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Anti-ccr4 antibody and its application

A technology of antibodies and antagonists, applied in the direction of antibodies, anti-inflammatory agents, anti-bacterial drugs, etc., can solve problems such as lack of applications

Inactive Publication Date: 2016-04-27
CANCER RES TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0025] Accordingly, the art remains lacking in anti-CCR4 antibodies that can be used in the safe and effective treatment of patients with disorders involving CCR4, including chronic administration, and presents challenges in developing such antibodies

Method used

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  • Anti-ccr4 antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0476] Example 1: New Antibodies

[0477] Nine human antibodies capable of specifically binding CCR4 have been identified. The single chain form of the antibody was cloned into the pHOG21 plasmid, which contains c-myc and 6 his-tagged epitopes. TG1 bacteria were transfected and scFv expression was induced with IPTG. Binding of purified scFv was confirmed by EasyCyte (see Example 2).

[0478] The nucleotide sequences of the heavy and light chains of the resulting cloned antibodies were sequenced. The antibodies are designated as 208, 306, 308, 406, 501, 503, 601, 603 and 803. The CDR regions of the light and heavy chains of 208, 306, 308, 406, 501, 503, 601, 603 and 803 The iso-sequences are shown in Tables 1-9, respectively.

[0479] Antibodies 208, 306, 308, 406, 501, 503, 601, 603 and 803 were also prepared in IgG form. IgG was prepared using standard protocols. Briefly, the genes encoding the corresponding variable domains were cloned into the mammalian expression v...

Embodiment 2

[0481] Example 2: Binding of Anti-CCR4 Antibodies to Target Expressing Cells

[0482] To demonstrate the CCR4 specificity of the antibodies disclosed in Example 1, scFv208, 306, 308, 406, 501, 503, 601, 603 and 803 were used in flow cytometry for internally transfected with CCR4 and not transfected with CCR4. HEK293T cells, DT40 cells and native CCR4 + CCRF-CEM cell line staining. An internally cloned and expressed KM3060var scFv was used as a positive control. Anti-GFPscFv antibody (raised against green fluorescent protein) was used as a negative control.

[0483] CCRF-CEM (acute lymphocytic leukemia, ATCC No. CCL-119), HEK293T / 17 (human kidney, ATCC No. CRL-11268), and DT40 (chicken lymphoma, ATCC No. CRL-2111) cell lines were cultured from American models Acquired from the Art Collection (ATCC, Rockville, MD).

[0484] For transient transfection with human CCR4, Hek293T / 17 cells were seeded in T75 (NUNC) flasks at 2 × 10 6 cells. 48 hours after seeding, the cells we...

Embodiment 3

[0503] Example 3: Anti-CCR4 Antibodies Interfering with Ligand Binding

[0504] To determine whether the anti-CCR4 antibodies from Example 1 interfere with CCR4 ligand and receptor binding, competition experiments were performed. To this end, CCR4-positive CCRF-CEM target cells were incubated at a fixed concentration of MDC-SNAP in the presence of increasing concentrations of scFv. The MDC is genetically fused to the N-terminus of the SNAP tag such that the SNAP tag is fused to the C-terminus of the MDC. The SNAP tag is derived from the 20 kDa DNA repair protein O6 alkylguanine-DNA alkyltransferase. The gene encoding the MDC-SNAP fusion protein was custom made at Geneart (Regensburg, Germany) and HEK293 / T cells were transiently transfected with the gene. After 5-6 days, the fusion protein was purified by a Ni-NTA affinity column, followed by isolation of a single-section MDC-SNAP fragment by steric exclusion. MDC-SNAP was labeled with Alexa647 according to the procedure i...

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Abstract

The present disclosure provides epitopes that bind to the extracellular domain of human CC chemokine receptor 4 (CCR4) and are capable of inhibiting macrophage-derived chemokine (MDC) and / or thymus activation-regulated chemokine (TARC) Antibodies that bind to CCR4. The invention also provides immunoconjugates and compositions comprising such antibodies, as well as methods and uses involving such antibodies, particularly in the fields of medicine and diagnostics.

Description

technical field [0001] The present invention relates generally to antibodies, CCR4 biology and related therapeutic fields. More specifically, the invention provides antibodies that bind to CCR4. Such anti-CCR4 antibodies have applications in the diagnosis and treatment of diseases and conditions associated with CCR4, such as imaging tumor blood vessels, treating cancer and treating viral and other infections, and inflammatory and immune diseases. The antibody-based compositions and methods of the invention also extend to the use of immunoconjugates and other therapeutic compositions, kits and methods. Background technique [0002] With more than 800 members, G protein-coupled receptors (GPCRs) represent the largest family of cell surface molecules involved in signaling and account for >2% of all genes encoded in the human genome. Members of the GPCR superfamily share a common membrane topology: an extracellular N-terminus, an intracellular C-terminus, and a seven-transm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28G01N33/574A61K39/395A61P35/00A61P35/02
CPCA61K2039/505C07K2317/21C07K2317/33C07K2317/41C07K2317/732C07K2317/76G01N2333/7158C07K16/2866A61P1/00A61P1/04A61P1/16A61P1/18A61P11/00A61P11/02A61P11/06A61P13/08A61P13/12A61P15/00A61P15/02A61P17/00A61P17/04A61P17/06A61P19/02A61P19/06A61P21/00A61P25/00A61P25/28A61P27/02A61P27/14A61P27/16A61P29/00A61P31/00A61P31/04A61P31/12A61P31/18A61P31/22A61P35/00A61P35/02A61P37/00A61P37/02A61P37/06A61P37/08A61P43/00A61P9/00A61P9/10A61P3/10C07K16/28G01N33/574A61K39/395C07K2317/14C07K2317/565C07K2317/622
Inventor U·B·哈格曼R·A·格里普H·雷厄瑟夫S·M·基普里亚诺夫
Owner CANCER RES TECH LTD
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