阅读说明：本技术 抗ccr4抗体及其在治疗癌症中的应用 (anti-CCR 4 antibodies and their use in treating cancer ) 是由 彭菲 顾超 于 2020-06-27 设计创作，主要内容包括：本发明涉及一种特异性结合CCR4的抗体及其在治疗癌症中的应用。所述抗体能够以较高的亲和力结合CCR4阳性细胞,阻断CCL17、CCL22与肿瘤细胞表面的CCR4结合,促进效应细胞对肿瘤细胞的ADCC作用,为癌症的治疗和/或预防提供新的可能。(The present invention relates to an antibody that specifically binds to CCR4 and its use in the treatment of cancer. The antibody can be combined with CCR4 positive cells with higher affinity, block the combination of CCL17 and CCL22 with CCR4 on the surface of tumor cells, promote the ADCC effect of effector cells on the tumor cells, and provide new possibility for the treatment and/or prevention of cancers.)

1. An anti-CCR 4 antibody consisting of a heavy chain and a light chain, wherein the heavy chain comprises the VH-CDR1 amino acid sequence shown as SEQ ID NO. 3, the VH-CDR2 amino acid sequence shown as SEQ ID NO. 4, and the VH-CDR3 amino acid sequence shown as SEQ ID NO. 5; the light chain comprises the amino acid sequence of VL-CDR1 shown in SEQ ID NO. 6, the amino acid sequence of VL-CDR2 shown in SEQ ID NO. 7, and the amino acid sequence of VL-CDR3 shown in SEQ ID NO. 8.

2. The anti-CCR 4 antibody according to claim 1, characterized in that: the heavy chain comprises a heavy chain variable region as shown in SEQ ID No. 1; the light chain comprises a light chain variable region shown as SEQ ID No. 2.

3. An expression vector for replication in a prokaryotic or eukaryotic cell line, characterized in that: encoding an antibody according to any one of claims 1-2.

4. A pharmaceutical composition comprising an antibody according to any one of claims 1-2.

5. Use of an anti-CCR 4 antibody according to any one of claims 1-2 for the manufacture of a medicament for the treatment of cancer.

6. The use of claim 5, wherein the cancer is selected from cutaneous T-cell lymphoma (CTCL), Mycosis Fungoides (MF), primary cutaneous anaplastic large cell lymphoma (cutaneous ALCL), adult T-cell leukemia/lymphoma (ATLL), renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, renal cancer, gastric cancer, or acute lymphatic leukemia.

7. The use of claim 6, wherein the cancer is acute lymphocytic leukemia.

Technical Field

The invention relates to the field of biomedicine, in particular to an anti-CCR 4 antibody and application thereof in treating various hematological malignancies and solid tumors.

Background

Chemokine receptors are seven transmembrane receptors expressed on the surface of some specific cells, and their encoded proteins belong to the G protein-coupled receptor family, which can be classified into four classes according to their ligands: CXC type, XC type, CC type, CX₃And (3) type C. Type CC chemokine receptor 4(CCR4), also known as CD194, is expressed predominantly on T cells (especially Th2 cells and Treg cells), and is also found expressed on dendritic cells, macrophages, NK cells, platelets, mast cells and certain tumor cells. CCR4 has three natural specific ligands: thymus activation-regulated chemokine (TRAC/CCL17), macrophage-derived chemokine (MDC/CCL22), and chemokine-like factor 1(CKLF 1).

With the discovery of CCR4 and its three ligands, more and more studies indicate that the binding of CCR4 and its ligands CCL17, CCL22, CKLF1 is closely related to the biological behavior, metastasis, of T-cell leukemia, lymphoma cell tumors, and solid tumors. Lee et al examined and studied gastric cancer cell lines, found that they all expressed CCR4, but no expression of CCR4 was found in the epithelium of normal gastric tissues, and that CCR4 positive expression has significant correlation with gross typing, depth of invasion, lymph node metastasis, pathological staging, recurrence, overall survival of gastric cancer. And also demonstrates that CCR4 overexpression can promote tumor enlargement and knockout of CCR4 gene can induce tumor reduction in a mouse breast cancer model. In vitro mouse gastric cancer peritoneal metastasis model shows that gastric cancer cells with positive CCR4 expression can stimulate macrophages of omentum majus to secrete CCL22 after entering the abdominal cavity, and the combination of the gastric cancer cells and the CCL can chemotactic and induce gastric cancer peritoneal metastasis. It has also been shown that chemokine receptor CCR4 is associated with breast cancer HER2 expression, lymph node metastasis. In addition, Biragyn et al report that targeting to interfere with CCR4 + Tregs cells can reduce the metastatic potential of breast cancer cells, even block breast cancer lung metastasis. Through blocking the combination of CCR4 and CCL17 and CCL22, Treg cells near the tumor are reduced, so that the self anti-tumor immunity of the body is maintained, and therefore CCR4 becomes a potential ideal target of an anti-tumor drug.

Disclosure of Invention

Based on the above findings, the primary object of the present invention is to provide a novel CCR4 antibody that has high affinity and can block the binding of CCR4 to a ligand.

The invention provides the following technical scheme:

a humanized anti-CCR 4 antibody consisting of a heavy chain and a light chain comprising a variable region and a constant region, respectively, wherein the heavy chain variable region sequence is SEQ ID NO 1 and the light chain variable region sequence is SEQ ID NO 2.

The heavy and light chain variable regions of the CCR4 antibody of the present invention each have 3 Complementarity Determining Regions (CDRs). 3 CDR sequences of a heavy chain variable region of the CCR4 antibody are respectively SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 5; the variable region of light chain has 3 CDR sequences of SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8.

The heavy and light chains of the CCR4 antibodies of the invention further comprise constant regions, and the antibody light chain constant regions comprise human kappa and lambda chain sequences, and further preferably kappa chains. The antibody heavy chain constant region comprises human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM, and more preferably IgG 1.

In some embodiments, the CCR4 antibody is capable of antagonizing CCR4 activity, wherein antagonism blocks binding between CCR4 and ligands CCL17, CCL22, blocking CCR4 activity.

In some embodiments, the CCR4 antibody antagonists of the present invention can be used to treat cancer, including but not limited to: cutaneous T Cell Lymphoma (CTCL), mycosis granulomatosis (MF), primary cutaneous anaplastic large cell lymphoma (cutaneous ALCL), adult T cell leukemia/lymphoma (ATLL), renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney cancer, or stomach cancer.

The present invention provides a method of treating cancer, characterized by: the anti-CCR 4 antibody is used for treating cancers such as Cutaneous T Cell Lymphoma (CTCL), mycosis granulosa (MF), primary cutaneous anaplastic large cell lymphoma (cutaneous ALCL), adult T cell leukemia/lymphoma (ATLL), renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, prostatic cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney cancer, stomach cancer or acute lymphocytic leukemia.

The CCR4 antibody can be applied to preparation of a medicine for treating and/or preventing cancers, wherein the cancers are selected from Cutaneous T Cell Lymphoma (CTCL), mycosis granulomatosis (MF), primary cutaneous anaplastic large cell lymphoma (cutaneous ALCL), adult T cell leukemia/lymphoma (ATLL), renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, prostatic cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney cancer or stomach cancer and the like.

The term "antibody" as used herein includes any moiety having immunoglobulin-like binding function. The term includes whole antibody molecules and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof, camelid antibodies including, for example, nanobodies, phage display binding constructs, and the like. Natural bovine antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region consisting of three domains: CH1, CH2 and CH3 only two types of light chains: λ and κ. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions may also be subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). As found in nature, each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. Antibodies can be monoclonal or polyclonal.

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules, all of which share a single molecular component. Monoclonal antibodies thus exhibit unique binding characteristics and affinities for a particular epitope.

The term "polyclonal antibodies" as used herein refers to antibody compositions having a heterogeneous population of antibodies. Polyclonal antibodies are typically derived from collected sera of immunized animals or from selected humans.

The term "hypervariable region" or "CDR region" or "complementarity determining region" as used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. CDR region sequences can be defined by Kabat, Chothia method definition or the field of any known CDR region sequence determination method and identification of the variable region within amino acid residues. The methods used in the present invention may utilize or be defined according to CDRs defined by any of these methods, including but not limited to any of the Kabat definitions, Chothia definitions. In particular, the CDR sequences provided herein are according to the Kabat definition.

The term "humanized antibody" as used herein generally refers to a chimeric antibody that contains fewer sequences from non-human immunoglobulins, thereby reducing the immunogenicity of the xenogenous antibody when introduced into humans, while maintaining the full antigen-binding affinity and specificity of the antibody. For example, CDR grafting and variants thereof can be used; the method comprises technical means such as remodeling, high-degree addition, veneering, surface reconstruction and the like, and humanizes the non-human-derived binding domain. Other regions, such as the hinge region and constant region domains, may also be humanized if they are also derived from non-human sources.

The term "blocking binding" as used herein refers to the ability of an antibody or antigen-binding fragment thereof to inhibit binding between two molecules (e.g., human CCR4 and ligand CCL17 or CCL 22). In certain embodiments, an antibody or antigen-binding fragment that blocks binding between two molecules can inhibit the interaction of binding between two molecules by at least 85% or at least 90%.

The term "ADCC" as used herein refers to the biological activity of an Fc region of an antibody to bind to an Fc receptor. Antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis induced by binding of the Fc region of an antibody to Fc receptors on effector cells.

Advantageous effects

The invention has the following beneficial effects: the antibody can bind to CCR4 protein with higher affinity, block the binding of ligands CCL17 and CCL22 to CCR4 on the surface of a tumor cell, promote the ADCC effect of an effector cell on the tumor cell, and provide new possibility for the treatment and/or prevention of cancer.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

FIG. 1 shows ADCC activity of Hu-11F6-IgG1 against CCRF-CEM cells.

FIG. 2 is a NSG mouse CCRF-CEM tumor model.

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.