CN111704668A

CN111704668A - anti-CCR 4 antibodies and their use in treating cancer

Info

Publication number: CN111704668A
Application number: CN202010595527.9A
Authority: CN
Inventors: 彭菲; 顾超
Original assignee: Beijing Yuehao Technology Development Co ltd
Current assignee: Tianjin Jinyu Medical Laboratory Co.,Ltd.
Priority date: 2020-06-27
Filing date: 2020-06-27
Publication date: 2020-09-25
Anticipated expiration: 2040-06-27
Also published as: CN111704668B

Abstract

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.

Description

anti-CCR 4 antibodies and their use in treating cancer

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.

There are some CCR4 antibodies disclosed in the prior art, such as CN107250160A, CN104436191A, CN103328513B, US20170290911, etc., but there is still a great need for new anti-CCR 4 antibodies.

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.

Example 1 anti-CCR 4 antibody preparation and purification

Immunization of C57BL with high levels and stable CCR4 expressing CHO cells expressing CCR4 on their cell membraneAnd 6 mice. Will 10⁷Individual CCR4⁺CHO cells were injected into the abdominal cavity of female C57BL/6 mice. Immunizing for 1 time every two weeks, and immunizing for 5-8 times; last immunization CCR4⁺Killing the mice 4 days after CHO cells, taking spleens of the mice, fusing the cells, mixing the cells according to the proportion of splenocytes to SP2/0 cells to 1:1, centrifuging at 1500rpm for 7min, then discarding supernatant, resuspending the cells by using 20mL of electrotransformation buffer, centrifuging at 1500rpm for 7min, discarding supernatant, repeating the steps once, and respectively resuspending the cells by using proper amount of electrotransformation buffer to ensure the cell concentration to be 2 × 10⁷Adding the cell suspension into 9mL electrical transfusing tank to fuse, transferring the cell suspension into 15mL RPMI 1640 complete culture medium containing 20% FBS after fusing, standing at room temperature for 20min, suspending the fused cells by using RPMI 1640 medium containing 1 × HAT, 1 × BIOMYC3 and 20% FBS, adding the cell suspension into a plurality of 96-well cell culture plates according to 100 mul/well, and ensuring that the cell amount per well is about 4 × 10⁴The method comprises the following steps of culturing the cells/hole in a 37 ℃ cell culture box, supplementing 100 mu L/hole RPMI 1640 complete culture medium (containing 20% FBS, 1 × HAT and 1 × BIOMYC-3) after 5 days, fusing for one week, taking cell supernatant, obtaining hybridoma cell strains which are positive in 2 screening experiments by using a direct ELISA (enzyme-linked immunosorbent assay) combination method and a ligand combination blocking test from the harvested supernatant, subcloning the positive cell strains by using a limiting dilution method, detecting the combination activity of the subcloned supernatant and CCR4 molecules and the combination activity of blocking CCR4 and ligand by using ELISA after culturing for one week, and finally obtaining a double-positive cell strain which is marked as 11F6.

Example 2 antibody humanization

The subcloned positive hybridoma cells were expanded and cultured, and a suitable amount of cells were extracted for total RNA according to the instructions of RNeasy PlusMini Kit (Qiagen, 74134), and first strand cDNA was synthesized using TransScript One-Step gDNAremoval and cDNA Synthesis Supermix (TRANS, AT311-02) reverse transcription Kit. Specific primers (the 5' end contains a homologous arm sequence for homologous recombination with a eukaryotic expression vector) are designed according to the mouse antibody subtype variable region, and the cDNA is taken as a template to carry out PCR amplification on antibody variable region genes, so that gene fragments of mouse antibody light chain and heavy chain variable regions are respectively obtained. Humanization of antibodies was first performed by aligning the amino acid sequences of the VH and VK domains of the antibody with the amino acid sequences of all known human germline VH and VK domains using currently available public databases (i.e., Blast for IgG from NCBI and V-base from MRC). By observing the alignment of amino acids within the framework regions, highly homologous human germline VH and VK domains are determined. On the basis of the framework structure, the computer model is used for analyzing the variable region steric structure of the mouse anti-CCR 4 antibody 11F6, and the corresponding framework amino acid residues of the mouse antibody required to be reserved for supporting CDR configuration are analyzed. The mouse frameworks are transformed or mutated in an iterative manner to match the corresponding human germline frameworks. The synthetic VH and VK domains were cloned into a specialized mammalian expression vector that enabled expression of the respective domains in the complete human IgG1 or kappa antibody backbone. The lgG 1 construct was co-transfected with the kappa construct into 293F cells for small-scale preparation of humanized antibodies. The supernatant of the transient transfection was passed through protein a or G resin to obtain purified CCR4 antibody. Finally, the humanized and transformed antibody is named as Hu-11F6-IgG1, the amino acid sequence of the VH chain of the antibody Hu-11F6-IgG1 is shown as SEQ ID NO. 1, and the amino acid sequence of the VL chain is shown as SEQ ID NO. 2; wherein the amino acid sequences of the heavy chain hypervariable region VH-CDR1, VH-CDR2 and VH-CDR3 are respectively shown as SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5; the amino acid sequences of the light chain hypervariable regions VL-CDR1, VL-CDR2 and VL-CDR3 are shown in SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8, respectively.

Example 3 specific binding of Hu-11F6-IgG1 to CCR4 on the surface of tumor cells

The CCR4 positive cell line, human breast cancer cell line MCF-7, was selected and tested for binding activity against the CCR4 antibody to the CCR4 protein expressed on the surface of tumor cells, a cell-based ELISA method was used MCF-7 cells were plated in 96 well cell culture plates, each well plated with 1 × 10⁵The cells were cultured overnight at 37 ℃. The CCR4 antibody Hu-11F6-IgG1 to be tested and the control antibody 503 (the control antibody sequence is obtained on page 23 of the CN103328513B patent specification, and the control antibody 503 is obtained according to the method in example 2) are treated with 10% FBS-containing solutionThe DMEM medium was subjected to 3-fold gradient dilution at a concentration of 1.83ng/mL to 4. mu.g/m L, and then added to the corresponding wells in a volume of 50. mu.L per well, respectively, and the cell culture plates were incubated at 4 ℃ for 1 h. The primary antibody was removed and washed 5 times with PBS containing 1% Tween 20, pH 7.4. HRP-labeled goat anti-human IgG antibody was reacted as a secondary antibody. mu.L of the suspension was added to each well and incubated at 4 ℃ for 40 min. The secondary antibody was removed and washed 5 times with 1% Tween 20 in PBS pH 7.4. Adding color development solution, incubating for 15-25min in dark, adding stop solution, and stopping color development reaction. The absorbance at 492/630nm was read using a microplate reader. The data were processed to finally determine the EC50 value for each sample, and the results are shown in the table below.

TABLE I determination of the binding Capacity of anti-CCR 4 antibodies to CCR4 on the surface of tumor cells

Name (R)	EC50(ng/ml)
		Hu-11F6-IgG1	43.2±11.5
Control antibody-503	109.4±23.7

Example 4 detection of anti-CCR 4 antibody blocking Activity

FACS detection of the ability of the anti-CCR 4 antibody prepared in example 2 to block the binding of CCL17 or CCL22 ligand to the cell surface CCR4 receptor non-Hodgkin lymphoma cells L-428 were resuspended and added to 96-well plates, 1 × 10 per well⁵And (4) cells. The diluted anti-CCR 4 antibody Hu-11F6-IgG1 and control antibody-503 were added to respective wells at a concentration of 100. mu.L per well, ranging from 0.128ng/mL to 10. mu.g/mL. The antibody was bound to the cells for 1h at 4 ℃. After removing the primary antibody, the mixture was washed with a solution containing 0.2% BSA and 0.09% NaN3 were washed three times in PBS, 150ng/mL FITC labeled CCL17 or CCL22 ligand protein was added to each sample, incubated at 4 ℃ for 40min, and similarly washed three times in PBS containing 0.2% BSA and 0.09% NaN 3. And finally, carrying out flow detection. The results are shown in Table two, which indicates that anti-CCR 4 antibody Hu-11F6-IgG1 blocks the binding of ligand CCL17 or CCL22 to CCR4 receptor, and thus anti-CCR 4 antibody Hu-11F6-IgG1 has CCR4 blocking activity.

Watch two

Antibodies	CCL17 IC₅₀(ng/mL)	CCL22 IC₅₀(ng/mL)
			Hu-11F6-IgG1	7.8	25.3
Control antibody-503	7.4	36.2

Example 5 ADCC assay

The in vitro ADCC assay was performed to determine whether Hu-11F6-IgG1 kills tumor cells with NK cells through Fc-Fc γ R interaction mediated cytotoxicity. Human peripheral blood mononuclear cells were isolated in vitro as effector cells (Ec) and human acute lymphoblastic leukemia T lymphocytes CCRF-CEM as target cells (Tc). Target cells CCRF-CEM were dispensed into 96-well plates at 2X10 per well⁴(ii) individual cells; then, 10. mu.L of anti-CCR 4 antibody Hu-11F6-IgG1 diluted in a concentration gradient was added to each well. After incubation at 37 ℃ for 15 minutes,add 4 × 10 to each well⁵PBMCs to give 20: an E/T ratio of 1. After incubation at 37 ℃ for 4 hours, the 96-well plate was centrifuged at 400g for 5min, and 120. mu.l of the supernatant from each well was added to a new 96-well plate and subjected to cytotoxicity assay using a lactate dehydrogenase cytotoxicity detection kit (Roche). As a result, as shown in FIG. 1, the anti-CCR 4 antibody Hu-11F6-IgG1 induced potent ADCC activity on CCRF-CEM cells.

Example 6 NSG mouse CCRF-CEM tumor model

Culturing sufficient CCRF-CEM tumor cells in logarithmic growth phase, suspending in 1640 medium at a cell concentration of 5 × 10⁷One cell/ml. each NSG mouse was injected with 200. mu.l CCRF-CEM tumor cells via tail vein, i.e., one mouse was injected with 1 × 10⁷The NSG mice are randomly divided into 3 groups, 6 mice are divided into a negative control group injected with IgG, an experimental group injected with Hu-11F6-IgG1 and an experimental group injected with 503, the injection concentration of the antibody is 30 mg/kg., the injection concentration of the antibody is injected once every three days, the antibody treatment is carried out for 10 times, the length and the short diameter of the tumor are measured simultaneously, the size of the tumor is calculated, the tumor volume calculation formula is that the volume is (the long diameter × short diameter × short diameter)/2, the result is shown in figure 2, after the mice are treated with the anti-CCR 4 antibody, the growth speed of the tumor of the mice is obviously slowed, and the anti-CCR 4 antibody Hu-11F6-IgG1 can inhibit the growth of the tumor.

Sequence listing

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Claims

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.