Antibodies to l-type voltage gated channels and related methods

Abstract

Provided are antibodies, and antigen-binding fragments thereof, which specifically bind to an extracellular poor loop of an alpha 1a subunit of L-type voltage gated calcium channel, and related compositions, kits, and methods of use thereof, for instance, administration to a subject in need thereof to modify an immune response, for example, in the treatment of cancer.

1 . An isolated antibody, or antigen-binding fragment thereof, which specifically binds to an alpha 1 subunit of an L-type voltage-gated calcium channel, wherein the antibody or antigen binding fragment thereof, (a) specifically binds to an amino acid sequence of an extracellular domain of a pore loop between transmembrane segments S5 and S6 of motif I of the alpha 1 subunit, or (b) competitively inhibits the binding of (a) to the alpha 1 subunit. 
     
     
         2 . The isolated antibody, or antigen-binding fragment thereof, of  claim 1 , wherein the L-type voltage-gated calcium channel is from human or mouse. 
     
     
         3 . The isolated antibody, or antigen-binding fragment thereof, of any of  claim 1  or  2 , wherein the binding of the antibody or antigen binding fragment thereof to the alpha 1 subunit alters activity of the L-type voltage-gated calcium channel. 
     
     
         4 . The isolated antibody, or antigen-binding fragment thereof, of any of  claims 1 - 3 , wherein the L-type voltage-gated calcium channel is Cav1.4. 
     
     
         5 . The isolated antibody, or antigen-binding fragment thereof, of  claim 4 , wherein the amino acid sequence of the extracellular domain is GPGRPGDAPHTG [SEQ ID NO: 1], or is at least 90% identical to GPGRPGDAPHTG [SEQ ID NO: 1]. 
     
     
         6 . The antibody, or antigen-binding fragment thereof, of any of  claims 1 - 3 , wherein the L-type voltage-gated calcium channel is Cav1.3. 
     
     
         7 . The isolated antibody, or antigen-binding fragment thereof, of  claim 6 , wherein the amino acid sequence of the extracellular domain is LTKETEGGNHSSGKSG [SEQ ID NO 2] or is at least 90% identical to LTKETEGGNHSSGKSG [SEQ ID NO 2]. 
     
     
         8 . The antibody, or antigen-binding fragment thereof, of any of  claims 1 - 3 , wherein the L-type voltage-gated calcium channel is Cav1.2. 
     
     
         9 . The isolated antibody, or antigen-binding fragment thereof, of  claim 8 , wherein the amino acid sequence of the extracellular domain is ATKADGANALGGKGA [SEQ ID NO: 3] at least 90% identical to ATKADGANALGGKGA [SEQ ID NO: 3]. 
     
     
         10 . The antibody, or antigen-binding fragment thereof, of any of  claims 1 - 4 , wherein the L-type voltage-gated calcium channel is Cav1.1. 
     
     
         11 . The isolated antibody, or antigen-binding fragment thereof, of  claim 10 , wherein the amino acid sequence of the extracellular domain is PMQIELRHREWVH [SEQ ID NO 4] or is at least 90% identical to PMQIELRHREWVH [SEQ ID NO 4]. 
     
     
         12 . The isolated antibody, or antigen-binding fragment thereof, of any of  claims 1 - 11 , wherein the antibody or antigen-binding fragment binds to any of Cav1.4, Cav1.3, Cav1.2, or Cav1.1. 
     
     
         13 . The isolated antibody, or antigen-binding fragment thereof, of any of  claims 1 - 11 , wherein the antibody or antigen-binding fragment thereof binds to any three of Cav1.4, Cav1.3, Cav1.2, or Cav1.1. 
     
     
         14 . The isolated antibody, or antigen-binding fragment thereof, of  claim 13 , wherein the antibody or antigen-binding fragment thereof binds to any of Cav1.4, Cav1.3, or Cav1.2. 
     
     
         15 . The isolated antibody, or antigen-binding fragment thereof, of  claim 13 , wherein the antibody or antigen-binding fragment thereof binds to any of Cav1.4, Cav1.3, or Cav1.1. 
     
     
         16 . The isolated antibody, or antigen-binding fragment thereof, of  claim 13 , wherein the antibody or antigen-binding fragment thereof binds to any of Cav1.4, Cav1.2, or Cav1.1. 
     
     
         17 . The isolated antibody, or antigen-binding fragment thereof, of  claim 13 , wherein the antibody or antigen-binding fragment thereof binds to any of Cav1.3, Cav1.2, or Cav1.1. 
     
     
         18 . The isolated antibody, or antigen-binding fragment thereof, of any of  claims 1 - 11 , wherein the antibody or antigen-binding fragment thereof binds to any two of Cav1.4, Cav1.3, Cav1.2, or Cav1.1. 
     
     
         19 . The isolated antibody, or antigen-binding fragment thereof, of  claim 18 , wherein the antibody or antigen-binding fragment thereof binds Cav1.4 or Cav1.3. 
     
     
         20 . The isolated antibody, or antigen-binding fragment thereof, of  claim 18 , wherein the antibody or antigen-binding fragment thereof binds Cav1.4 or Cav1.2. 
     
     
         21 . The isolated antibody, or antigen-binding fragment thereof, of  claim 18 , wherein the antibody or antigen-binding fragment thereof binds Cav1.4 or Cav1.1. 
     
     
         22 . The isolated antibody, or antigen-binding fragment thereof, of  claim 18 , wherein the antibody or antigen-binding fragment thereof binds Cav1.3 or Cav1.2. 
     
     
         23 . The isolated antibody, or antigen-binding fragment thereof, of  claim 18 , wherein the antibody or antigen-binding fragment thereof binds Cav1.3 or Cav1.1. 
     
     
         24 . The isolated antibody, or antigen-binding fragment thereof, of  claim 18 , wherein the antibody or antigen-binding fragment thereof binds Cav1.2 or Cav1.1. 
     
     
         25 . The isolated antibody, or antigen-binding fragment thereof, of any of  claims 1 - 11 , wherein the antibody or antigen-binding fragment thereof binds Cav1.4 and does not significantly bind Cav1.1, Cav1.2, or Cav1.3. 
     
     
         26 . The isolated antibody, or antigen-binding fragment thereof, of any of  claims 1 - 11 , wherein the antibody or antigen-binding fragment thereof binds Cav1.3 and does not significantly bind Cav1.1, Cav1.2, or Cav1.4. 
     
     
         27 . The isolated antibody, or antigen-binding fragment thereof, of any of  claims 1 - 11 , wherein the antibody or antigen-binding fragment thereof binds Cav1.2 and does not significantly bind Cav1.1, Cav1.3, or Cav1.4. 
     
     
         28 . The isolated antibody, or antigen-binding fragment thereof, of any of  claims 1 - 11 , wherein the antibody or antigen-binding fragment thereof binds Cav1.1 and does not significantly bind Cav1.2, Cav1.3, or Cav1.4. 
     
     
         29 . The isolated antibody, or antigen-binding fragment thereof, of any one of the preceding claims, which comprises a heavy chain variable region (V H ) that comprises V H CDR1, V H CDR2 and V H CDR3 amino acid sequences; and/or a light chain variable region (L H ) that comprises V L CDR1, V L CDR2 and V L CDR3 amino acid sequences, which are selected from:
 (A) (i) V H CDR1, V H CDR2 and V H CDR3 comprise, respectively, the amino acid sequences of SEQ ID NOS:14-16; and (ii) V L CDR1, V L CDR2 and V L CDR3 comprise, respectively, the amino acid sequences of (i) SEQ ID NOS:18-20; including variants thereof where at least one of said V H CDR or V L CDR amino acid sequences is modified by about 1, 2, or 3 amino acid substitutions, additions, or deletions;   (B) (i) V H CDR1, V H CDR2 and V H CDR3 comprise, respectively, the amino acid sequences of SEQ ID NOS:22-24; and (ii) V L CDR1, V L CDR2 and V L CDR3 comprise, respectively, the amino acid sequences of (i) SEQ ID NOS:26-28; including variants thereof where at least one of said V H CDR or V L CDR amino acid sequences is modified by about 1, 2, or 3 amino acid substitutions, additions, or deletions;   (C) (i) V H CDR1, V H CDR2 and V H CDR3 comprise, respectively, the amino acid sequences of SEQ ID NOS:30-32; and (ii) V L CDR1, V L CDR2 and V L CDR3 comprise, respectively, the amino acid sequences of (i) SEQ ID NOS:34-36; including variants thereof where at least one of said V H CDR or V L CDR amino acid sequences is modified by about 1, 2, or 3 amino acid substitutions, additions, or deletions;   (D) (i) V H CDR1 and V H CDR2 comprise, respectively, the amino acid sequences of SEQ ID NOS:38-39; and (ii) V L CDR1, V L CDR2 and V L CDR3 comprise, respectively, the amino acid sequences of (i) SEQ ID NOS:42-44; including variants thereof where at least one of said V H CDR or V L CDR amino acid sequences is modified by about 1, 2, or 3 amino acid substitutions, additions, or deletions;   (E) (i) V H CDR1, V H CDR2 and V H CDR3 comprise, respectively, the amino acid sequences of SEQ ID NOS:46-48; and (ii) V L CDR1, V L CDR2 and V L CDR3 comprise, respectively, the amino acid sequences of (i) SEQ ID NOS:50-52; including variants thereof where at least one of said V H CDR or V L CDR amino acid sequences is modified by about 1, 2, or 3 amino acid substitutions, additions, or deletions;   (F) (i) V H CDR1 and V H CDR2 comprise, respectively, the amino acid sequences of SEQ ID NOS:54-55; and (ii) V L CDR1, V L CDR2 and V L CDR3 comprise, respectively, the amino acid sequences of (i) SEQ ID NOS:58-60; including variants thereof where at least one of said V H CDR or V L CDR amino acid sequences is modified by about 1, 2, or 3 amino acid substitutions, additions, or deletions;   (G) (i) V H CDR1, V H CDR2 and V H CDR3 comprise, respectively, the amino acid sequences of SEQ ID NOS:62-64; including variants thereof where at least one of said V H CDR or V L CDR amino acid sequences is modified by about 1, 2, or 3 amino acid substitutions, additions, or deletions;   (H) (i) V H CDR1, V H CDR2 and V H CDR3 comprise, respectively, the amino acid sequences of SEQ ID NOS:70-72; and (ii) V L CDR1, V L CDR2 and V L CDR3 comprise, respectively, the amino acid sequences of (i) SEQ ID NOS:74-76; including variants thereof where at least one of said V H CDR or V L CDR amino acid sequences is modified by about 1, 2, or 3 amino acid substitutions, additions, or deletions;   (I) (i) V H CDR1, V H CDR2 and V H CDR3 comprise, respectively, the amino acid sequences of SEQ ID NOS:78-80; and (ii) V L CDR1, V L CDR2 and V L CDR3 comprise, respectively, the amino acid sequences of (i) SEQ ID NOS:82-84; including variants thereof where at least one of said V H CDR or V L CDR amino acid sequences is modified by about 1, 2, or 3 amino acid substitutions, additions, or deletions;   (J) (i) V H CDR1, V H CDR2 and V H CDR3 comprise, respectively, the amino acid sequences of SEQ ID NOS:86-88; and (ii) V L CDR1, V L CDR2 and V L CDR3 comprise, respectively, the amino acid sequences of (i) SEQ ID NOS:90-92; including variants thereof where at least one of said V H CDR or V L CDR amino acid sequences is modified by about 1, 2, or 3 amino acid substitutions, additions, or deletions;   (K) (i) V L CDR1, V L CDR2 and V L CDR3 comprise, respectively, the amino acid sequences of (i) SEQ ID NOS:98-100; including variants thereof where at least one of said V H CDR or V L CDR amino acid sequences is modified by about 1, 2, or 3 amino acid substitutions, additions, or deletions; and   (L) (i) V L CDR1, V L CDR2 and V L CDR3 comprise, respectively, the amino acid sequences of (i) SEQ ID NOS:106-108; including variants thereof where at least one of said V H CDR or V L CDR amino acid sequences is modified by about 1, 2, or 3 amino acid substitutions, additions, or deletions.   
     
     
         30 . The antibody, or antigen-binding fragment thereof, of any one of the preceding claims, comprising a V H  sequence that is at least 90% identical to SEQ ID NO:13, 21, 29, 37, 45, 53, 61, 69, 77, or 85. 
     
     
         31 . The antibody, or antigen-binding fragment thereof, of any one of the preceding claims, comprising a V L  sequence that is at least 90% identical to SEQ ID NO:17, 25, 33, 41, 49, 57, 73, 81, 89, 97, or 105. 
     
     
         32 . The antibody, or antigen-binding fragment thereof, of any one of the preceding claims, comprising a V H  sequence that is at least 90% identical to SEQ ID NO: 13, 21, 29, 37, 45, 53, 61, 69, 77, or 85, and a V L  sequence that is at least 90% identical to SEQ ID NO: 17, 25, 33, 41, 49, 57, 73, 81, 89, 97, or 105. 
     
     
         33 . The antibody, or antigen-binding fragment thereof, of any one of the preceding claims, comprising a V H  sequence and a V L  sequence selected from:
 (A) the V H  sequence of SEQ ID NO:13 and the V L  sequence of SEQ ID NO:17;   (B) the V H  sequence of SEQ ID NO:21 and the V L  sequence of SEQ ID NO:25;   (C) the V H  sequence of SEQ ID NO:29 and the V L  sequence of SEQ ID NO:33;   (D) the V H  sequence of SEQ ID NO:37 and the V L  sequence of SEQ ID NO:41;   (E) the V H  sequence of SEQ ID NO:45 and the V L  sequence of SEQ ID NO:49;   (F) the V H  sequence of SEQ ID NO:53 and the V L  sequence of SEQ ID NO:57;   (G) the V H  sequence of SEQ ID NO:69 and the V L  sequence of SEQ ID NO:73;   (H) the V H  sequence of SEQ ID NO:77 and the V L  sequence of SEQ ID NO:81;   (I) the V H  sequence of SEQ ID NO:85 and the V L  sequence of SEQ ID NO:89; and   (J) a variant V H  sequence and a variant V L  sequence that is at least 90% identical to any of (A)-(I).   
     
     
         34 . A polynucleotide encoding the antibody, or antigen-binding fragment thereof, of any one of the preceding claims. 
     
     
         35 . A vector encoding the polynucleotide of  claim 34 . 
     
     
         36 . A cell expressing the polynucleotide of  claim 34  or the vector of  claim 35 . 
     
     
         37 . A method for modulating a function of a cell expressing an L-type voltage-gated calcium channel comprising contacting the cell with an antibody, or antigen-binding fragment thereof, which specifically binds to (a) an amino acid sequence of an extracellular domain of a pore loop between transmembrane segments S5 and S6 of domain 1 of an alpha 1 subunit of the L-type voltage-gated calcium channel, or (b) competitively inhibits the binding of (a) to the alpha 1 subunit, wherein binding of the agent to the alpha I subunit modulates the activity of the L-type voltage-gated calcium channel. 
     
     
         38 . The method of  claim 37 , wherein the antibody or binding fragment thereof is the antibody or binding fragment thereof of any one of the preceding claims. 
     
     
         39 . The method of  claim 37  or  38 , wherein the cell is a hematopoietic cell. 
     
     
         40 . The method of any one of the preceding claims, wherein the antibody or antigen binding-fragment thereof inhibits the activity of the L-type voltage-gated calcium channel. 
     
     
         41 . The method any one of the preceding claims, wherein the antibody or antigen binding-fragment thereof increases the activity of the L-type voltage-gated calcium channel. 
     
     
         42 . The method of any of  claims 39 - 41 , wherein the cell is a hematopoietic cell of the lymphoid lineage. 
     
     
         43 . The method of  claim 42 , wherein the cell is a T cell. 
     
     
         44 . The method of  claim 43 , wherein the function of the cell comprises T cell maturation. 
     
     
         45 . The method of  claim 43 , wherein the function of the T cell comprises antigen binding. 
     
     
         46 . The method of  claim 42 , wherein the cell is a B cell. 
     
     
         47 . The method of  claim 46 , wherein the function of the cell comprises B cell maturation. 
     
     
         48 . The method of  claim 46 , wherein the function of the cell comprises B cell receptor-induced activation. 
     
     
         49 . The method of any one of the preceding claims, wherein the L-type voltage-gated calcium channel is Cav1.4. 
     
     
         50 . The method of any one of the preceding claims, wherein the L-type voltage-gated calcium channel is Cav1.3. 
     
     
         51 . The method of any one of the preceding claims, wherein the L-type voltage-gated calcium channel is Cav1.2. 
     
     
         52 . The method of any one of the preceding claims, wherein the L-type voltage-gated calcium channel is Cav1.1. 
     
     
         53 . A method of modulating an immune response in a subject comprising administering to the subject an effective amount of an antibody, or antigen-binding fragment thereof, which specifically binds to (a) an amino acid sequence of an extracellular domain of a pore loop between transmembrane segments S5 and S6 of domain 1 of an alpha 1 subunit of the L-type voltage-gated calcium channel, or (b) competitively inhibits the binding of (a) to the alpha 1 subunit, wherein the L-type voltage-gated calcium channel is expressed in a hematopoietic cell. 
     
     
         54 . The method of  claim 53 , wherein the antibody or binding fragment thereof is the antibody or binding fragment thereof of any of  claims 1 - 10 . 
     
     
         55 . The method of  claim 53  or  54 , wherein the hematopoietic cell is of the lymphoid lineage. 
     
     
         56 . The method of any one of  claims 53 - 55 , wherein the hematopoietic cell is a T cell or a B cell. 
     
     
         57 . The method of any one of  claims 53 - 56 , wherein the L-type voltage-gated calcium channel is Cav1.4. 
     
     
         58 . The method of any one of  claims 53 - 57 , wherein the L-type voltage-gated calcium channel is Cav1.3. 
     
     
         59 . The method of any one of  claims 53 - 58 , wherein the L-type voltage-gated calcium channel is Cav1.2. 
     
     
         60 . The method of any one of  claims 53 - 59 , wherein the L-type voltage-gated calcium channel is Cav1.1. 
     
     
         61 . A method of inhibiting an immune response in a subject comprising administering to the subject an effective amount of an antibody, or antigen-binding fragment thereof, which specifically binds to (a) an amino acid sequence of an extracellular domain of a pore loop between transmembrane segments S5 and S6 of domain 1 of an alpha 1 subunit of the L-type voltage-gated calcium channel, or (b) competitively inhibits the binding of (a) to the alpha 1 subunit, wherein the L-type voltage-gated calcium channel is expressed in a hematopoietic cell. 
     
     
         62 . The method of  claim 61 , wherein the antibody or binding fragment thereof is the antibody or binding fragment thereof of any one of the preceding claims. 
     
     
         63 . The method according to  claim 61  or  62 , wherein the hematopoietic cell is of the lymphoid lineage. 
     
     
         64 . The method according any one of  claims 61 - 63 , wherein the hematopoietic cell is a T cell. 
     
     
         65 . The method of  claim 64 , wherein the administering the effective amount of the antibody or antigen-binding fragment thereof decreases T cell receptor-induced Ca2+ fluxes. 
     
     
         66 . The method of  claim 64 , wherein the administering the effective amount of the antibody or antigen-binding fragment thereof reduces naïve T cell survival. 
     
     
         67 . The method of  claim 64 , wherein the administering the effective amount of the antibody or antigen-binding fragment reduces CD3/CD28 induced T cell proliferation. 
     
     
         68 . The method according any of  claims 61 - 63 , wherein the hematopoietic cell is a B cell. 
     
     
         69 . The method of  claim 68 , wherein the administering the effective amount of the antibody or antigen-binding fragment inhibits B cell receptor-induced activation. 
     
     
         70 . The method of any one of  claims 61 - 69 , wherein the L-type voltage-gated calcium channel is Cav1.4. 
     
     
         71 . The method of any one of  claims 61 - 70 , wherein the L-type voltage-gated calcium channel is Cav1.3. 
     
     
         72 . The method of any one of  claims 61 - 71 , wherein the L-type voltage-gated calcium channel is Cav1.2. 
     
     
         73 . The method of any one of  claims 61 - 72 , wherein the L-type voltage-gated calcium channel is Cav1.1. 
     
     
         74 . A method of treating a disease in a subject comprising administering to the subject an effective amount of an antibody, or antigen-binding fragment thereof, which specifically binds to (a) an amino acid sequence of an extracellular domain of a pore loop between transmembrane segments S5 and S6 of domain 1 of an alpha 1 subunit of the L-type voltage-gated calcium channel, or (b) competitively inhibits the binding of (a) to the alpha 1 subunit. 
     
     
         75 . The method of  claim 74 , wherein the antibody or binding fragment thereof is the antibody or binding fragment thereof of any one of the preceding claims. 
     
     
         76 . The method of  claim 74  or  75 , wherein the disease is an inflammatory disease. 
     
     
         77 . The method of  claim 76 , wherein the inflammatory disease is X-linked agammaglobulinemia, systemic lupus erythematosus, inflammatory (rheumatoid) arthritis, Hashimoto's thyroiditis, pernicious anemia, inflammatory bowel disease (Crohn's disease and ulcerative colitis), psoriasis, renal fibroses, pulmonary fibroses, hepatic fibroses, Addison's disease, Type I diabetes, systemic lupus erythematosus (SLE), dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, Reiter's syndrome, asthma, or Grave's disease. 
     
     
         78 . The method of  claim 74  or  75 , wherein the disease is a cancer. 
     
     
         79 . The method of  claim 78 , wherein the cancer is a hematopoietic cancer. 
     
     
         80 . The method of  claim 79 , wherein hematopoietic cancer is a lymphoma, leukemia, or multiple myeloma. 
     
     
         81 . The method of  claim 80 , wherein the lymphoma is a T-cell lymphoma, B-cell lymphoma, small lymphocytic lymphoma, mangle cell lymphoma, anaplastic large cell lymphoma (ALCL), follicular lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma. 
     
     
         82 . The method of  claim 80 , wherein the leukemia is chronic lymphocytic leukemia (CLL), hairy cell leukemia, acute lymphoblastic leukemia, myelocytic leukemia, acute myeloid or myelogenous leukemia, or chronic myelogenous leukemia. 
     
     
         83 . The method of  claim 78 , wherein the cancer is selected from one or more of breast cancer, cervical cancer, prostate cancer, gastrointestinal cancer, lung cancer, ovarian cancer, testicular cancer, head and neck cancer, bladder cancer, kidney cancer (e.g., renal cell carcinoma), soft tissue sarcoma, squamous cell carcinoma, CNS or brain cancer, melanoma, non-melanoma cancer, thyroid cancer, endometrial cancer, an epithelial tumor, and bone cancer. 
     
     
         84 . The method of any one of  claims 74 - 83 , wherein the cancer expresses or overexpresses Cav1.1, Cav1.2, Cav1.3, Cav1.4, or any combination thereof. 
     
     
         85 . The method of  claim 84 , wherein the cancer expresses or overexpresses Cav1.1 and the antibody, or antigen-binding fragment thereof, specifically binds to Cav1.1. 
     
     
         86 . The method of  claim 84 , wherein the cancer expresses or overexpresses Cav1.2 and the antibody, or antigen-binding fragment thereof, specifically binds to Cav1.2. 
     
     
         87 . The method of  claim 84 , wherein the cancer expresses or overexpresses Cav1.3 and the antibody, or antigen-binding fragment thereof, specifically binds to Cav1.3. 
     
     
         88 . The method of  claim 84 , wherein the cancer expresses or overexpresses Cav1.4 and the antibody, or antigen-binding fragment thereof, specifically binds to Cav1.4.