the others inFIGS.13A-13C, an alternative consensus sequence is VH CDR3 Motif 3:

Noting in particular that the VH CDR3 sequence of SEQ ID NO: 129 is derived from an antibody that may recognize a conformational Kv1.3 epitope distinct from the others inFIGS.10A-10C, an alternative consensus sequence is VH CDR3 Motif 4:

CDR Canonical Structures

Prediction of CDR structures in light and heavy chain variable regions is based on the work of Chothia and colleagues (e.g., Chothia et al. (1987),J. Mol. Biol.196:901-17; Al-Lazikani et al. (1997),J. Mol. Biol.273:927-48), and focuses on conserved canonical CDR structures displayed by immunoglobulin hypervariable regions (North et al. (2011),J. Mol. Biol.406(2):228-56). To determine potential canonical structures associated with the disclosed anti-Kv1.3 antibodies derived from chickens (Table 4), light and heavy chain variable regions from each anti-Kv1.3 clone were submitted for sequence analysis in the “SAbDAb” structural antibody database (Dunbar et al. (2014),Nucleic Acids Res.42:D1140-D1146). The RSCB Protein Data Bank (PDB) structures with the highest percentage identity were further analyzed in the PyIgClassify database to identify the associated CDR loop conformations. In some instances more than one PDB structure with the same percent identity was identified by SAbDAb analysis. In those cases, the canonical structures for each PDB hit was determined with the PyIgClassify database (Table 5).

TABLE 5PDB structure and CDR canonical structure assignments of anti-Kv1.3antibodies derived from chickens.SAbDAb Sequence AnalysisPercentIdentity(fullPyIgClassify CDR CanonicalvariableStructure AssignmentsmAb ClonePDB Structureregion)H1H2H3L1L2L319724p2.A25d7260.28H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*5d7060.28H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p2.A35d7065.09H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p2.A54qci63.01H1-13-1H2-10-2H3-13-*L1-11-3L2-8-1L3-9-*19724p2.A75d7s63.33H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*5c7x63.33H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*19724p2.B55d7160.56H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*5d7060.56H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p2.C45d7s63.5H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*5c7x63.5H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*19724p2.D15d7263.67H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*5d7063.67H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p2.D25d7264.31H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*5d7064.31H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p2.D94qhu62.79H1-13-1H2-10-*H3-8-2L1-11-3L2-8-1L3-11-119724p2.E65d7264.28H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*19724p2.F75d7265.72H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*5d7065.72H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p2.G95d7065.09H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p2.H105d7162.73H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p2.H125d7264.31H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*5d7064.31H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p2.H45d7264.78H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*5d7064.78H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p2.H65d7263.67H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*5d7063.67H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p1_A14qhu62.79H1-13-1H2-10-*H3-8-2L1-11-3L2-8-1L3-11-119724p1_A55d7164.31H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p1_A95d7262.61H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*5d7062.61H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p1_A115d7063.03H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p1_B15d7264.92H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*5d7064.92H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p1_B115d7066.19H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p 1_C45d7262.61H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*5d7062.61H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p1_C125d7266.82H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*19724p1_D25d7262.73H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*19724p1_D85d7263.38H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*19724p1_D115d7062.91H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p1_E25d7261.5H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*19724p1_E35d7163.38H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p1_E65d7064.95H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p1_F35d7066.19H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p1_F65d7260.37H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*19724p1_F75d7266.35H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*5d7066.35H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p1_F85d7262.08H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*5d7062.08H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p1_F95d7266.82H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*5d7066.82H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p1_G65d7263.67H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*5d7s63.67H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*5c7x63.67H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*19724p1_H25d7261.97H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*19724p1_H44qhu62.79H1-13-1H2-10-*H3-8-2L1-11-3L2-8-1L3-11-119724p1_H75d7062.85H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p1_H125d7063.84H1-13-1H2-10-2H3-8-2L1-11-3L3-9-**SAbDAb: Sequence searches based on full variable domain

Based on this analysis, anti-Kv1.3 antibodies may be comprised of the following PDB structures: 5d72, 5d70, 4qci, 5d7s, 5c7x, 5d71 and 4qhu.

Based on the PDB structures, anti-Kv1.3 antibodies may have the following canonical CDR structures: H1CDR is H1-13-1; H2 CDR is H2-12-1, H2-10-2 or H2-10-*; H3 CDR is H3-8-2 or H3-13-*; L1 CDR is L1-11-3; L2 CDR is L2-6-* or L2-8-1; and L3 CDR is L3-9-* or L3-11-1.

Noting in particular that anti-Kv1.3 antibody clones 19724p1_F8, 19724p1_A1, 19724p2_A3, 19724p2_G9, 19724p1_A11, 19724p1_D8, 19724p1_E6, 19724p1_H4 and 19724p1_H7 inhibit Kv1.3 activity, distinct from other discovered antibodies, the associated PDB structures of anti-Kv1.3 antibodies may comprise 5d72, 5d70 and 4qhu (Table 6). Based on these PDB structure assignments, functionally inhibiting Kv1.3 antibodies may comprise the following canonical CDR sequences: H1 CDR is H1-13-1; H2 CDR is H2-12-1, H2-10-2 or H2-10-*; H3 CDR is H3-8-2; L1 CDR is L1-11-3; L2 CDR is L2-6-* or L2-8-1 and L3 CDR is L3-9-* or L3-11-1.

Noting in particular that anti-Kv1.3 antibody clones 19724p1_A11, 19724p1_H7, 19724p2.G9, 19724p2.A3, 19724p1.E6 and 19724_D8 are FACS positive for Jurkat binding, distinct from other discovered antibodies, the associated PDB structures of anti-Kv1.3 antibodies may comprise 5d70 and 5d72 (Table 6). Based on these PDB structure assignments, FACS positive Jurkat binding Kv1.3 antibodies may comprise the following canonical CDR sequences: H1 CDR is H1-13-1; H2 CDR is H2-10-2 or H2-12-1; H3 CDR is H3-8-2; L1 CDR is L1-11-3; L2 CDR is L2-6-* and L3 CDR is L3-9-*.

Noting in particular that anti-Kv1.3 antibody clone 19724p1_E6 may recognize a conformational Kv1.3 epitope, distinct from other discovered antibodies, the associated PDB structures of anti-Kv1.3 antibodies may be comprised of 5d70 (Table 6). Based on this PDB structure assignment, conformational Kv1.3 antibodies may comprise the following canonical CDR sequences: H1 CDR is H1-13-1; H2 CDR is H2-10-2; H3 CDR is H3-8-2; L1 CDR is L1-11-3; and L3 CDR is L3-9-*.

TABLE 6PDB structure and CDR canonical structure assignments of select anti-Kv1.3 antibodies derived from chickens.PyIgClassify CDR CanonicalmAbPDBStructure AssignmentsCharacteristicClonesStructureH1H2H3L1L2L3Inhibit Kv1.319724p1_F85d72H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*Activity5d70H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p1_A14qhuH1-13-1H2-10-*H3-8-2L1-11-3L2-8-1L3-11-119724p2_A35d70H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p2_G95d70H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p1_A115d70H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p1_D85d72H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*19724p1_E65d70H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p1_H44qhuH1-13-1H2-10-*H3-8-2L1-11-3L2-8-1L3-11-119724p1_H75d70H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*FACS Positive19724p1_A115d70H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*Jurkat Binding19724p1_H75d70H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p2.G95d70H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p2.A35d70H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*19724p1_D85d72H1-13-1H2-12-1H3-8-2L1-11-3L2-6-*L3-9-*19724p1_E65d70H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*Recognizes19724p1E65d70H1-13-1H2-10-2H3-8-2L1-11-3L3-9-*Conformationalepitope

Additional clones producing potentially useful anti-Kv1.3 antibodies were DNA sequenced and the corresponding amino acid sequences of light and heavy chain variable domains were deduced. Sequences are disclosed for ten antibodies derived from the GEM screen described above.

Variable Light Chain Sequences

Alignments of all of the VL sequences described above are shown inFIG.19. The figure indicates the approximate locations of the three CDR regions (bold, underscore) and the SEQ ID NO corresponding to each sequence.

Unique VL CDR Sequences.

Alignments of the unique CDR sequences of the VLs ofFIG.19are shown inFIG.20. Of the 10 VL sequences, there are 6 unique CDR1 sequences, 5 unique CDR2 sequences and 7 unique CDR3 sequences, as shown inFIG.20.

Variable Heavy Chain Sequences

Alignments of all of the VH sequences described above are shown inFIG.21. The figure indicates the approximate locations of the three CDR regions (bold, underscore) and the SEQ ID NO corresponding to each sequence.

Unique VH CDR Sequences.

Alignments of the unique CDR sequences of the VHs ofFIG.21are shown inFIG.22. Of the 10 VH sequences, there are 7 unique CDR1 sequences, 7 unique CDR2 sequences and 7 unique CDR3 sequences, as shown inFIG.22.

Antibodies Derived from Llamas:

Clones producing potentially useful anti-Kv1.3 antibodies were DNA sequenced and the corresponding amino acid sequences of light and heavy chain variable domains were deduced. Sequences are disclosed for nineteen antibodies derived from Kv1.3 immunized llamas as described above.

Variable Light Chain Sequences

Alignments of all of the VL sequences described above are shown inFIG.15. The figure indicates the approximate locations of the three CDR regions (bold, underscore) and the SEQ ID NO corresponding to each sequence.

Unique VL CDR Sequences.

Alignments of the unique CDR sequences of the VLs ofFIG.15are shown inFIG.16. Of the 19 VL sequences, there are 19 unique CDR1 sequences, 13 unique CDR2 sequences and 17 unique CDR3 sequences, as shown inFIG.16.

Based on the sequences disclosed inFIG.16, as well as structure/function characteristics of the naturally occurring amino acids, consensus sequences for the VL CDRs can be determined.

One consensus sequence is VL CDR1 Motif 5:

Noting in particular that the VL CDR1 sequence of SEQ ID NO: 231 is derived from an antibody that inhibits Kv1.3 function distinct from the others inFIG.15, an alternative consensus sequence is VL CDR1 Motif 6:

For the VL CDR2, one consensus sequence is VL CDR2 Motif 5:

Noting in particular that the VL CDR2 sequences of SEQ ID NO: 231 is derived from an antibody that inhibits Kv1.3 function distinct from the others inFIG.15, an alternative consensus sequence is VL CDR2 Motif 6:

For the VL CDR3, one consensus sequence is VL CDR3 Motif 5:

Noting in particular that the VL CDR3 sequences of SEQ ID NO 231 is derived from an antibody that inhibits Kv1.3 function distinct from the others inFIG.15, an alternative consensus sequence is VL CDR3 Motif 6:

Variable Heavy Chain Sequences

Alignments of all of the VH sequences described above are shown inFIG.17. The figure indicates the approximate locations of the three CDR regions (bold, underscore) and the SEQ ID NO corresponding to each sequence.

Unique VH CDR Sequences.

Alignments of the unique CDR sequences of the VHs ofFIG.17are shown inFIG.18. Of the 19 VH sequences, there are 18 unique CDR1 sequences, 18 unique CDR2 sequences and 18 unique CDR3 sequences, as shown inFIG.18.

Based on the sequences disclosed inFIG.18, as well as structure/function characteristics of the naturally occurring amino acids, consensus sequences for the VH CDRs can be determined.

For the VH CDR1, one consensus sequence is VH CDR1 Motif 5:

Noting in particular that the VH CDR1 sequence of SEQ ID NO: 306 is derived from an antibody that inhibit Kv1.3 function distinct from the others inFIG.17, an alternative consensus sequence is VH CDR1 Motif 6:

For the VH CDR2, one consensus sequence is VH CDR2 Motif 5:

Noting in particular that the VH CDR2 sequence of SEQ ID NO: 306 is derived from an antibody that inhibits Kv1.3 function distinct from the others inFIG.17, an alternative consensus sequence is VH CDR2 Motif 6:

For the VH CDR3, one consensus sequence is VH CDR3 Motif 5:

Noting in particular that the VH CDR3 sequence of SEQ ID NO: 306 is derived from an antibody that inhibits Kv1.3 function distinct from the others inFIG.17, an alternative consensus sequence is VH CDR3 Motif 6:

CDR Canonical Structures

To determine potential canonical structures associated with the disclosed anti-Kv1.3 antibodies derived from llamas (Table 7), light and heavy chain variable regions from each anti-Kv1.3 clone were submitted for sequence analysis in the “SAbDAb” structural antibody database (Dunbar et al. (2014),Nucleic Acids Res.42:D1140-D1146). The RSCB Protein Data Bank (PDB) structures with the highest percentage identity were further analyzed in the PyIgClassify database to identify the associated CDR loop conformations. In some instances more than one PDB structure with the same percent identity was identified by SAbDAb analysis. In those cases, the canonical structures for each PDB hit was determined with the PyIgClassify database (Table 8).

TABLE 8PDB structure and CDR canonical structure assignments of anti-Kv1.3antibodies derived from llama's.SAbDAb SequenceAnalysisPercentIdentity (fullmAbPDBvariablePyIgClassify CDR Canonical Structure AssignmentsCloneStructureregion)H1H2H3L1L2L33A125i1d74.77H1-13-1H2-10-12H3-12-*L1-17-1L2-8-1L3-9-cis7-13qos74.771A35i1d73.79H1-13-1H2-10-12H3-12-*L1-17-1L2-8-1L3-9-cis7-13qos73.793B125i1d74.23H1-13-1H2-10-12H3-12-*L1-17-1L2-8-1L3-9-cis7-13qos74.233B65kna73.215kmv73.213A45tzt70.223B25kna70.985kmv70.983F94lkx73.66H1-14-1H2-9-1H3-10-*L1-16-1L2-8-2L3-9-cis7-12A104ojf77.31H1-13-1H2-10-6H3-12-*L1-16-1L2-8-1L3-9-cis7-14hix77.31H1-13-1H2-10-6H3-12-*L1-16-1L2-8-1L3-9-cis7-13.00E+125kna755kmv754ojf75H1-13-1H2-10-6H3-12-*L1-16-1L2-8-1L3-9-cis7-14hix75H1-13-1H2-10-6H3-12-*L1-16-1L2-8-1L3-9-cis7-13F44ojf75.89H1-13-1H2-10-6H3-12-*L1-16-1L2-8-1L3-9-cis7-14hix75.89H1-13-1H2-10-6H3-12-*L1-16-1L2-8-1L3-9-cis7-13E55kna77.235kmv77.232A25kna79.015kmv79.013F25kna77.575kmv77.573G104zs779.74H1-15-cis11-*H2-9-1H3-14-*L1-14-2L2-8-1L3-10-cis6-*3C74zs778.44H1-15-cis11-*H2-9-1H3-14-*L1-14-2L2-8-1L3-10-cis6-*2E25f6i76.88H1-13-1H2-10-2H3-10-*L1-14-2L2-8-1L3-10-*4rav76.88H1-13-1H2-10-2H3-8-2L1-14-2L2-8-2L3-9-*3H54zs782.17H1-15-cis11-*H2-9-1H3-14-*L1-14-2L2-8-1L3-10-cis6-*1E64o9h69.91H1-13-1H2-10-2H3-11-*L1-14-1L2-8-1L3-9-*3C9409h70.35H1-13-1H2-10-2H3-11-*L1-14-1L2-8-1L3-9-*

Noting in particular that anti-Kv1.3 antibody clones 1A3 inhibits Kv1.3 activity, distinct from other discovered antibodies, the associated PDB structures of anti-Kv1.3 antibodies may comprise 5i1d or 3qos (Table 8). Based on these PDB structure assignments, functionally inhibiting Kv1.3 antibodies may comprise the following canonical CDR sequences: H1 CDR is H1-13-1; H2 CDR is H2-10-12; H3 CDR is H3-12-*; L1 CDR is L1-17-1; L2 CDR is L2-8-1 and L3 CDR is L3-9-cis7-1.