WO2024255467A1

WO2024255467A1 - Anti-ccr8 antibody and anti-ccr8/ctla4 bispecific antibody

Info

Publication number: WO2024255467A1
Application number: PCT/CN2024/090136
Authority: WO
Inventors: Qinglin Du; Tiantian ZHOU; Yiqi CAO; Fei Peng; Xueyan YANG; Fei HU; Shuhua Han
Original assignee: GENOR BIOPHARMA CO Ltd
Current assignee: GENOR BIOPHARMA CO Ltd
Priority date: 2023-06-16
Filing date: 2024-04-26
Publication date: 2024-12-19
Anticipated expiration: 2025-12-16
Also published as: CN121152808A

Abstract

An anti-CCR8 antibody or antigen-binding fragment thereof, or an anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof, nucleic acid encoding the same, pharmaceutical compositions comprising the same, a method for producing the same, and the uses thereof.

Description

Anti-CCR8 Antibody and anti-CCR8/CTLA4 Bispecific Antibody

FIELD OF THE INVENTION

The present disclosure relates to biomedicine field, and in particular to an anti-CCR8 antibody or antigen-binding fragment thereof that specifically binds to CCR8, an anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof that specifically binds to CCR8 and CTLA4, a method for producing the same and the use thereof.

BACKGROUND

Tumor immunotherapy has achieved revolutionary success, but tumor tissues are infiltrated with a large number of immunosuppressive cells, resulting in a low response rate to tumor immunotherapy in patients with different types of tumors. For the treatment of solid tumors, the most important aspect is to remove the inhibition of tumor microenvironment (TME) on immune effector cells in order to improve the therapeutic effect.

Regulatory T cells (Tregs) in the tumor microenvironment are associated with tumor progression and may be a major obstacle to tumor immunotherapy. Therefore, removal of tumor-infiltrating Tregs (TI-Tregs) is a promising approach to overcome the resistance to immunotherapy while maintaining peripheral homeostasis. Regulatory T cells (Tregs) are a suppressive CD4+T-cell subset, which are indispensable for preventing autoimmunity, also suppress effective tumor immunity [1, 2] . Tregs abundantly infiltrate into tumor tissues, which is often associated with poor prognosis in cancer patients [3] . It has been reported that elimination of Tregs is also able to evoke strong antitumor immune responses in tumor-bearing animals [4-6] . However, removal of Tregs may also elicit autoimmunity [7] . How to specifically deplete Tregs infiltrating into tumor tissues without affecting tumor reactive effector T cells (Teffs) and suppressing autoimmunity, is a key issue in devising Tregs-targeting cancer immunotherapy.

CTLA-4 is a widely known Tregs target and anti-CTLA-4 antibodies have demonstrated potential and durable antitumor activity clinically both as monotherapy and in combination with PD-1 targeting therapies, but the severe immunotherapy-related adverse effects (irAEs) due to a systemic activation of T cells may limit their use. CTLA-4 is constitutively expressed on T regulatory cells (Tregs) and is upregulated on other T cells upon activation [8, 9] . CTLA-4 is highly expressed in the TME, especially on Tregs, which is considered to be a key molecule in controlling Treg function and modulating anti-tumor immunity [10, 11] . So far, several mechanisms of action of therapeutic CTLA-4 antibodies have been proposed, including activation of effector T cells by blocking the B7-CTLA-4 pathway and depletion of Tregs via antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) [12, 13] . However, the relative clinical importance of these mechanisms is still controversial [13] .

Ipilimumabis the best known anti-CTLA-4 antibody which is approved in 2011 for the treatment of advanced melanoma. Ipilimumab has exhibited strong and broad cancer immunotherapeutic effects (CITE) clinically both as monotherapy [14] and as part of combination therapy with Nivolumab [15] . However, CTLA-4 therapy demonstrates severe immunotherapy-related adverse effects (irAEs) [16] , especially in combination with Nivolumab due to a systemic activation of T cells by blocking the B7-CTLA-4 pathway, leading to limited doses tolerated by cancer patients [17, 18] .

One approach to improve the therapeutic window and reduce irAEs of anti-CTLA-4 antibodies is to direct the effect to the TME by bispecific antibodies (bsAbs) [19] . CCR8 is a chemokine receptor that has recently been identified as a potential specific marker for tumor-infiltrating Tregs (TI-Tregs) [20, 21] , since CCR8 is selectively up-regulated in these Tregs in multiple cancers, including breast, colorectal and lung cancers, while rarely expressed on Tregs and effector T cells (Teffs) in peripheral blood or in other tissues [22] .

Therefore, there is an unmet need for a bispecific antibody which simultaneously targets these two cell surface antigens (CCR8 and CTLA-4) with overlapping on TI-Tregs to reduce the exposure of antibodies in the periphery. By targeting two receptors overlapping on TI-Tregs, there is a potential to increase localization to the TME compared to monospecific antibodies, resulting in reduction of systemic T-cell activation and improvement of response rate.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides antibody or antigen-binding fragment thereof that specifically binds to human C-C Motif Chemokine Receptor 8 (CCR8) , comprising: a heavy chain complementarity determining region (HCDR) 1 having the amino acid sequence at least 80% identical to SEQ ID NO: 1, a HCDR2 having the amino acid sequence at least 80% identical to SEQ ID NO: 2, a HCDR3 having the amino acid sequence at least 80% identical to SEQ ID NO: 3, a light chain complementarity determining region (LCDR) 1 having the amino acid sequence at least 80%identical to SEQ ID NO: 4, a LCDR2 having the amino acid sequence at least 80% identical to SEQ ID NO: 5, and a LCDR3 having the amino acid sequence at least 80% identical to SEQ ID NO: 6.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 3, a LCDR 1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4, a LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 6.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region having the amino acid sequence at least 80% identical to SEQ ID NO: 7 or SEQ ID NO: 13, and a light chain variable region having the amino acid sequence at least 80% identical to SEQ ID NO: 8 or SEQ ID NO: 14.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 13, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 14.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15, and a light chain having the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the antibody or antigen-binding fragment thereof is a human antibody, humanized antibody, chimeric antibody, monoclonal antibody, polyclonal antibody, recombinant antibody, diabody, triabody, tetrabody, Fab fragment, F (ab') ₂ fragment, scFv fragment, Fv fragment, Fab'fragment or domain antibody.

In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that competes for binding to CCR8 with the antibody or antigen-binding fragment thereof as described above.

In one aspect, the present disclosure provides a bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof, comprising a first domain specifically binding CCR8 and a second domain specifically binding CTLA-4, wherein the first domain comprises the antibody or antigen-binding fragment thereof as described above.

In some embodiments, the first domain is the antibody or antigen-binding fragment thereof as described above.

In some embodiments, the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof has at least one of the following properties:

a) strong induction of ADCC response to kill human Treg cells or CHOK1-human CCR8/CTLA-4 over expressing cells;

b) reduced tumor growth and significantly prolonged survival than anti-CCR8 antibody;

c) partial blocking of CD80 or CD86-CTLA4 interaction on CHOK1-human CTLA-4 overexpressing cells but significant blocking of the binding of CD80 or CD86 to CTLA-4 on CHOK1-human CCR8/CTLA-4 overexpressing cells;

d) better tolerance and longer survival than ipilimumab;

e) better in vivo anti-tumor efficacy compared to anti-CCR8 antibody and ipilimumab in a dose-dependent manner.

In some embodiments, the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof is afucosylated.

In some embodiments, the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof is a human antibody or humanized antibody.

In some embodiments, the second domain comprises an anti-CTLA-4 nanobody.

In some embodiments, the second domain comprises the amino acid sequence at least 80% identical to SEQ ID NO: 22 or SEQ ID NO: 23.

In some embodiments, the second domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23.

In some embodiments, the bispecific anti-CCR8/CTLA-4 antibody further comprises an Fc region of IgG. In one embodiment, the Fc region is of IgG1.

In some embodiments, the first domain specifically binding to CCR8 and the second domain specifically binding to CTLA-4 are connected to each other directly or via one or more linkers. In one embodiment, the linkers are same or different. In one embodiment, the linkers are a flexible connection. In one embodiment, the linker is a peptide linker.

Accordingly, the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof further comprises one or more linkers, optionally, the linker comprises one or more amino acids selected from glycine and serine.

In some embodiments, the one or more linkers are inserted at the C-terminal, N-terminal, or both terminals of the second domain.

In some embodiments, the linker comprises one or more of the following amino acid sequences: GGGGSGGGGS (SEQ ID No: 24) and GS.

In some embodiments, the linker comprises GGGGSGGGGS and GS.

In some embodiments, the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof comprises: a heavy chain having the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 20, and a light chain having the amino acid sequence of SEQ ID NO: 10.

In one aspect, the present disclosure provides a nucleic acid encoding the antibody or antigen-binding fragment thereof or the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof as described above.

In some embodiments, the nucleic acid comprises: a heavy chain encoding nucleic acid having the nucleotide sequence ofSEQ ID NO: 11 or 16; and a light chain encoding nucleic acid having the nucleotide sequence of SEQ ID NO: 12 or 17.

In some embodiments, the nucleic acid comprises: a heavy chain encoding nucleic acid having the nucleotide sequence of SEQ ID NO: 19 or 21; and a light chain encoding nucleic acid having the nucleotide sequence of SEQ ID NO: 17.

In one aspect, the present disclosure provides an expression vector comprising the nucleic acid as described above.

In one aspect, the present disclosure provides a host cell comprising the expression vector as described above.

In one aspect, the present disclosure provides a pharmaceutical composition comprising: the antibody or antigen-binding fragment thereof, the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof, the nucleic acid, or the expression vector as described above, and a pharmaceutically acceptable carrier.

In one aspect, the present disclosure provides a method of producing the antibody or antigen-binding fragment thereof, or the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof as described above, comprising culturing the host cell as described above under conditions that allow the expression of the antibody or antigen-binding fragment thereof.

In one aspect, the present disclosure provides a method of treating a disease or condition associated with the abnormal expression of CCR8 and/or CTLA-4 in a subject, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof, the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof, the nucleic acid, the expression vector, the host cell, or the pharmaceutical composition as described above to the subject.

In one aspect, the present disclosure provides use of the antibody or antigen-binding fragment thereof, or the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof as described above, in the manufacture of a medicament for treating a disease associated with the abnormal expression of CCR8 and/or CTLA-4 in a subject.

In some embodiments, the disease is a cancer.

In some embodiments, the cancer is: a solid tumor selected from the group consisting of squamous cell carcinoma, small cell lung cancer (SCLC) , non-small cell lung cancer (NSCLC) , squamous NSCLC, non-squamous NSCLC, head and neck cancer, breast cancer, cancer of the esophagus, gastric cancer, gastrointestinal cancer, cancer of the small intestine, liver cancer, hepatocellular carcinoma (HCC) , pancreatic cancer (PAC) , kidney cancer, renal cell carcinoma (RCC) , bladder cancer, cancer of the urethra, cancer of the ureter, colorectal cancer (CRC) , colon cancer, colon carcinoma, cancer of the anal region, endometrial cancer, prostate cancer, a fibrosarcoma, neuroblastoma, glioma, glioblastoma, germ cell tumor, pediatric sarcoma, sinonasal natural killer, melanoma, skin cancer, bone cancer, cervical cancer, uterine cancer, carcinoma of the endometrium, carcinoma of the fallopian tubes, ovarian cancer, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, testicular cancer, cancer of the endocrine system, thyroid cancer, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the penis, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS) , primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain cancer, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, solid tumors of childhood, environmentally-induced cancers, virus-related cancers, cancers of viral origin, advanced cancer, unresectable cancer, metastatic cancer, refractory cancer, recurrent cancer, and any combination thereof; or a hematological malignancy selected from the group consisting of acute lymphoblastic leukemia (ALL) , acute myelogenous leukemia (AML) , chronic lymphocytic leukemia (CLL) , chronic myelogenous leukemia (CML) , a T cell lymphoma, Hodgkin’s lymphoma (HL) , non-Hodgkin’s lymphomas (NHLs) , multiple myeloma, smoldering myeloma, monoclonal gammopathy of undetermined significance (MGUS) , advanced, metastatic, refractory and/or recurrent hematological malignancies, and any combinations of said hematological malignancies.

In some embodiments, the cancer is bladder cancer.

These and other aspects are described in more detail herein. Each of the aspects provided may include a variety of embodiments provided herein. It is therefore expected that each of the aspects described may include each of the embodiments involving an element or a combination of elements, and all such combinations of the aspects and embodiments are explicitly taken into account.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. Binding of anti-CCR8 antibodies to cell surface human CCR8 protein.

Fig. 2. Inhibition of anti-CCR8 antibodies on the binding of CCR8 to its ligand CCL1.

Fig. 3. Binding of anti-CCR8 antibodies to in-vitro induced human Treg cell.

Fig. 4. Inhibition of anti-CCR8 antibodies on the binding of CCR8 to its ligand CCL1 on in-vitro induced human Treg cell.

Fig. 5. Binding of anti-CCR8 antibodies to cell surface cyno-CCR8 protein.

Fig. 6. ADCC (antibody dependent cellular cytotoxicity) bioassay using PBMC as effector cells incubation with afucosylated (AF) anti-CCR8 antibodies and in-vitro induced human Treg target cells (E: T=20: 1) .

Fig. 7. Changes in tumor growth in MC38 model after anti-CCR8 antibodies administration.

Fig. 8. Survival proportions of anti-CCR8 antibodies administered MC38 model.

Fig. 9. Schematic diagram of bsAb-38-64-05-3 tetravalent structure.

Fig. 10A-C Binding of bsAb-38-64-05-2 or bsAb-38-64-05-3, parental CCR8 mAb (HC64-23-AF) or parental CTLA-4 nanobody (GBD008-hS005-3-2) to CCR8 single-positive cells (A) , CTLA-4 single-positive cells (B) and CCR8/CTLA-4 double-positive cells (C) .

Fig. 11A-B. Inhibition of bsAb-38-64-05-2 or parental CCR8 mAb (HC64-23-AF) on the binding of CCR8 to its ligand CCL1 on CCR8 single-positive cells (A) and CCR8/CTLA-4 double-positive cells (B) .

Fig. 12A-B. Inhibition of bsAb-38-64-05-2 or bsAb-38-64-05-3 or parental CTLA-4 nanobody (GBD008-hS005-3-2) on the binding of CTLA-4 to its ligand CD80 on CTLA-4 single-positive cells (A) and CCR8/CTLA-4 double-positive cells (B) .

Fig. 13A-B. Inhibition of bsAb-38-64-05-2 or parental CTLA-4 nanobody (GBD008-hS005-3-2) on the binding of CTLA-4 to its ligand CD86 on CTLA-4 single-positive cells (A) and CCR8/CTLA-4 double-positive cells (B) .

Fig. 14A-B. Binding of bsAb-38-64-05-2 or bsAb-38-64-05-3 or parental nanobody to cell surface cyno-CCR8 protein (A) or coated cyno-CTLA-4 protein (B) .

Fig. 15. ADCC (antibody dependent cellular cytotoxicity) bioassay using PBMC as effector cells incubation with bsAb-38-64-05-2, parental CCR8 mAb (AF) (HC64-23-AF) or parental CTLA-4 nanobody (GBD008-hS005-3-2) and CHOK1-hCCR8/hCTLA-4 target cells (E: T=20: 1) .

Fig. 16. Changes of body weight gain in ten-days old C57BL/6 CCR8/CTLA-4 double knock-in mice after bsAb-38-64-05-2 or ipilimumab combination with anti-mPD1 administration.

Fig. 17. Survival proportions of bsAb-38-64-05-2 or ipilimumab combination with anti-mPD1 administered ten-days old C57BL/6 CCR8/CTLA-4 double knock-in mice.

Fig. 18. Changes in tumor growth in MB49 model after bsAb-38-64-05-2or bsAb-38-64-05-3 antibodies administration in C57BL/6 CCR8/CTLA-4 double knock-in mice.

DETAILED DESCRIPTION OF THE INVETION

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though fully set forth.

Unless otherwise defined herein, the scientific and technical terms used herein should have the meaning generally understood by those skilled in the art. In addition, singular terms are intended to include plural terms and vice versa unless expressly indicated otherwise in the context. In order to facilitate reading the present application, certain terms are defined below.

Definitions

As used herein, the indefinite articles “a” or “an” should be understood to refer to “one or more” of any recited or enumerated component.

As used herein, the term “about” , when applied to a numeric value, refers to a value that is reasonably close to the stated value and within an acceptable error range as determined by those skilled in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean a range of plus or minus 50% of a stated reference value, preferably a range of plus or minus 25%, or more preferably a range of plus or minus 10%. When particular values are provided in the application, the meaning of “about” , unless otherwise stated, should be understood to be within an acceptable error range for that particular value according to the practice in the art.

An “antibody” (Ab) shall include, without limitation, a glycoprotein immunoglobulin (Ig) which binds specifically to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding fragment thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as V_H) and a heavy chain constant region. The heavy chain constant region of an IgG Ab comprises three constant domains, CH₁, CH₂ and CH₃. Each light chain comprises a light chain variable region (abbreviated herein as V_L) and a light chain constant region. The light chain constant region of an IgG Ab comprises one constant domain, C_L. The V_H and V_L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) , interspersed with regions that are more conserved, termed framework regions (FR) . Each V_H and V_L comprises three CDRs (LCDRs including LCDR1, LCDR2, and LCDR3, heavy chain CDRs including HCDR1, HCDR2, HCDR3) and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. A variety of methods have been used to delineate the CDR domains within an Ab, including the Rabat, Chothia, AbM, contact, and IMGT definitions. Unless specifically indicated, Kabat numbering is used in the present disclosure as a default. The constant regions of the Abs may mediate the binding of the Ig to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM. Several of the major antibody classes are divided into subclasses such as IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

As used herein, the term “antibody” includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, bispecific antibody that binds to a specific antigen. An intact antibody or an antibody fragment having the antigen binding portion of the antibody can be used. The term “antigen-binding fragment” as used herein refers to an antibody fragment formed from a portion of an intact antibody comprising one or more CDRs, or any other antibody fragment that can bind to an antigen but does not comprise an intact native antibody structure. The term “antibody or antigen-binding fragment thereof” as used herein refers to an intact antibody or an antibody fragment having an antigen-binding portion. The antigen-binding portion can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of an intact antibody. The antibody or antigen-binding fragment thereof includes, without limitation, Fab, Fab', F (ab') ₂, Fv, domain antibody (dAb) , fragments comprising complementarity determining regions (CDRs) , single chain variable fragment (scFv) , chimeric antibody, diabody, triabody, tetrabody, and a polypeptide containing at least a portion of the immunoglobulin that is sufficient to impart specific antigen-binding to the polypeptide.

“Fab fragment” is a monovalent fragment having the V_L, V_H, C_L, and CH₁ domains. “F (ab') ₂ fragment” is a bivalent fragment having two Fab fragments connected in the hinge region by a disulfide bond. “Fv fragment” has the V_L and V_H domains derived from a single arm of an antibody. “domain antibody (dAb) ” consists of the V_H or V_L domains. “Single-chain variable fragment (scFv) ” is an antibody in which the V_L and V_H regions are connected to form a continuous protein chain via a linker (e.g., a synthetic sequence of amino acid residues) , wherein the linker is sufficient in length to allow the protein chain to form a monovalent antigen binding site. The term “diabody” is a divalent antibody comprising two polypeptide chains, wherein each polypeptide chain comprises the V_H and V_L domains connected by a linker that is too short to allow the two domains on the same chain to be paired, thereby allowing each domain to be paired with the complementary domain on the other polypeptide chain. If the two polypeptide chains of the diabody are same, the resulted diabody will have two identical antigen binding sites. The polypeptide chains having different sequences can be used to produce a diabody or a bispecific antibody having two different antigen binding sites. The diabody or bispecific antibody also refers to an artificial antibody or an antigen-binding fragment which has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes. The two epitopes may present on the same antigen, or they may present on two different antigens. Similarly, the triabody, tetrabody or other multispecific antibody are antibodies comprising three, four or multiple polypeptide chains that may be same or different, and thus form three, four or multiple antigen binding sites that may be same or different, respectively.

As used herein, the term “human” Ab refers to an Ab having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the Ab contains a constant region, the constant region also is derived from human germline immunoglobulin sequences.

As used herein, the term “humanized” Ab refers to an Ab in which some, most or all of the amino acids outside the CDR domains of a non-human Ab are replaced with corresponding amino acids derived from human immunoglobulins. In one embodiment of a humanized form of an Ab, some, most or all of the amino acids outside the CDR domains have been replaced with amino acids from human immunoglobulins, whereas some, most or all amino acids within one or more CDR regions are unchanged. Minor additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not abrogate the ability of the Ab to bind to a particular antigen. A “humanized” Ab retains an antigenic specificity similar to that of the original Ab.

As used herein, the term “monoclonal” Ab (mAb) refers to a non-naturally occurring preparation of Ab molecules of single molecular composition, i.e., Ab molecules whose primary sequences are essentially identical and which exhibit a single binding specificity and affinity for a particular epitope. MAbs may be produced by hybridoma, recombinant, transgenic or other techniques known to those skilled in the art.

As used herein, the term “chimeric” Ab refers to an Ab in which the variable regions are derived from one species and the constant regions are derived from another species, such as an Ab in which the variable regions are derived from a mouse Ab and the constant regions are derived from a human Ab. In an illustrative example, a chimeric antibody may comprise a constant region derived from human and a variable region from a non-human animal such as mouse. In some embodiments, the non-human animal is a mammal, for example, a mouse, a rat, a rabbit, a goat, a sheep, a guinea pig, or a hamster.

As used herein, the term “specific binding” or “specifically binds” refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen. Binding affinity of the antibody and antigen-binding fragment provided herein can be represented by K_D value, which represents the ratio of dissociation rate to association rate (koff/kon) when the binding between the antigen and antigen-binding molecule (e.g. the antibody and antigen-binding fragment) reaches equilibrium. The antigen-binding affinity (e.g. K_D) can be appropriately determined using suitable methods known in the art, including, for example, Biacore techniques, Kinexa techniques, and flow cytometry.

As used herein, the term “compete for binding” refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g. human CCR8 and an anti-CCR8 antibody) to any detectable degree (e.g. by at least 85%, or at least 90%, or at least 95%) . Those skilled in the art will recognize that it is possible to determine, without undue experimentation, if a given antibody competes for binding to CCR8 with the antibody of present disclosure.

As used herein, the term “epitope” refers to the specific group of atoms or amino acids on an antigen to which an antibody or an antigen-binding portion binds. The minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen's primary structure, as the epitope may depend on an antigen's three-dimensional configuration based on the antigen's secondary and tertiary structure. The CDRs are important for recognizing an epitope of an antigen.

“Multispecific” refers to an antibody that specifically binds at least two distinct antigens or two distinct epitopes within the antigens, for example three, four or five distinct antigens or epitopes.

“Bispecific” refers to an antibody that specifically binds two distinct antigens or two distinct epitopes within the same antigen. Bispecific antibody may have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs) , such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno) , Pan troglodytes (chimpanzee, chimp) or Callithrix jacchus (common marmoset, marmoset) , or may bind an epitope that is shared between two or more distinct antigens.

“Bispecific anti-CCR8/CTLA-4 antibody” , “CCR8/CTLA-4 antibody” , “anti-CCR8/CTLA-4 antibody” or “antibody that specifically binds CCR8 and CTLA-4” refers to a molecule comprising at least one domain specifically binding CCR8 and at least one domain specifically binding CTLA-4. The domains specifically binding CCR8 and CTLA-4 are typically VH/VL pairs. The bispecific anti-CCR8/CTLA-4 antibody may be monovalent in terms of its binding to either CCR8 or CTLA-4.

As used herein, “percent (%) identical to” with respect to amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences. Sequence identity refers to exact matches between the nucleotides or amino acids of two sequences which are being compared. Sequence identity can be determined by those skilled in the art through conventional means, such as BLAST algorithm.

As used herein, “administering” , “administered” or “administration” refers to the physical introduction of a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. A preferred route for administration of therapeutic antibodies is intravenous (IV) administration. Other routes of administration include subcutaneous (SC) , intraperitoneal (IP) , intramuscular (IM) , spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, the antibody or antigen-binding fragment thereof according to the present disclosure can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.

As used herein, the term “C-C Motif Chemokine Receptor 8” ( “CCR8” , also known as CY6, TER1, CCR-8, CKRL1, CDw198, CMKBR8, GPRCY6, CMKBRL2, or CC-CKR-8) is a seven-transmembrane GPCR which has been shown to be expressed primarily on intratumoral FOXP3^hi Tregs. The term “CCR8” as used herein includes human CCR8 (hCCR8) , variants, isoforms, species homologs of hCCR8 such as mouse CCR8 (mCCR8) , and analogs having at least one common epitope with hCCR8. The complete hCCR8 and mCCR8 amino acid sequences can be found underAccession Nos. AAI07160.1 and NP_031746.1, respectively.

The term “CTLA4 protein” as used herein, includes a full-length CTLA4 protein, CTLA4 protein fragments, CTLA4 protein variants, and CTLA4 fusion proteins (e.g., CTLA4/Fc fusion protein) , which an anti-CTLA4 antibody (e.g., MDX-010) can bind.

As used herein, the term “cancer” refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth result in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term “tumor” as used herein refers to cancerous cells, e.g., a mass of cancerous cells. Cancers that can be treated or diagnosed using the methods described herein include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In some embodiments, the antibody or antigen-binding fragment thereof provided herein are designed for treating or diagnosing a carcinoma in a subject. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the cancer is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

As used herein, “subject” includes any human or non-human animal. The term “non-human animal” includes, without limitation, vertebrates such as non-human primates, sheep, dogs, monkey, chimpanzee, gorilla, and rodents such as mice, rats and guinea pigs. In preferred embodiments, the subject is a human. The terms “subject” and “patient” are used interchangeably herein.

As used herein a “vector” refers to a polynucleotide molecule which enables replicating/cloning of a desired nucleic acid fragment contained therein, or enables expressing of a protein encoded by such desired nucleic acid fragment as introduced into an appropriate cell host. Examples of vectors include both cloning vectors and expression vectors. The term “expression vector” as used herein refers to a vehicle into which apolynucleotide encoding a protein maybe operably inserted so as to bring about the expression of that protein. An expression vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. A vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran) , transformation, transfection, and infection and/or transduction (e.g., with recombinant virus) . Non-limiting examples of vectors include viral vectors (which can be used to generate recombinant virus) , naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

As used herein, the “host cell” means a cell that has been transformed or is capable of being transformed with a nucleic acid sequence and thus expresses a target gene. The host cell can be prokaryotic (e.g., E. coli) , eukaryotic (e.g., yeast, plant including tobacco and tomato, animals including human, monkey, hamster, rat, mouse, or insect) , or hybridomas.

As used herein, “therapeutically effective amount” or “therapeutically effective dosage” of a drug or therapeutic agent is any amount of the drug or agent that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, a prevention or reduction of impairment or disability due to the disease affliction, or otherwise an amelioration of disease symptoms in the subject. In addition, the terms “effective” and “effectiveness” with regard to a treatment includes both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of the drug to promote disease regression, e.g., cancer regression, in the patient. Physiological safety refers to an acceptable level of toxicity, or other adverse physiological effects at the cellular, organ and/or organism level (adverse effects) resulting from administration of the drug. The efficacy of a therapeutic agent can be evaluated using a variety of methods known to the practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

As used herein, the term “treating” or “treatment” of a disease or condition as used herein includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof.

As used herein, the term “pharmaceutically acceptable carrier” indicates that the designated carrier, vehicle, diluent, excipient (s) , and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.

Antibodies and Antigen-binding Fragments

The present disclosure provides a novel anti-CCR8 antibody (e.g., HC64, HC64- 23) or antigen-binding fragment thereof, that specifically binds to CCR8, such as human CCR8 expressed the surface of a cell.

The present disclosure also provides a novel anti-CCR8/CTLA4 bispecific antibody (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) or antigen-binding fragment thereof, that specifically binds to CCR8 and CTLA4, such as human CCR8 and CTLA4 expressed the surface of a cell.

In particular embodiments, bsAb-38-64-05-2/bsAb-38-64-05-3 was designed by inserting anti-CTLA-4 nanobody (e.g., GBD008-hS005-3-2) ) between anti-CCR8 Fab and hinged region in an identical afucosylated IgG1 format. The bispecific antibody bsAb-38-64-05-2/bsAb-38-64-05-3 simultaneously targets these two cell surface antigens (CCR8 and CTLA-4) with overlapping on TI-Tregs to reduce the exposure of antibodies in the periphery. By targeting two receptors overlapping on TI-Tregs, there is a potential to increase localization to the TME compared to monospecific antibodies, resulting in reduction of systemic T-cell activation and improvement of response rate.

The experiments showed increased binding of the anti-CCR8/CTLA4 bispecific antibody of the present disclosure (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) to cells when both target antigens were engaged compared to single antigen engagement. The experiments further demonstrated that the anti-CCR8/CTLA4 bispecific antibody of the present disclosure (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) -mediated TI-Treg depletion in several mouse tumor models and enabled potent antitumor responses. Additionally, the anti-CCR8/CTLA4 bispecific antibody of the present disclosure (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) didn’ t cause toxicity in mice irAEs model compared with ipilimumab by combination of anti-PD-1 antibody. These data indicate the potential and safe clinical utility of selective depletion of tumor resident T regulatory cells by the anti-CCR8/CTLA4 bispecific antibody of the present disclosure (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) .

Our preclinical mouse tumor modeling showed that Fc-optimized afucosylated (AF) anti-CCR8/CTLA4 bispecific antibody of the present disclosure (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) exhibited significant tumor suppression through specific depletion of TI-Tregs but not Teffs. In addition, the anti-CCR8/CTLA4 bispecific antibody of the present disclosure (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) showed better tolerance compared with ipilimumab combination of anti-PD-1 antibody in irAEs model. These findings suggest that the anti-CCR8/CTLA4 bispecific antibody of the present disclosure (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) may deliver optimal TI-Tregs depletion in the clinic, providing anti-tumor immunity while limiting peripheral toxicities.

The antibody or antigen-binding fragment thereof provided herein comprises: a heavy chain complementarity determining region (HCDR) 1 having the amino acid sequence at least 80% (e.g., at least85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 1 or having up to 1 amino acid addition, substitution and/or deletion compared with SEQ ID NO: 1, a HCDR2 having the amino acid sequence at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 2 or having up to 3 (e.g., 1, 2 or 3) amino acid additions, substitutions and/or deletions compared with SEQ ID NO: 2, and a HCDR3 having the amino acid sequence at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 3 or having up to 1 amino acid addition, substitution and/or deletion compared with SEQ ID NO: 3, a LCDR1 having the amino acid sequence at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 4 or having up to 2 (e.g., 1 or 2) amino acid additions, substitutions and/or deletions compared with SEQ ID NO: 4; a LCDR2 having the amino acid sequence at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 5 or having up to 1 amino acid addition, substitution and/or deletion compared with SEQ ID NO: 5; and a LCDR3 having the amino acid sequence at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 6 or having up to 1 amino acid addition, substitution and/or deletion compared with SEQ ID NO: 6.

In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises: a HCDR1 having the amino acid sequence of SEQ ID NO: 1; a HCDR2 having the amino acid sequence of SEQ ID NO: 2; and a HCDR3 having the amino acid sequence of SEQ ID NO: 3, a LCDR1 having the amino acid sequence of SEQ ID NO: 4; a LCDR2 having the amino acid sequence of SEQ ID NO: 5; and a LCDR3 having the amino acid sequence of SEQ ID NO: 6.

In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises the CDR sequences of HC64 (see Table 1) .

Table 1. The CDR Sequences of HC64.

In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises: a HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; a LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 6.

CDRs are known to be responsible for antigen binding, however, it has been found that not all of the six CDRs are indispensable or unchangeable. In other words, it is possible to replace or change or modify one or more CDRs in HC64 or HC64-23, yet substantially retain the specific binding affinity to CCR8.

In some embodiments, the antibody or antigen-binding fragment thereof provided herein may comprise one or more modifications or substitutions in one or more CDR regions as provided in Table 1. Such variants retain specific binding affinity to CCR8 of their parent antibody, but may have one or more improvement in properties such as higher antigen-binding affinity or reduced likelihood of glycosylation.

In some embodiments, the antibody or antigen-binding fragment thereof provided herein may be modified to remove one or more Asn or Asp hotspots within the CDR regions (or within the variable regions) . Such Asn and Asp hotspots may lead to degradation of the antibodies and consequently reduce the stability of the antibodies.

In some embodiment, the one or more modification or substitution is a conservative substitution.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 7 or SEQ ID NO: 13 or having up to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acid additions, substitutions and/or deletions compared with SEQ ID NO: 7 or SEQ ID NO: 13 (i.e., the heavy chain variable regions for HC64 or HC64-23) ; and a light chain variable region having the amino acid sequence at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 8 or SEQ ID NO: 14 or having up to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acid additions, substitutions and/or deletions compared with SEQ ID NO: 8 or SEQ ID NO: 14 (i.e., the light chain variable regions for HC64 or HC64-23) . In a preferred embodiment, the substitution is a conservative substitution.

In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 13; and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 14.

The antibody or antigen-binding fragment thereof provided herein further comprises an immunoglobulin constant region, optionally a constant region of human immunoglobulin, optionally a constant region of human IgG. In some embodiments, an immunoglobulin constant region comprises a heavy chain and/or a light chain constant region. The heavy chain constant region comprises CH₁, hinge, and/or CH₂-CH₃ regions. In some embodiments, the heavy chain constant region comprises a Fc region. In some embodiments, the light chain constant region comprises C_κ or C_λ. In a preferred embodiment, the constant region is derived from human IgG1 (hIgG1) . In a preferred embodiment, the constant region is a constant region of human IgG1.

In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises at least one heavy chain and/or at least one light chain. In an embodiment, the heavy chain having the amino acid sequence at least 85% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 9 or SEQ ID NO: 15 or having up to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50) amino acid additions, substitutions and/or deletions compared with SEQ ID NO: 9 or SEQ ID NO: 15 (i.e., the full-length heavy chain sequence of HC64 or HC64-23) . In an embodiment, the light chain having the amino acid sequence at least 85% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 10 or having up to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50) amino acid additions, substitutions and/or deletions compared with SEQ ID NO: 10 (i.e., the full-length light chain sequence of HC64 or HC64-23) . In a preferred embodiment, the substitution is a conservative substitution.

In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15; and a light chain having the amino acid sequence of SEQ ID NO: 10.

Table 2 shows the amino acid sequences and nucleotide sequences of HC64.

Table 2. The amino acid sequences and nucleotide sequences of HC64.

Table 3 shows the amino acid sequences and nucleotide sequences of HC64-23.

Table 3. The amino acid sequences and nucleotide sequences of HC64-23

The present disclosure provides a bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof, comprising a first domain specifically binding CCR8 and a second domain specifically binding CTLA-4, wherein the first domain comprises the antibody or antigen-binding fragment thereof as described above.

In some embodiments, the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof is isolated.

b) reduced tumor growth and significantly prolonged survival than anti-CCR8 antibody,

d) better tolerance and longer survival than ipilimumab; and

In some embodiments, the bispecific anti-CCR8/CTLA-4 antibody or antigen- binding fragment thereof is afucosylated.

In some embodiments, the second domain comprises an anti-CTLA-4 nanobody, e.g., GBD008-hS005-3-2.

In some embodiments, the second domain comprises the amino acid sequence at least 80% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 22 or SEQ ID NO: 23.

In some embodiments, the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof may be generated by covalently linking first domain and the second domain of the invention directly or via a linker.

In some embodiments, the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof further comprises one or more linkers, optionally, the linker comprises one or more amino acids selected from glycine and serine. The linkers can be determined by those skilled in the art via routine experimentation.

In some embodiments, the linker comprises GGGGSGGGGS and GS.

In some embodiments, the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof comprises: a heavy chain having the amino acid sequence at least 85% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 18 or SEQ ID NO: 20 or having up to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50) amino acid additions, substitutions and/or deletions compared with SEQ ID NO: 18 or SEQ ID NO: 20 (i.e., the full-length heavy chain sequence of bsAb-38-64-05-2 or bsAb-38-64-05-3) ; and a light chain having the amino acid sequence at least 85% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 10 or having up to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50) amino acid additions, substitutions and/or deletions compared with SEQ ID NO: 10 (i.e., the full-length light chain sequence of bsAb-38-64-05-2 or bsAb-38-64-05-3) . In a preferred embodiment, the substitution is a conservative substitution.

In a preferred embodiment, the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof comprises a heavy chain having the amino acid sequence of SEQ ID NO: 18or SEQ ID NO: 20; and a light chain having the amino acid sequence of SEQ ID NO: 10.

Tables 4-5 show the amino acid sequences and nucleotide sequences of bsAb-38-64-05-2 and bsAb-38-64-05-3, respectively.

Table 4. The amino acid sequences and nucleotide sequences of bsAb-38-64-05-2

Table 5. The amino acid sequences and nucleotide sequences of bsAb-38-64-05-3

In some embodiments, the anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof provided herein may contain one or more modifications or substitutions in one or more sequences provided herein, yet retaining specific binding affinity to CCR8. Various methods known in the art can be used to achieve this purpose. For example, computer software can be used to virtually simulate the binding of the antibodies to CCR8, and identify the amino acid residues on the antibodies which form the binding interface. Such residues may be either avoided in the substitution so as to prevent reduction in binding affinity, or targeted for substitution to provide for a stronger binding.

“Conservatively modified variants” or “conservative substitution” as used herein refers to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc. ) , such that the changes can frequently be made without altering the biological activity of the protein. Those skilled in this art would recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Ed. ) ) . In addition, substitutions of structurally and/or functionally similar amino acids are less likely to disrupt biological activity. Various embodiments of the anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof according to the present disclosure comprise polypeptide chains with sequences that include up to 0 (no changes) , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50 or more conservative amino acid substitutions when compared with the specific amino acid sequences disclosed herein, e.g. SEQ ID NOs: 7, 8, 9 or 10. As used herein, the phrase “up to X” conservative amino acid substitutions includes 0 substitutions and any number of substitutions up to and including X substitutions. Such exemplary substitutions are preferably made in accordance with those set forth in the following table:

Table 6. Exemplary Conservative Amino Acid Substitutions

Functionally conservative variants of the anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof according to the present disclosure are also contemplated by the present disclosure. "Functionally conservative variants" are those in which one or more amino acid residues in a protein have been changed without altering the overall conformation and function of the polypeptide, including, without limitation, replacement of an amino acid with one having similar properties.

The anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof provided herein also comprises a constant region capable of inducing effector function. In some embodiments, the constant region comprises one or more modifications which enhances antibody-dependent cellular cytotoxicity (ADCC) . In some embodiments, the anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof is afucosylated.

In some embodiments, the afucosylated antibody can increase effector functions (e.g., ADCC) of an antibody or antigen binding fragment thereof by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or100-fold, as compared to those of a wild-type antibody or antigen-binding fragment thereof.

As described above, “antibody or antigen-binding fragment thereof” as used herein refers to an intact antibody or an antibody fragment having the antigen-binding portion. Various types of antibodies or antigen-binding fragments are known in the art and can be developed based on the antigen-binding portion of an anti-CCR8 antibody (e.g., HC64) provided herein.

In some embodiments, the anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof of the present disclosure is a human antibody, humanized antibody, chimeric antibody, monoclonal antibody, polyclonal antibody, recombinant antibody, diabody, triabody, tetrabody, Fab fragment, F (Fab') 2 fragment, scFv fragment, Fv fragment, Fab'fragment, or domain antibody.

Antibody Characteristics

The bispecific antibody bsAb-38-64-05-2/bsAb-38-64-05-3 simultaneously targets these two cell surface antigens (CCR8 and CTLA-4) with overlapping on TI-Tregs to reduce the exposure of antibodies in the periphery. By targeting two receptors overlapping on TI-Tregs, localization to the TME has been increased compared to monospecific antibodies, resulting in reduction of systemic T-cell activation and improvement of response rate.

The bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof according to the present disclosure (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) has at least one of the following properties:

a) strong induction ofADCC response to kill human Treg cells or CHOK1-human CCR8/CTLA-4 over expressing cells;

c) partial blocking of CD80or CD86-CTLA4 interaction on CHOK1-human CTLA-4 overexpressing cells but significant blocking of the binding of CD80 or CD86 to CTLA-4 on CHOK1-human CCR8/CTLA-4 overexpressing cells;

d) better tolerance and longer survival than ipilimumab; and

In some embodiments, the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof provided herein specifically binds to CCR8 with a binding affinity (K_D) less than 1 x 10^-8 M, less than 1 x 10^-9 M, or less than 1 x 10^-10 M. In some embodiments, the K_D is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof provided herein specifically binds to CTLA-4 with a binding affinity (K_D) less than 1 x 10^-8 M, less than 1 x 10^-9 M, or less than 1 x 10^-10 M. In some embodiments, the K_D is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM.

General techniques for measuring the affinity of an antibody for an antigen include, e.g., ELISA, RIA, and surface plasmon resonance (SPR) .

Polynucleotides and Production Methods

The present disclosure also provides a nucleic acid that encode the anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof provided herein.

The term “nucleic acid” or “polynucleotide” as used herein refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses polynucleotides containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) , alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19: 5081 (1991) ; Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985) ; and Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994) ) .

In some embodiments, the nucleic acid encoding the anti-CCR8 antibody or antigen-binding fragment thereof comprises: a heavy chain encoding nucleic acid having the nucleotide sequence at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 11 or 16; and a light chain encoding nucleic acid having the nucleotide sequence at least80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 12 or 17.

In some embodiments, the nucleic acid encoding the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof comprises: a heavy chain encoding nucleic acid having the nucleotide sequence at least 80% (e.g., at least85%, 90%, 95%, 96%, 97%, 98%, 99%or100%) identical to SEQ ID NO: 19 or 21; and/or a light chain encoding nucleic acid having the nucleotide sequence at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 17.

DNA encoding the anti-CCR8 antibody or the anti-CCR8/CTLA4 bispecific antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) . The encoding DNA may also be obtained by synthetic methods.

The anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof provided herein can be produced by any method known in the art for synthesis of proteins (e.g., antibodies) , especially by chemical synthesis or preferably by recombinant expression techniques.

The recombinant expression of an antibody requires the construction of an expression vector containing a nucleic acid encoding the antibody. Once the nucleic acid encoding the antibody is obtained, a vector for producing the antibody can be produced by recombinant DNA techniques. In the present disclosure, an expression vector is constructed to contain an antibody-coding sequence and appropriate transcription and translation regulatory elements. These methods include, without limitation, in vitro recombinant DNA technologies, synthesis techniques, and in vivo genetic recombination.

The expression vector is transferred to the host cell by conventional techniques, and then the transfected cells were cultured by conventional techniques to produce the antibody or antigen-binding fragment thereof according to the present disclosure.

In an embodiment, a method of producing the anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof according to the present disclosure comprises culturing the host cell according to the present disclosure under conditions that allow the expression of the anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof. In a preferred embodiment, the method further comprises recovering and/or purifying the resulting anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof from the host cell and/or the culture medium.

Pharmaceutical Composition

The present disclosure provides a pharmaceutical composition comprising the anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof provided herein, the nucleic acid provided herein, the expression vector provided herein, or the antibody-drug conjugate provided herein, and one or more pharmaceutically acceptable carriers. In a preferred embodiment, the pharmaceutical composition comprises a therapeutically effective amount of the anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof provided herein, and one or more of additional components, such as a pharmaceutically acceptable carrier, vehicle, or medium. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier can include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

In some embodiments, the compositions can include a sterile diluent (e.g., sterile water or saline) , a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose) , polyalcohols (e.g., mannitol or sorbitol) , or salts (e.g., sodium chloride) , or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811) . Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations) , proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant. Absorption of the anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin) . Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc. ) .

The pharmaceutical composition can be administered by any suitable method known to those skilled in the art, such as those parenteral and non-parenteral roots as described above. In a preferred embodiment, the pharmaceutical composition can be administered by intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural or intrasternal injection.

The present disclosure also provides a kit comprising the anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof provided herein. In a preferred embodiment, the kit according to the present disclosure further comprises an instruction for guiding the use of the anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof of the present disclosure, such as in treating or preventing a disease associated with the abnormal expression of CCR8 in a subject, such as a cancer.

In some embodiments, the anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof provided herein, the nucleic acid provided herein, the expression vector provided herein, or the antibody-drug conjugate provided herein and at least one additional therapeutic agent are administered in a same composition. In some embodiments, the anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof provided herein, the nucleic acid provided herein, the expression vector provided herein, or the antibody-drug conjugate provided herein and at least one additional therapeutic agent are administered in two different compositions.

In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of B-Raf, an EGFR inhibitor, an inhibitor of a MEK, an inhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-Met, an inhibitor of anaplastic lymphoma kinase (ALK) , an inhibitor of a phosphatidylinositol 3-kinase (PI3K) , an inhibitor of an Akt, an inhibitor of mTOR, a dual PI3K/mTOR inhibitor, an inhibitor of Bruton's tyrosine kinase (BTK) , and an inhibitor of Isocitrate dehydrogenase 1 (IDH1) and/or Isocitrate dehydrogenase 2 (IDH2) . In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2, 3-dioxygenase-1) (IDO1) (e.g., epacadostat) .

In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of HER3, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.

In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a Selectin agonist.

In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, or an anti-GITR antibody.

Therapeutic method & Use

The anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof, nucleic acid, expression vector, host cell and the pharmaceutical composition according to the present disclosure can be used in treating or preventing a disease associated with the abnormal expression of CCR8 and/or CTLA-4 in a subject, such as a cancer. In an embodiment, a method for treating or preventing a disease associated with the abnormal expression of CCR8 and/or CTLA-4 in a subject comprises administering a therapeutically effective amount of the anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof, nucleic acid, expression vector, host cell, or the pharmaceutical composition according to the present disclosure to the subject.

Further, the anti-CCR8 antibody, the anti-CCR8/CTLA4 bispecific antibody or antigen-binding fragment thereof, nucleic acid, expression vector, host cell and the pharmaceutical composition according to the present disclosure can be used in the manufacture of a medicament and/or kit, such as for treating or preventing a disease associated with the abnormal expression of CCR8 and/or CTLA-4 in a subject.

In an embodiment, the disease is a cancer. In a preferable embodiment, the cancer is a solid tumor selected from the group consisting of squamous cell carcinoma, small cell lung cancer (SCLC) , non-small cell lung cancer (NSCLC) , squamous NSCLC, non-squamous NSCLC, head and neck cancer, breast cancer, cancer of the esophagus, gastric cancer, gastrointestinal cancer, cancer of the small intestine, liver cancer, hepatocellular carcinoma (HCC) , pancreatic cancer (PAC) , kidney cancer, renal cell carcinoma (RCC) , bladder cancer, cancer of the urethra, cancer of the ureter, colorectal cancer (CRC) , colon cancer, colon carcinoma, cancer of the anal region, endometrial cancer, prostate cancer, a fibrosarcoma, neuroblastoma, glioma, glioblastoma, germ cell tumor, pediatric sarcoma, sinonasal natural killer, melanoma, skin cancer, bone cancer, cervical cancer, uterine cancer, carcinoma of the endometrium, carcinoma of the fallopian tubes, ovarian cancer, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, testicular cancer, cancer of the endocrine system, thyroid cancer, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the penis, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS) , primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain cancer, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, solid tumors of childhood, environmentally-induced cancers, virus-related cancers, cancers of viral origin, advanced cancer, unresectable cancer, metastatic cancer, refractory cancer, recurrent cancer, and any combination thereof. In another preferable embodiment, the cancer is a hematological malignancy selected from the group consisting of acute lymphoblastic leukemia (ALL) , acute myelogenous leukemia (AML) , chronic lymphocytic leukemia (CLL) , chronic myelogenous leukemia (CML) , a T cell lymphoma, Hodgkin’s lymphoma (HL) , non-Hodgkin’s lymphomas (NHLs) , multiple myeloma, smoldering myeloma, monoclonal gammopathy of undetermined significance (MGUS) , advanced, metastatic, refractory and/or recurrent hematological malignancies, and any combinations of said hematological malignancies.

EXAMPLES

The present invention is now illustrated by the following examples which should not be construed as limiting.

Example 1: Generation of anti-human CCR8 antibodies

Anti-human CCR8 antibodies were generated by immunizing the SJL strains of inbred mice with one or more of the following antigens: 1) Recombinant, Human CCR8 overexpressing HEK293F cells; 2) Full length human CCR8 expressing vector in pCP (pCP-hCCR8) .

Plasmid DNA immunization was conducted by intraperitoneal (i.p. ) injection. Cellular immunization was performed by intraperitoneal (i.p. ) injection of human CCR8 transfected HEK293F cells. A cohort of animals was immunized with Human CCR8 overexpressing HEK293F cells followed by DNA boosting as follows. Mice were injected intraperitoneally with prepared antigen every two weeks. Animals that developed anti-CCR8 titers were given an intraperitoneal (i. p. ) injection of 1 × 10⁷ human CCR8 overexpressing HEK293F cells or pCP-hCCR8 plus 1 × 10⁷ human CCR8 overexpressing HEK293F cells. Spleens and/or lymph nodes were harvested, and the splenocytes and/or lymph node cells were used for hybridoma preparation.

To select an animal that produced CCR8-binding antibodies, serum from immunized animals was tested by ELISA to determine binding to CHOK1-human CCR8 overexpressing cells and CHOK1-blank. Briefly, the binding of polyclonal serum was assessed by incubating CHOK1-human CCR8 overexpressing cells with the diluted serum samples. The cells were then washed, and binding was detected with a Aleax488 donkey anti-mouse IgG (H+L) . Flow cytometric analyses were performed using a fluorescence microplate cytometry (Acumen Explorer microplate cytometer) . Mice that developed the highest titers of anti-CCR8 antibodies were used for fusions. Fusions were performed as described below. Hybridoma supernatants were tested for anti-CCR8 activity by flow cytometry.

The splenocytes and/or lymphocytes from mice were fused with a Sp2/0-Ag14 cell line by electrofusion. The supernatants that were scored positive for mouse IgG antibodies were then subsequently screened by flow cytometry for anti-CCR8 IgG antibodies. The anti-CCR8 antibody-secreting hybridomas were then undergone subcloning process by limiting dilution. The stable subclones were cultured in vitro to determine binding to human CCR8 by flow cytometry. The confirmed positive clones were scaled up to generate small amounts of antibodies in tissue culture medium for further characterization. The antibodies from hybridoma supernatants were purified by protein A column chromatography.

A number of anti-CCR8 antibodies were obtained including HC64.

Example 2: Humanization of anti-CCR8 antibodies

Heavy and light chain variable region (VH and VL) sequences of murine anti-human CCR8 antibody were used to search the corresponding variable region sequences of human CCR8 antibody in IMGT database. MOE software was used to compare the variable regions of heavy and light chains of mouse antibodies and the variable regions of heavy and light chains of human antibodies. Germline genes with high homology with mouse antibodies were selected as templates, and CDRs of mouse antibodies were grafted into corresponding human templates to form the sequence of variable regions FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. According to the structure analysis of the heavy chain variable region and light chain variable region of the selected human antibody and the mouse antibody to ensure the affinity of the original mouse antibody, the key amino acids in the skeleton sequence were restored and mutated to the corresponding amino acids of the mouse antibody, namely, the humanized anti-CCR8 antibody. The determination of amino acid residues in CDR regions was determined and annotated by Kabat numbering system.

The humanized antibody of antibody HC64is designated HC64-23.

Example 3: Generation of bsAb-38-64-05-2, bsAb-38-64-05-3 and afucosylated (AF) anti-CCR8 mAbs

To generate the afucosylated (AF) bsAb-38-64-05-2, bsAb-38-64-05-3 or anti-CCR8 mAbs, expression plasmids pCDNA3.4 provided by Biometas (Shanghai) Limited were transformed to E. coli for propagation respectively in appropriate scale. NucleoBond Xtra Maxi Plus EF kit were used for large scale plasmid generation. The constructs containing heavy chain and light chain of each antibody were co-transfected into FUT8-KO CHO cells with PEI. Harvest conditioned medium 9-11 days post transfection. Conditioned medium expressing target Abs were harvested by centrifugation and filtration, then loaded onto Protein A affinity column. Purified Abs were analyzed by SDS-PAGE, SEC-HPLC and endotoxin measurement.

Example 4: Binding of anti-CCR8 or anti-CTLA-4 antibodies to cell surface antigens

For CCR8 binding, the binding of the antibodies of the present disclosure was assessed by incubating Raji-human CCR8 over expressing cells with the serially diluted (1: 5) antibodies. The cells were washed, and binding was detected with an APC anti-human IgG Fc antibody (Jackson lab, Catalog#309-605-008) by flow cytometry (BD LSRFortessa) . The antibodies of the present disclosure showed comparable or better binding abilities to Raji-human CCR8 overexpressing cell line as compared to benchmark (i.e., 4A19 (anti-CCR8, BMS) ) (Fig. 1 and 10A) .

For CTLA-4 binding, the binding of the antibodies of the present disclosure was assessed by incubating CHOK1-human CTLA-4 over expressing cells with the serially diluted (1: 5) antibodies. Similar to the procedure of CCR8 binding, the cells were finally analyzed by flow cytometry. The antibodies of the present disclosure showed weaker binding abilities to CHOK1-human CTLA-4 overexpressing cell line as compared to benchmark (i.e., ipilimumab) (Fig. 10B) .

For CCR8/CTLA-4 binding, the binding of the antibodies of the present disclosure was assessed by incubating CHOK1-human CCR8/CTLA-4 over expressing cells with the serially diluted (1: 5) antibodies. Similar to the procedure of CCR8 binding, the cells were finally analyzed by flow cytometry. The antibodies of the present disclosure showed comparable or better binding abilities to CHOK1-human CCR8/CTLA-4 overexpressing cell line as compared to parental anti-CCR8 antibody (GBD008-hS005-3-2) (Fig. 10C) .

Example 5: Blocking of CCR8-CCL1 binding

The blocking on CCL1 ligand by the antibodies of the present disclosure was assessed by incubating Raji-human CCR8 or Raji-human CCR8/CTLA-4 overexpressing cells with the serially diluted (1: 5) antibodies. The cells were washed twice, and added 0.4 μg/mL Human I-309-AF647 to the designated row of the assay plate, mixed well and incubated at 4 ℃ for 30 min. After incubation, the cells were washed once and re-suspended in 120 μL FACS buffer (BioLegend, Catalog#420201) . The assay plate was detected by flow cytometry. The results are shown in Fig. 2 and 11. The antibodies of the present disclosure significantly blocked the binding of CCL1 to CCR8.

Example 6: Binding and blocking of anti-CCR8 antibodies on human Treg cells

For human Treg cells separation, Treg cells (CD4+CD127lowCD25+) were separated from human peripheral blood mononuclear cells (PBMCs) (Biotechnologies) using EasySep^TM Human CD4+CD127lowCD25+Regulatory T Cell Isolation Kit (Stem Cell, Catalog#18063) .

For Treg cells induction, human monocytes were separated using StemSep ^TMHuman CD14 Positive Selection Kit (Stem Cell, Catalog#14758) from PBMCs and were mixed with Treg cells at the ratios 1: 1 in the presence of human T Cell TransAct ^TM(10 μl/mL, Miltenyi Biotec, Catalog#130-111-160) and hIL-2 (10 ng/ml, PeproTech, Catalog#200-02) . After a 3 days incubation, monocytes were removed and Treg cells were cultured sequentially for another 10-13 days expansion.

For the binding and blocking of anti-CCR8 antibodies, the antibodies of the present disclosure showed better binding abilities to human Treg cells as compared to benchmark (i.e., 4A19 (anti-CCR8, BMS) ) (Fig. 3) and significantly blocked the binding of CCL1 to CCR8 (Fig. 4) .

Example 7: Cross binding of anti-CCR8 antibodies to cyno CCR8

The cross binding of the anti-CCR8 antibodies to cyno CCR8 was assessed by incubating cyno CCR8-overexpressing 293T cells with the serially diluted (1: 10) antibodies. The cells were washed, and binding was detected with an APC anti-human IgG Fc antibody by flow cytometry. The result is shown in Fig. 5 and 14A. The anti-CCR8 antibodies of the present disclosure exhibited significant binding abilities to cyno CCR8. This is conducive to later toxicological evaluation.

Example 8: Cross binding of anti-CTLA-4 antibodies to cyno CTLA-4

The cross binding of the anti-CTLA-4 antibodies to cyno CTLA-4 was measured by ELISA assay. Each step of each assay was performed by room temperature incubation with the appropriate reagent for 1 hour, except the initial plate coating step was done overnight at 4℃. Between each step, plates were washed 3 times in PBS containing 0.05% Tween 20.

Plates were coated with 1 μg/ml cyno CTLA-4 (Sino Biological, Catalog#90213-C08H) , and followed by blocking with 2% BSA. The plates were next incubated with serial diluted (1: 5) anti-CTLA-4 mAbs, and the bound antibodies were detected with HRP-hFc (Sigma, Catalog # A0170) and TMB substrate (Cell Signaling, Catalog #7004P6) . The result is shown in Fig. 14B. The anti-CTLA-4 antibodies of the present disclosure exhibited significant binding abilities to cyno CTLA-4. This is conducive to later toxicological evaluation.

Example 9: ADCC for bsAb-38-64-05-2 or anti-CCR8 antibodies

Cytotoxic activity was assessed using FACS analysis. The effect cell human PBMCs were obtained from individual human donors and cultured with 10ng/ml hIL-2 overnight. The target human Treg cells or CHOK1-human CCR8/CTLA-4 over expressing cells were labeled with Celltrace Far red (Thermo, Catalog#C34564) for 10 minutes at 37℃, washed twice with RPMI 1640 media (Gibco, Catalog#A10491-01) with 10 % FBS (Gibco, Catalog#10099-141) , and plated at the effector-to-target cell ratios (20: 1) in 96-well round-bottom plates. The serial diluted (1: 5) antibodies were added to the designated row of the assay plate. After a 2-hour incubation at 37℃, 2 μl Propidium Iodide Staining Solution (BD biosciences, Catalog#556547) was added to each well to dye dead cells at room temperature for 10 minutes. Cells were analyzed by flow cytometry directly. The result is shown in Fig. 6 and 15, the antibody of the present disclosure including HC64-23-AF and bsAb-38-64-05-2 induced strong ADCC response to kill human Treg cells or CHOK1-human CCR8/CTLA-4 over expressing cells.

Example 10: Efficacy study in MC38 model

The anti-tumor activity of the anti-CCR8 antibodies (afucosylated hIgG1) was measured in the MC38 mouse colon adenocarcinoma model. 6-8 weeks old female C57BL/6-CCR8^em3 ^(hCCR8) ^/Smoc mice (Shanghai Model Organisms Center, Inc) were each implanted SC with 1×10⁶ MC38 tumor cells and randomized into treatment groups of 6 mice/group 6 days post-implantation. Abs (anti-hCCR8-hIgG1-AF or a control hIgG1-AF Ab) were administered at 3 mpk/mouse in a volume of 200 μL via IP injection at day 0, 4, 7 and 11 post-implantation. Tumor measurements were recorded twice per week for up to 16 days post-implantation, after which the mice were euthanized. The result is shown in Fig. 7. Compared with the negative control (human IgG1-AF) , both 4A19 (anti-CCR8, BMS) -AF and HC64-23-AF reduced tumor growth and significantly prolonged survival (Fig. 8) , and HC64-23-AF had better effect than 4A19 (anti-CCR8, BMS) -AF.

Example 11: Kinetics parameters of antibodies detected by Octet

Kinetic assays were performed by first capturing mAb using anti-human Fc (AHC) Octet (Sartorius) biosensors followed by at least two baseline steps of 30 s each in HBS-EBT buffer. The mAb-captured biosensors were then submerged in wells containing different concentrations of antigen for 4–6 min followed by 10–15 min of dissociation time in HBS-EBT buffer. The mAb-captured sensors were also dipped in wells containing HBS-EBT buffer to allow single reference subtraction in order to compensate for the natural dissociation of captured mAb. The binding sensorgrams were collected using the high sensitivity 16-channel detection mode on the Octet HTX biosensor. Unless specified, fresh AHC biosensors were used without any regeneration step. Kinetics parameters of the antibodies of the present disclosure were shown in Table 7.

Table 7. Kinetics parameters of antibodies detected by Octet

Example 12: Blocking of CTLA4-CD80/86 binding

The blocking on CD80 or CD86 ligands by the antibodies of the present disclosure was assessed by incubating CHOK1-human CTLA-4 or CHOK1-human CCR8/CTLA-4 overexpressing cells with the serially diluted (1: 5) antibodies, in the presence of CD80 Fc-AF647 (Kactus, Catalog#B71-HM280) or CD86 Fc-AF647 (Kactus, Catalog#B72-HM286) . After incubation for 30 min at 4 ℃, the cells were washed once and re-suspended in 120 μL FACS buffer. The assay plate was detected by flow cytometry. The antibodies of the present disclosure only partially blocked CD80 or CD86-CTLA4 interaction on CHOK1-human CTLA-4 overexpressing cells (Fig. 12A and 13A) but significantly blocked the binding of CD80 or CD86 to CTLA-4 on CHOK1-human CCR8/CTLA-4 overexpressing cells (Fig. 12B and 13B) .

Example 13: irAE model in ten-days old C57BL/6 CCR8/CTLA-4 double knock-in mice

Young CCR8/CTLA-4 double knock-in mice were treated, respectively, with indicated antibodies at a dose of 100 or 300 μg/mouse/injection combination with anti-mPD1 antibody (BioXcell, Catalog#BE0146) at a dose of 100 μg/mouse/injection every 3 days for total seven injections, starting on day 10 of birth. To avoid cage variation, mice in the same cages were individually tagged and treated with different antibodies. To avoid gender and weight variation, female mice with similar weight (4.5–5.3 g) were used for all the study, although similar trends were observed in male mice. For Kaplan–Meier survival analyses, mice are considered to have reached endpoint if they become moribund or died. The experiments were performed double blind. As shown in Fig. 16 and 17, the antibodies of the present disclosure including bsAb-38-64-05-2 exhibited better tolerance and longer survival than ipilimumab.

Example 14: Efficacy study in MB49 model

The anti-tumor activity of the bsAb-38-64-05-3 was measured in the MB49 mouse bladder cancer model. 6-8 weeks old male C57BL/6-CCR8^em3 ^(hCCR8) ^/SmocCTLA-4^em1 ^(hCTLA4) ^Smocmice (Shanghai Model Organisms Center, Inc) were each implanted SC with 1×10⁶ MB49 tumor cells and randomized into treatment groups of 6 mice/group 6 days post-implantation. Abs (bsAb-38-64-05-3 or a control hIgG1-AF Ab) were administered at 10 mpk/mouse in a volume of 200 μL via IP injection at day 0, 4, 7 and 11 post-implantation. Tumor measurements were recorded twice per week for up to 32 days post-implantation, after which the mice were euthanized. Compared with the negative control (human IgG1-AF) , bsAb-38-64-05-3 reduced tumor growth (Fig. 18) . In addition, bsAb-38-64-05-3 showed better in vivo anti-tumor efficacy compared to 4A19 (anti-CCR8) -AF and ipilimumab in a dose-dependent manner (Fig. 18) .

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

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Claims

An antibody or antigen-binding fragment thereof that specifically binds to human C-C Motif Chemokine Receptor 8 (CCR8) , comprising:

a heavy chain complementarity determining region (HCDR) 1 having the amino acid sequence at least 80%identical to SEQ ID NO: 1,

a HCDR2 having the amino acid sequence at least 80%identical to SEQ ID NO: 2,

a HCDR3 having the amino acid sequence at least 80%identical to SEQ ID NO: 3,

a light chain complementarity determining region (LCDR) 1 having the amino acid sequence at least 80%identical to SEQ ID NO: 4,

a LCDR2 having the amino acid sequence at least 80%identical to SEQ ID NO: 5, and

a LCDR3 having the amino acid sequence at least 80%identical to SEQ ID NO: 6.
The antibody or antigen-binding fragment thereof of claim 1, comprising: a HCDR1comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1,

a HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2,

a HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 3,

a LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4,

a LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 5, and

a LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 6.
The antibody or antigen-binding fragment thereof of claim 1, comprising:

a heavy chain variable region having the amino acid sequence at least 80%identical to SEQ ID NO: 7 or SEQ ID NO: 13, and

a light chain variable region having the amino acid sequence at least 80%identical to SEQ ID NO: 8 or SEQ ID NO: 14.
The antibody or antigen-binding fragment thereof of claim 3, comprising:

a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 13, and

a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 14.
The antibody or antigen-binding fragment thereof of any of the preceding claims, comprising:

a heavy chain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15, and

a light chain having the amino acid sequence of SEQ ID NO: 10.
The antibody or antigen-binding fragment thereof of any of the preceding claims, which is a human antibody, humanized antibody, chimeric antibody, monoclonal antibody, polyclonal antibody, recombinant antibody, diabody, triabody, tetrabody, Fab fragment, F (ab') ₂ fragment, scFv fragment, Fv fragment, Fab' fragment or domain antibody.
An antibody or antigen-binding fragment thereofthat competes for binding to CCR8 with the antibody or antigen-binding fragment thereof of any one of claims 1-6.
An bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof, comprising

a first domain specifically binding CCR8 and a second domain specifically binding CTLA-4, wherein the first domain comprises the antibody or antigen-binding fragment thereof of any one of claims 1-7.
The bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof of claim 8, wherein the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereofhas at least one of the following properties:

a) strong induction of ADCC response to kill human Treg cells or CHOK1- human CCR8/CTLA-4 over expressing cells;

b) reduced tumor growth and significantly prolonged survival than anti-CCR8 antibody, and/or

c)partial blocking of CD80 or CD86-CTLA4 interaction on CHOK1-human CTLA-4 overexpressing cells but significant blocking of the binding of CD80 or CD86 to CTLA-4 on CHOK1-human CCR8/CTLA-4 overexpressing cells.
The bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof of claim 8, which is afucosylated.
The bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof of any of claims 8-10, which is a human antibody or humanized antibody.
The bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof of any of claims 8-11, wherein the second domain comprises the amino acid sequence at least 80%identical to SEQ ID NO: 22 or SEQ ID NO: 23.
The bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof of any of claims 8-12, wherein the second domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23.
The bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof of any of claims 8-13, further comprising one or more linkers, optionally, the linker comprises one or more amino acids selected from glycine and serine.
The bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof of claim 14, wherein the linker comprises one or more of the following amino acid sequences: GGGGSGGGGS (SEQ ID No: 24) and GS.
The bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof of claim 15, wherein the linker comprises GGGGSGGGGS and GS.
The bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof of any of claims 8-16, comprising:

a heavy chain having the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 20, and

a light chain having the amino acid sequence of SEQ ID NO: 10.
A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-7 or the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof of any one of claims 8-17.
The nucleic acid of claim 18, comprising:

a heavy chain encoding nucleic acid having the nucleotide sequence of SEQ ID NO: 11 or 16; and

a light chain encoding nucleic acid having the nucleotide sequence of SEQ ID NO: 12 or 17.
The nucleic acid of claim 18, comprising:

a heavy chain encoding nucleic acid having the nucleotide sequence of SEQ ID NO: 19 or 21; and

a light chain encoding nucleic acid having the nucleotide sequence of SEQ ID NO: 17.
An expression vector comprising the nucleic acid of any one of claims 18-20.
A host cell comprising the expression vector of claim 21.
A pharmaceutical composition comprising:

the antibody or antigen-binding fragment thereof of any one of claims 1-7, the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof of any one of claims 8-17, the nucleic acid of any one of claims 18-20, or the expression vector of claim 21, and

a pharmaceutically acceptable carrier.
A method of producing the antibody or antigen-binding fragment thereof of any one of claims 1-7, or the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof of any one of claims 8-17, comprising culturing the host cell of claim 22 under conditions that allow the expression of the antibody or antigen-binding fragment thereof.
A method of treating a disease or condition associated with the abnormal expression of CCR8 and/or CTLA-4 in a subject, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-7, the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof of any one of claims 8-17, the nucleic acid of any one of claims 18-20, the expression vector of claim 21, the host cell of claim 22, or the pharmaceutical composition of claim 23to the subject.
Use of the antibody or antigen-binding fragment thereof of any one of claims 1-7, or the bispecific anti-CCR8/CTLA-4 antibody or antigen-binding fragment thereof of any one of claims 8-17, in the manufacture of a medicament for treating a disease associated with the abnormal expression of CCR8 and/or CTLA-4 in a subject.
The method or use of claim 25 or 26, wherein the disease is a cancer.
The method or use of claim 27, wherein the cancer is:

a solid tumor selected from the group consisting of squamous cell carcinoma, small cell lung cancer (SCLC) , non-small cell lung cancer (NSCLC) , squamous NSCLC, non-squamous NSCLC, head and neck cancer, breast cancer, cancer of the esophagus, gastric cancer, gastrointestinal cancer, cancer of the small intestine, liver cancer, hepatocellular carcinoma (HCC) , pancreatic cancer (PAC) , kidney cancer, renal cell carcinoma (RCC) , bladder cancer, cancer of the urethra, cancer of the ureter, colorectal cancer (CRC) , colon cancer, colon carcinoma, cancer of the anal region, endometrial cancer, prostate cancer, a fibrosarcoma, neuroblastoma, glioma, glioblastoma, germ cell tumor, pediatric sarcoma, sinonasal natural killer, melanoma, skin cancer, bone cancer, cervical cancer, uterine cancer, carcinoma of the endometrium, carcinoma of the fallopian tubes, ovarian cancer, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, testicular cancer, cancer of the endocrine system, thyroid cancer, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the penis, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS) , primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain cancer, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, solid tumors of childhood, environmentally-induced cancers, virus-related cancers, cancers of viral origin, advanced cancer, unresectable cancer, metastatic cancer, refractory cancer, recurrent cancer, and any combination thereof; or

a hematological malignancy selected from the group consisting of acute lymphoblastic leukemia (ALL) , acute myelogenous leukemia (AML) , chronic lymphocytic leukemia (CLL) , chronic myelogenous leukemia (CML) , a T cell lymphoma, Hodgkin’s lymphoma (HL) , non-Hodgkin’s lymphomas (NHLs) , multiple myeloma, smoldering myeloma, monoclonal gammopathy of undetermined significance (MGUS) , advanced, metastatic, refractory and/or recurrent hematological malignancies, and any combinations of said hematological malignancies.
The method or use of claim 27, wherein the cancer is bladder cancer.