WO2024062019A1 - Anti-ccr8 antibodies and uses thereof - Google Patents

Anti-ccr8 antibodies and uses thereof Download PDF

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Publication number
WO2024062019A1
WO2024062019A1 PCT/EP2023/076042 EP2023076042W WO2024062019A1 WO 2024062019 A1 WO2024062019 A1 WO 2024062019A1 EP 2023076042 W EP2023076042 W EP 2023076042W WO 2024062019 A1 WO2024062019 A1 WO 2024062019A1
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Prior art keywords
antigen
antibody
binding fragment
seq
isolated antibody
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PCT/EP2023/076042
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French (fr)
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Yannick Nizet
Julien ISOARD
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Synabs
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Publication of WO2024062019A1 publication Critical patent/WO2024062019A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • the present invention relates to antibodies specific for the chemokine (C-C motif) receptor 8 (CCR8) and their use for depleting or inhibiting regulatory T cells in tumors.
  • C-C motif chemokine receptor 8
  • Chemokine (C-C motif) receptor 8 (CCR8) is a G protein-coupled receptor (GPCR) chemokine receptor.
  • GPCR G protein-coupled receptor
  • the CCR8 ligands are Chemokine (C-C motif) ligand 1, 16 and 18 (CCL1, CCL16 and CCL18).
  • CCR8 is mainly expressed by T reg cells and sometimes by T lymphomas or leukemias.
  • cells involved in the immune response such as macrophages produce CCL1.
  • the CCL1-CCR8 binding permits the Treg homing. It provokes activation of the cytoskeleton, which allows the T reg cells to migrate up the CCL1 gradient to the site of the immune reaction where T reg cells negatively regulate the immune reaction by secreting cytokines such as IL- 10.
  • T reg cells infiltrate tumors and promote their progression by inhibiting the activity of immune cells, such as cytotoxic T lymphocytes.
  • antibodies against CCR8 are useful for the treatment of cancer and more generally the treatment of diseases associated with an immune response dysfunction.
  • WO2021194942 discloses an anti-CCR8 antibody exerting antibody-dependent cellular cytotoxicity (ADCC)-mediate depletion of CCR8-expressing cells (such as T reg cells) and use thereof in mouse model of colon cancer, alone or in combination with a mouse anti-mPD-1 Ab.
  • ADCC antibody-dependent cellular cytotoxicity
  • WO2021163064 discloses an anti-CCR8 antibody that may comprise one mutation/or modification (e.g., afucosylation) or may also be chimeric, that may favor depletion of Treg and reduces tumor growth in MC38 syngeneic model in mice.
  • this anti-CCR8 antibody may be used as monotherapy or in combination with an anticancer agent such as an immune checkpoint inhibitor (e.g., PD-1).
  • an immune checkpoint inhibitor e.g., PD-1).
  • WO2021142002 discloses human or cyno antibodies that specifically bind the N- terminal extracellular domain of human CCR8 and inducing ADCC in 293T cells, and therapeutic use thereof.
  • the inventors provide a new antibody targeting CCR8, with specific CDRs sequences, having the capacity to inhibit the binding of CCL1 to CCR8 and to induce CCR8 internalization.
  • This new anti-CCR8 antibody will target tumor-infiltrating immune cells, thereby reducing tumor progression and thus treat subjected afflicted with cancer.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds the chemokine (C-C motif) receptor 8 (CCR8).
  • the isolated antibody or antigen-binding fragment thereof is monoclonal. [0014] In some embodiments, the isolated antibody or antigen-binding fragment thereof binds human CCR8 and rabbit CCR8.
  • the isolated antibody or antigen-binding fragment thereof recognizes an epitope having or consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the isolated antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region which comprises at least one, preferably at least two, more preferably the three following complementary-determining regions (CDRs):
  • the antibody or antigen-binding fragment thereof comprises a light chain variable region which comprises at least one, preferably at least two, more preferably the three following complementary- determining regions (CDRs):
  • the epitope is post-transcriptionally modified.
  • the epitope is sulfonated.
  • the epitope is sulfonated at the amino acid position 7 and/or 9, wherein position numbering is with respect to SEQ ID NO: 1 or SEQ ID NO: 2.
  • the isolated antibody or antigen-binding fragment thereof according to invention comprises a heavy chain variable region (VH) which comprises at least one, preferably at least two, more preferably the three following complementary- determining regions (CDRs):
  • VH heavy chain variable region
  • CDRs complementary- determining regions
  • VH-CDR1 SEQ ID NO : 10
  • VH-CDR2 SEQ ID NO : 11 , and/or
  • VH-CDR3 SEQ ID NO: 12.
  • the isolated antibody or antigen-binding fragment thereof according to invention comprises a light chain variable region (VL) which comprises at least one, preferably at least two, more preferably the three following complementary- determining regions (CDRs):
  • VL light chain variable region
  • CDRs complementary- determining regions
  • VL-CDR1 SEQ ID NO: 13
  • VL-CDR2 SEQ ID NO: 14, and/or
  • VL-CDR3 SEQ ID NO : 15.
  • the antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain variable region (VH) which comprises the three following complementary-determining regions (CDRs):
  • VH-CDR3 SEQ ID NO : 12, and further comprises a light chain variable region (VL) which comprises the three following complementary-determining regions (CDRs):
  • VL-CDR1 SEQ ID NO: 13
  • VL-CDR2 SEQ ID NO: 14, and/or VL-CDR3 : SEQ ID NO : 15.
  • the isolated antibody or antigen-binding fragment thereof to CCR8 inhibits the binding of chemokine (C-C motif) ligand 1 (CCL1) and/or chemokine (C-C motif) ligand 18 (CCL18) to CCR8.
  • the present invention further relates to a nucleic acid encoding the isolated antibody or antigen-binding fragment thereof according to the invention.
  • the present invention further relates to a vector containing a nucleic acid according to the invention.
  • the present invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof, the nucleic acid or the vector according to the invention, and at least one pharmaceutically acceptable excipient.
  • the pharmaceutical composition further comprises at least on additional therapeutic agent.
  • the additional therapeutic agent is a programmed cell death protein 1 (PD-1) inhibitor.
  • PD-1 programmed cell death protein 1
  • the present invention further relates to an isolated antibody or antigen-binding fragment thereof, a nucleic acid, a vector or a pharmaceutical composition according to the invention, for use as a medicament.
  • the present invention further relates to the isolated antibody or antigen-binding fragment thereof, nucleic acid, vector or pharmaceutical composition for use according to the invention, for treating and/or preventing a CCR8-related disease in a subject in need thereof.
  • the disease is cancer.
  • the cancer is caused by cells expressing CCR8, whether cancerous or non-cancerous cells.
  • the present invention further relates to a method for treating and/or preventing a disease, preferably a CCR8-related disease, comprising administering to a subject in need thereof a therapeutically effective amount of the isolated antibody or antigen-binding fragment thereof according to the invention.
  • the present invention further relates to the use of an isolated antibody or antigenbinding fragment thereof, a nucleic acid, a vector or a pharmaceutical composition according to the invention, for the manufacture of a medicament, preferably for the treatment and/or prevention of a CCR8-related disease.
  • affinity and “avidity” are well-known in the art and are used to defined the strength of an antibody-antigen complex. Affinity measures the strength of interaction between an epitope and an antigen binding site on an antibody. It may be expressed by an affinity constant KA or by a dissociation constant KD. Avidity (or functional affinity) gives a measure of the overall strength of an antibody-antigen complex. It may depend on different parameters, including in particular the affinity of the antibody or antigen-binding fragment thereof for an epitope, (ii) the valency of both the antibody and the antigen and (iii) structural arrangement of the parts that interact.
  • antibody and “immunoglobulin” may be used interchangeably and refer to a protein having a combination of two heavy and two light chains whether or not it possesses any relevant specific immunoreactivity.
  • Antibodies refers to such assemblies which have significant known specific immunoreactive activity to an antigen of interest (e.g., human CCR8).
  • anti-hCCR8 antibodies is used herein to refer to antibodies which exhibit immunological specificity for human CCR8 protein.
  • specificity for human CCR8 does not exclude crossreaction with species homologues of hCCR8, such as, for example, with simian CCR8.
  • Antibodies and immunoglobulins comprise light and heavy chains, with or without an interchain covalent linkage between them.
  • Basic immunoglobulin structures in vertebrate systems are relatively well understood.
  • the generic term “immunoglobulin” comprises five distinct classes of antibody that can be distinguished biochemically. Although the following discussion will generally be directed to the IgG class of immunoglobulin molecules, all five classes of antibodies are within the scope of the present invention.
  • immunoglobulins comprise two identical light polypeptide chains of molecular weight of about 23 kDa, and two identical heavy chains of molecular weight of about 53-70 kDa.
  • the four chains are joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.
  • the light chains of an antibody are classified as either kappa (K) or lambda (X).
  • K kappa
  • X lambda
  • Each heavy chain class may be bonded with either a K or X light chain.
  • the light and heavy chains are covalently bonded to each other, and the “tail” regions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells.
  • heavy chains In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
  • heavy chains are classified as gamma (y), mu (p), alpha (a), delta ( ⁇ ) or epsilon (a) with some subclasses among them (e.g., yl-y4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgD or IgE, respectively.
  • variable region of an antibody allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the light chain variable domain (VL domain) and heavy chain variable domain (VH domain) of an antibody combine to form the variable region that defines a three-dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site presents at the end of each arm of the “Y”.
  • antigen-binding fragment refers to a part or region of the antibody according to the present invention, which comprises fewer amino acid residues than the whole antibody.
  • An “antigen-binding fragment” binds antigen and/or competes with the whole antibody from which it was derived for antigen binding (e.g., specific binding to human CCR8).
  • Antibody antigen-binding fragments encompasses, without any limitation, single chain antibodies, Fv, Fab, Fab', Fab'-SH, F(ab)’2, Fd, defucosylated antibodies, diabodies, triabodies and tetrabodies.
  • effector T cells refers to a group of cells that includes several T cells types (e.g., CD4+ and CD8+ T cells). It includes helpers T cells (Th cells) that help other leukocytes in immunologic processes, including maturation of B cells into plasma cells and memory B cells and cytotoxic T cells (Tc cells, CTLs, T-killer cells, killer T cells) that destroy virus-infected cells and tumor cells, and are also implicated in transplant rejection.
  • Th cells helpers T cells
  • epitope refers to a specific arrangement of amino acids located on a protein or proteins to which an antibody or antigen-binding fragment thereof or an antibody mimetic bind.
  • Epitopes often consist of a chemically active surface grouping of molecules such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • Epitopes can be linear (or sequential) or conformational, i.e., involving two or more sequences of amino acids in various regions of the antigen that may not necessarily be contiguous.
  • Fc domain refers to a C-terminal fragment of an antibody heavy chain, e.g., from about amino acid (aa) 230 to about aa 450 of human gamma heavy chain or its counterpart sequence in other types of antibody heavy chains (e.g., a, 6, a and p for human antibodies), or a naturally occurring allotype thereof.
  • Fv refers to the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one HCVR and one LCVR in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the heavy and light chain) that contribute to antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the term “heavy chain region” includes amino acid sequences derived from the constant domains of an immunoglobulin heavy chain.
  • a protein comprising a heavy chain region comprises at least one of a CHI domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof.
  • the antibody or antigen-binding fragment thereof according to the present invention may comprise the Fc region of an immunoglobulin heavy chain (e.g., a hinge portion, a CH2 domain, and a CH3 domain).
  • the antibody or antigen-binding fragment thereof according to the present invention lacks at least a region of a constant domain (e.g., all or part of a CH2 domain).
  • at least one, and preferably all, of the constant domains are derived from a human immunoglobulin heavy chain.
  • the heavy chain region comprises a fully human hinge domain.
  • the heavy chain region comprising a fully human Fc region (e.g., hinge, CH2 and CH3 domain sequences from a human immunoglobulin).
  • the constituent constant domains of the heavy chain region are from different immunoglobulin molecules.
  • a heavy chain region of a protein may comprise a CH2 domain derived from an IgGl molecule and a hinge region derived from an IgG3 or IgG4 molecule.
  • the constant domains are chimeric domains comprising regions of different immunoglobulin molecules.
  • a hinge may comprise a first region from an IgGl molecule and a second region from an IgG3 or IgG4 molecule.
  • the constant domains of the heavy chain region may be modified such that they vary in amino acid sequence from the naturally occurring (wild-type) immunoglobulin molecule.
  • the antibody or antigen-binding fragment thereof according to the present invention may comprise alterations or modifications to one or more of the heavy chain constant domains (CHI, hinge, CH2 or CH3) and/or to the light chain constant domain (CL).
  • exemplary modifications include additions, deletions or substitutions of one or more amino acids in one or more domains.
  • hinge region includes the region of a heavy chain molecule that joins the CHI domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., 1998. J Immunol. 161 (8):4083 -90).
  • identity when used in a relationship between the sequences of two or more amino acid sequences, or of two or more nucleic acid sequences, refers to the degree of sequence relatedness between amino acid sequences or nucleic acid sequences, as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. “Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., “algorithms”). Identity of related amino acid sequences or nucleic acid sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Lesk A. M. (1988).
  • Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Genetics Computer Group, University of Wisconsin, Madison, WI; Devereux et al., 1984. Nucleic Acids Res. 12(1 Pt 1):387-95), BLASTP, BLASTN, and FASTA (Altschul et al., 1990. J Mol Biol. 215(3):403-10). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894). The well-known Smith Waterman algorithm may also be used to determine identity.
  • GAP Genetics Computer Group, University of Wisconsin, Madison, WI; Devereux et al., 1984. Nucleic Acids Res. 12(1 Pt 1):
  • the term "immunotoxin” refers to a bifunctional molecule comprising targeting moiety for delivery (a ligand) and a toxic moiety (toxin) for cytotoxicity.
  • the immunotoxin can be used to kill cells expressing receptors for the ligand.
  • the term “mammal” refers to any mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.
  • the mammal is human.
  • the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprised in the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies or antigen-binding fragment thereof according to the present invention may be prepared by the hybridoma methodology first described by Kohler et al., 1975. Nature. 256(5517):495-7, or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (Patent US4,816,567).
  • the “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., 1991. Nature. 352(6336):624-8 and Marks et al., 1991. J Mol Biol. 222(3):581-97, for example.
  • Nanobodies are well-known in the art and refer to antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally-occurring heavy chain antibodies (Muyldermans, 2013. Annu Rev Biochem. 82:775-97). These heavy chain antibodies may contain a single variable domain (VHH) and two constant domains (CH2 and CH3).
  • VHH variable domain
  • CH3 constant domain
  • nucleic acid or “polynucleotide” refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Nucleic acid or “Polynucleotides” include, without limitation single-and doublestranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double- stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • Nucleic acid refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term “nucleic acid” or “polynucleotide” also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • nucleic acid or “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
  • prevent refers to prophylactic and preventative measures, wherein the object is to reduce the chances that a subject will develop the pathologic condition or disorder over a given period of time. Such a reduction may be reflected, e.g., in a delayed onset of at least one symptom of the pathologic condition or disorder in the subject.
  • Treg cell refers to a specialized type of T cells, in particular of CD4+ T cell, that can suppress the responses of other T cells. Treg cells play a critical role in the induction and maintenance of peripheral self-tolerance to antigens, including those expressed by tumors.
  • single-chain Fv also abbreviated as “sFv” or “scFv” refers to antibody fragments that comprise the VH and VL antibody domains connected into a single amino acid chain.
  • the scFv amino acid sequence further comprises a peptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding (Pliickthun, 1994. Antibodies from Escherichia coli. In Rosenberg & Moore (Eds.), The pharmacology of monoclonal antibodies. Handbook of Experimental Pharmacology, 113:269-315. Springer: Berlin, Heidelberg).
  • the term “subject” refers to a mammal, preferably a human.
  • a subject may be a “patient”, i.e., a warm-blooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of a disease.
  • patient i.e., a warm-blooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of a disease.
  • mammal refers here to any mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.
  • the mammal is a primate, more preferably a human.
  • the term “therapeutically effective amount” refers to the level or amount of an antibody as described herein that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of a disease, disorder, or condition; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of the disease, disorder, or condition; (3) bringing about ameliorations of the symptoms of the disease, disorder, or condition; (4) reducing the severity or incidence of the disease, disorder, or condition; or (5) curing the disease, disorder, or condition.
  • a therapeutically effective amount may be administered prior to the onset of the disease, disorder, or condition, for a prophylactic or preventive action. Alternatively, or additionally, the therapeutically effective amount may be administered after initiation of the disease, disorder, or condition, for a therapeutic action.
  • treating or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder.
  • Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
  • a subject or mammal is successfully "treated” for a cancer or an infection if, after receiving a therapeutic amount of an antibody according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of cancer cells (or tumor size), or pathogenic cells; reduction in the percent of total cells that are cancerous or pathogenic; and/or relief to some extent, one or more of the symptoms associated with the specific disease or condition; reduced morbidity and mortality, and improvement in quality of life issues.
  • the above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.
  • variable refers to the fact that certain regions of the variable domains VH and VL differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its target antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called “hypervariable loops” in each of the VL domain and the VH domain which form part of the antigen binding site.
  • the first, second and third hypervariable loops of the V light chain domain are referred to herein as LI (X), L2 (X) and L3 (X) and may be defined as comprising residues 24-33 (L1(X), consisting of 9, 10 or 11 amino acid residues), 49-53 L2 (X), consisting of 3 residues) and 90-96 (L3(X), consisting of 6 residues) in the VL domain (Morea et al., 2000. Methods. 20(3):267-79).
  • the first, second and third hypervariable loops of the VK light chain domain are referred to herein as L1(K), L2(K) and L3(K) and may be defined as comprising residues 25-33 (L1(K), consisting of 6, 7, 8, 11, 12 or 13 residues), 49-53 (L2(K), consisting of 3 residues) and 90-97 (L3(K), consisting of 6 residues) in the VL domain (Morea et al., 2000. Methods. 20(3):267-79).
  • the first, second and third hypervariable loops of the VH domain are referred to herein as Hl, H2 and H3 and may be defined as comprising residues 25-33 (Hl, consisting of 7, 8 or 9 residues), 52-56 (H2, consisting of 3 or 4 residues) and 91-105 (H3, highly variable in length) in the VH domain (Morea et al., 2000. Methods. 20(3):267-79).
  • the terms LI, L2 and L3 respectively refer to the first, second and third hypervariable loops of a VL domain, and encompass hypervariable loops obtained from both VK and V isotypes.
  • Hl, H2 and H3 respectively refer to the first, second and third hypervariable loops of the VH domain, and encompass hypervariable loops obtained from any of the known heavy chain isotypes, including gamma (y), mu (p), alpha (a), delta (5) or epsilon (a).
  • This invention relates to an isolated antibody or antigen-binding fragment thereof that binds the chemokine (C-C motif) receptor 8 (CCR8).
  • the isolated antibody or antigen-binding fragment thereof binds to CCR8 from any animal species. In some embodiments, the isolated antibody or antigen-binding fragment thereof binds to any mammalian CCR8. In some embodiments, the isolated antibody or antigen-binding fragment thereof binds to both human and rabbit CCR8. In some embodiments, the isolated antibody or antigen-binding fragment thereof binds human CCR8, wherein the amino acid sequence of human CCR8 corresponds to SEQ ID NO: 3. In some embodiments, the isolated antibody or antigen-binding fragment thereof binds rabbit CCR8, wherein the amino acid sequence of rabbit CCR8 corresponds to SEQ ID NO: 18.
  • the isolated antibody or antigen-binding fragment thereof binds mouse CCR8, wherein the amino acid sequence of mouse CCR8 corresponds to SEQ ID NO: 19. In some embodiments, the isolated antibody or antigenbinding fragment thereof binds rat CCR8, wherein the amino acid sequence of rat CCR8 corresponds to SEQ ID NO: 20.
  • the isolated antibody or antigen-binding fragment thereof recognizes an epitope having at least 75% sequence identity with the amino acid sequence SEQ ID NO: 1 and/or SEQ ID NO: 2.
  • sequence identity means 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9, 99.99, 99.999 or 100% sequence identity.
  • the isolated antibody or antigen-binding fragment thereof recognizes an epitope having the amino acid sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2.
  • the isolated antibody or antigen-binding fragment thereof recognizes an epitope consisting of the amino acid sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2. [0066] In some embodiments, the isolated antibody or antigen-binding fragment thereof recognizes an epitope having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the isolated antibody or antigen-binding fragment thereof recognizes an epitope having the amino acid sequence of SEQ ID NO: 2.
  • the isolated antibody or antigen-binding fragment thereof recognizes an epitope consisting of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the isolated antibody or antigen-binding fragment thereof recognizes an epitope consisting of the amino acid sequence of SEQ ID NO: 2.
  • the epitope is post-transitionally modified. In some embodiments, the epitope is sulfonated.
  • sulfonated epitope refers to the addition of at least one sulfonic acid molecule (SO3H) on at least one moiety, e.g., an amine or a thiol, of the side chain of at least one amino acid, e.g., a tyrosine or a cysteine, within the amino acid sequence of the epitope.
  • SO3H sulfonic acid molecule
  • the epitope is sulfonated at the amino acid position 7 and/or 9, wherein position numbering is with respect to SEQ ID NO: 1 or SEQ ID NO: 2.
  • the amino acid sequence of the sulfonated epitope is VTTVTDY(SO3H)YY(SO3H)PDIFSSPC or VTAVTDY(SO3H)YY(SO3H)PDIFSSPC.
  • CDR numbering and definitions are according to the IMGT® numbering system.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (abbreviated herein as HCVR or VH) which comprises at least one, preferably at least two, more preferably the three following complementary-determining regions (CDRs):
  • HCVR heavy chain variable region
  • CDRs complementary-determining regions
  • VH-CDR1 EYTMH (SEQ ID NO: 10);
  • VH-CDR2 GINPKNGVTRYNQKFKG (SEQ ID NO: 11); and/or VH-CDR3: GYHGPFAY (SEQ ID NO: 12).
  • the antibody or antigen-binding fragment thereof according to the present invention comprises a HCVR which comprises the three following CDRs:
  • VH-CDR1 EYTMH (SEQ ID NO: 10);
  • VH-CDR2 GINPKNGVTRYNQKFKG (SEQ ID NO: 11); and VH-CDR3: GYHGPFAY (SEQ ID NO: 12).
  • the antibody or antigen-binding fragment thereof comprises a light chain variable region (abbreviated herein as LCVR or VL) which comprises at least one, preferably at least two, more preferably the three following complementary-determining regions (CDRs):
  • LCVR light chain variable region
  • CDRs complementary-determining regions
  • VL-CDR1 RASQEISGYLS (SEQ ID NO: 13);
  • VL-CDR2 AASTLDS (SEQ ID NO: 14); and/or VL-CDR3: LHYANAPYT (SEQ ID NO: 15).
  • the antibody or antigen-binding fragment thereof according to the present invention comprises a LCVR which comprises the three following CDRs:
  • VL-CDR1 RASQEISGYLS (SEQ ID NO: 13);
  • VL-CDR2 AASTLDS (SEQ ID NO: 14);
  • VL-CDR3 LHYANAPYT (SEQ ID NO: 15).
  • the antibody or antigen-binding fragment thereof according to the present invention comprises: a HCVR which comprises at least one, preferably at least two, more preferably the three following CDRs:
  • VH-CDR1 EYTMH (SEQ ID NO: 10);
  • VH-CDR2 GINPKNGVTRYNQKFKG (SEQ ID NO: 11); and/or VH-CDR3: GYHGPFAY (SEQ ID NO: 12); and a LCVR which comprises at least one, preferably at least two, more preferably the three following CDRs:
  • VL-CDR1 RASQEISGYLS (SEQ ID NO: 13);
  • VL-CDR2 AASTLDS (SEQ ID NO: 14); and/or
  • VL-CDR3 LHYANAPYT (SEQ ID NO: 15).
  • the antibody or antigen-binding fragment thereof according to the present invention comprises: a HCVR which comprises the three following CDRs:
  • VH-CDR1 EYTMH (SEQ ID NO: 10);
  • VH-CDR2 GINPKNGVTRYNQKFKG (SEQ ID NO: 11); and VH-CDR3: GYHGPFAY (SEQ ID NO: 12); and a LCVR which comprises the three following CDRs:
  • VL-CDR1 RASQEISGYLS (SEQ ID NO: 13);
  • VL-CDR2 AASTLDS (SEQ ID NO: 14); and VL-CDR3: LHYANAPYT (SEQ ID NO: 15).
  • any of CDR1, CDR2 and/or CDR3 of the HCVR with SEQ ID NOs 10-12 can be characterized as having 1, 2, 3 or more amino acids being substituted by a different amino acid.
  • any of CDR1, CDR2 and/or CDR3 of the LCVR with SEQ ID NOs 13-15 can be characterized as having 1, 2, 3 or more amino acids being substituted by a different amino acid.
  • any of CDR1, CDR2 and/or CDR3 of the HCVR with SEQ ID NOs 10-12 and/or of the LCVR with SEQ ID NOs 13-15 can be characterized as having 1, 2, 3 or more amino acids being substituted by a different amino acid.
  • conservative amino acid modifications refers to modifications that do not significantly affect or alter the binding characteristics of the antibody or antigen-binding fragment thereof containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antigen-binding fragment thereof by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions are typically those in which an amino acid residue is replaced with an amino acid residue having a side chain with similar physicochemical properties.
  • Specified variable region and CDR sequences may comprise 1, 2, 3 or more amino acid insertions, deletions and/or substitutions. Where substitutions are made, preferred substitutions will be conservative modifications. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • [3- branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • one or more amino acid residues within the CDRs and/or variable regions of the antibody or antigen-binding fragment thereof according to the present invention can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., the properties set forth herein, such as, e.g., the binding to CCR8) using the assays described herein.
  • a string of amino acids within the CDRs and/or variable regions of the antibody or antigen-binding fragment thereof according to the present invention can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
  • any of CDR1, CDR2 and/or CDR3 of the HCVR with SEQ ID Nos 10-12 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more of identity with the particular CDR or sets of CDRs listed in the corresponding SEQ ID NOs.
  • any of CDR1, CDR2 and/or CDR3 of the LCVR with SEQ ID NOs 13-15 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more of identity with the particular CDR or sets of CDRs listed in the corresponding SEQ ID NOs.
  • any of CDR1, CDR2 and/or CDR3 of the HCVR with SEQ ID NOs 10-12 and/or of the LCVR with SEQ ID NOs 13-15 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more of identity with the particular CDR or sets of CDRs listed in the corresponding SEQ ID NOs.
  • the LCVR and/or the HCVR further comprises a leader sequence, preferably located N terminally from the LCVR amino acid sequence or N terminally from the HCVR amino acid sequence respectively.
  • leader sequences include, but are not limited to, SEQ ID NO: 16 and 17.
  • the LCVR comprises an amino acid sequence leader sequence SEQ ID NO: 16 located N terminally from the HCVR amino acid sequence.
  • the LCVR comprises an amino acid leader sequence SEQ ID NO: 17 located N terminally from the LCVR amino acid sequence.
  • the antibody or antigen-binding fragment thereof according to the present invention comprises a fully or substantially fully human heavy chain constant region (abbreviated herein as HCCR or CH) and/or light chain constant region (abbreviated herein as LCCR or CL).
  • HCCR fully or substantially fully human heavy chain constant region
  • LCCR light chain constant region
  • the binding of the isolated antibody or antigen-binding fragment thereof to CCR8 inhibits the binding of chemokine (C-C motif) ligand 1 (CCL1) and/or chemokine (C-C motif) ligand 18 (CCL18) to CCR8.
  • an “isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other proteins or antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds CCR8 is substantially free of proteins or antibodies that specifically bind antigens other than CCR8).
  • An isolated antibody that specifically binds CCR8 may, however, have cross-reactivity to other antigens, such as CCR8 molecules from other species.
  • an isolated antibody may be substantially free of other cellular material and/or chemicals, in particular those that would interfere with diagnostic or therapeutic uses of the protein or antibody, including without limitation, enzymes, hormones, and other proteinaceous or non-proteinaceous components.
  • An antibody or antigen-binding fragment thereof is said to be “specific for”, “immunospecific” or to “specifically bind” an antigen if it reacts at a detectable level with said antigen (e.g., CCR8), preferably with an affinity constant (KA) of greater than or equal to about 10 6 M' 1 , preferably greater than or equal to about 10 7 M' 1 , 10 8 M' 1 , 5* 10 8 M' 1 , 10 9 M' 1 , 5* 10 9 M' 1 or more.
  • Affinity of an antibody or antigen-binding fragment thereof for its cognate antigen is also commonly expressed as an equilibrium dissociation constant (KD).
  • An antibody or antigen-binding fragment thereof is said to be “immunospecific”, “specific for” or to “specifically bind” an antigen if it reacts at a detectable level with said antigen (e.g., CCR8), preferably with a KD of less than or equal to 10' 6 M, preferably less than or equal to 10' 7 M, 5* 10' 8 M, 10' 8 M, 5* 10' 9 M, 10' 9 M or less.
  • said antigen e.g., CCR8
  • Binding properties of an antibody or antigenbinding fragment thereof to antigens, cells or tissues may generally be determined and assessed using immunodetection methods including, for example, ELISA, immunofluorescence-based assays, such as immuno-histochemistry (IHC) and/or fluorescence-activated cell sorting (FACS) or by surface plasmon resonance (SPR, e.g., using BIAcore®).
  • immunodetection methods including, for example, ELISA, immunofluorescence-based assays, such as immuno-histochemistry (IHC) and/or fluorescence-activated cell sorting (FACS) or by surface plasmon resonance (SPR, e.g., using BIAcore®).
  • the isolated antibody or antigen-binding fragment thereof is purified. [0101] In one embodiment, the antibody or antigen-binding fragment thereof is purified to:
  • the isolated antibody or antigen-binding fragment thereof is monoclonal. In another embodiment, the isolated antibody or antigen-binding fragment thereof is polyclonal.
  • the isolated antibody is whole. In some embodiments, the isolated antibody is truncated. In certain embodiments, the truncated antibody is an antigen-binding fragment; in practice, an antigen-binding fragment retains the ability to bind an epitope on an antigen, but lacks parts of its amino acid sequence that are not involved in epitope binding, e.g., fragment crystallizable region (Fc).
  • Fc fragment crystallizable region
  • the isolated antibody or antigen-binding fragment thereof is a recombinant antibody.
  • the isolated antibody or antigenbinding fragment thereof is a modified antibody, e.g., an antibody o with optimized Fc inducing an increased engagement of Fc receptors and improved isolated antibodydependent cellular cytotoxicity (ADCC).
  • ADCC isolated antibodydependent cellular cytotoxicity
  • the antibody or antigen-binding fragment thereof, or parts thereof is comprised in a fusion protein.
  • the antibody or antigen-binding fragment thereof according to the present invention is a molecule selected from the group comprising or consisting of a whole antibody, a humanized antibody, a single chain antibody, a dimeric single chain antibody, a Fv, a Fab, a Fab', a Fab'-SH, a F(ab)’2, a Fd, a defucosylated antibody, a bispecific antibody, a diabody, a triabody and a tetrabody.
  • Antigen-binding fragments of antibodies can be obtained using standard methods. For instance, Fab or F(ab')2 fragments may be produced by protease digestion of the isolated antibodies, according to conventional techniques.
  • antibodies or antigen-binding fragments thereof according to the present invention can be modified using known methods.
  • the antibody or antigen-binding fragment thereof may be modified with polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the antibody or antigen-binding fragment thereof according to the present invention is a molecule selected from the group comprising or consisting of a unibody, a domain antibody, and a nanobody.
  • the antibody or antigen-binding fragment thereof according to the present invention comprises a fully or substantially fully human heavy chain constant region (abbreviated herein as HCCR or CH) and/or light chain constant region (abbreviated herein as LCCR or CL).
  • HCCR fully or substantially fully human heavy chain constant region
  • LCCR light chain constant region
  • the constant region is of human origin.
  • the antibody or antigen-binding fragment thereof according to the present invention comprises a fully or substantially fully rabbit HCCR and/or LCCR.
  • the constant region is of rabbit origin.
  • the antibody or antigen-binding fragment thereof according to the present invention is a rabbit antibody or fragment thereof.
  • the antibody or antigen-binding fragment thereof according to the present invention comprises a fully or substantially fully murine HCCR and/or LCCR.
  • the constant region is of murine origin.
  • the antibody or antigen-binding fragment thereof according to the present invention is a murine antibody or fragment thereof.
  • the antibody or antigen-binding fragment thereof according to the present invention is a chimeric antibody or fragment thereof.
  • a “chimeric antibody”, as used herein, refers to an antibody or antigen-binding fragment thereof comprising a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked in nature.
  • the amino acid sequences may normally exist in separate proteins that are brought together in the fusion protein or they may normally exist in the same protein but are placed in a new arrangement in the fusion protein.
  • a chimeric protein may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
  • chimeric antibody encompasses herein antibodies and antigen-binding fragment thereof in which the constant region, or a portion thereof, is altered, replaced or exchanged so that the variable region is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region, or portion thereof, having a different or altered antigen specificity; or with corresponding sequences from another species or from another antibody class or subclass.
  • the antibody or antigen-binding fragment thereof according to the present invention is a humanized antibody or fragment thereof.
  • a “humanized antibody”, as used herein, refers to a chimeric antibody or antigenbinding fragment thereof which contains minimal sequence derived from a non-human immunoglobulin. It includes antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell, e.g., by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences.
  • Humanized antibodies or antigen-binding fragment thereof according to the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • a “humanized antibody” retains a similar antigenic specificity as the original antibody. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody may be increased.
  • Another method for humanizing the antibody or antigen-binding fragment thereof according to the present invention uses a particular framework from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.
  • the same framework can be used for several different humanized antibodies (Carter et al., 1992. Proc Natl Acad Sci USA. 89(10):4285-9; Presta et al., 1993. J Immunol. 151(5):2623-32). It is further important that antibodies be humanized with retention of high affinity for hCCR8 and other favorable biological properties.
  • humanized antibodies and antigen-binding fragments thereof are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
  • Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its epitope.
  • Another method for humanizing the antibody or antigen-binding fragment thereof according to the present invention is to use a transgenic or transchromosomic animal carrying parts of the human immune system for immunization. As a host, these animals have had their immunoglobulin genes replaced by functional human immunoglobulin genes. Thus, antibodies produced by these animals or in hybridomas made from the B cells of these animals are already humanized.
  • transgenic or transchromosomic animal examples include, without limitation: the XenoMouse (Abgenix, Fremont, CA), described in Patents US5,939,598, US6,075,181, US6,114,598, US6, 150,584 and US6, 162,963; the HuMAb Mouse® (Medarex, Inc.), described in Lonberg et al., 1994. Nature. 368(6474):856-859; Lonberg & Huszar, 1995. Int Rev Immunol. 13(l):65-93; Harding & Lonberg, 1995. Ann N Y Acad Sci. 764:536-46; Taylor et al., 1992. Nucleic Acids Res.
  • Humanized antibodies and antigen-binding fragments thereof may also be produced according to various other techniques, such as by using, for immunization, other transgenic animals that have been engineered to express a human antibody repertoire (Jakobovitz et al., 1993. Nature. 362(6417):255-8), or by selection of antibody repertoires using phage display methods. Such techniques are known to the skilled person and can be implemented starting from monoclonal antibodies or antigen-binding fragments thereof as disclosed in the present application.
  • the antibody or antigen-binding fragment thereof according to the present invention is produced in a mammalian species.
  • the mammalian species is selected from the group comprising or consisting of mouse, rat, goat, horse, pig, dog, cat, non-human primates such as chimpanzee, and the like.
  • the mammalian species is the mouse (e.g., mus musculus and the like).
  • the antibody or antigen-binding fragment thereof according to the present invention is from the IgG class.
  • the antibody or antigen-binding fragment thereof according to the present invention is from the human IgGl subclass. In another embodiment, the antibody or antigen-binding fragment thereof according to the present invention is from the human lgG2 subclass.
  • the Fc region of IgG antibodies interacts with cellular Fey receptors (FcyR) to stimulate and regulate downstream effector mechanisms.
  • Fey receptors There are five activating receptors, namely FcyRI (CD64), FcyRIIa (CD32a), FcyRIIc (CD32c), FcyRIIIa (CD 16a) and FcyRIIIb (CD 16b), and one inhibitory receptor FcyRIIb (CD32b).
  • FcyRI CD64
  • FcyRIIa CD32a
  • FcyRIIc CD32c
  • FcyRIIIa CD 16a
  • FcyRIIIb CD 16b
  • CD 16b FcyRIIIb
  • IgG subclasses vary in their ability to bind to FcyR and this differential binding determines their ability to elicit a range of functional responses.
  • FcyRIIIa is the major receptor involved in the activation of antibody-dependent cell-mediated cytotoxicity (ADCC) and IgG3 (followed closely by IgGl) display the highest affinities for this receptor, reflecting their ability to potently induce ADCC.
  • IgG2 have been shown to have weak binding for this receptor.
  • the antibody or antigen-binding fragment thereof according to the present invention binds FcyR with high affinity, preferably binds an activating receptor with high affinity.
  • the antibody or antigen-binding fragment thereof according to the present invention binds FcyRI and/or FcyRIIa and/or FcyRIIc and/or FcyRIIIa and/or FcyRIIIb with high affinity.
  • the IgGl antibody binds to at least one Fc activating receptor.
  • the antibody may bind to one or more receptor selected from FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa and FcyRIIIb.
  • the antibody is capable of binding to FcyRIIIa.
  • the antibody is capable of binding to FcyRIIa.
  • the antibody is capable of binding to FcyRIIIa, FcyRIIc and optionally FcyRI.
  • the antibody is capable of binding to FcyRIIIa, FcyRIIa and optionally FcyRI.
  • the antibody or antigen-binding fragment thereof according to the present invention binds to at least one activating Fey receptor with a dissociation constant of less than about 10-6M, 10-7M, 10-8M, 10-9M or 10-10M.
  • the IgGl antibody binds to FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and/or FcyRIIIb with a higher affinity than it binds to FcyRIIb, with low affinity.
  • the antibody or antigen-binding fragment thereof according to the present invention is an IgGl antibody, preferably a human IgGl antibody.
  • the antibody or antigen-binding fragment thereof according to the present invention may comprise human heavy chain constant regions sequences and allow to target, block, and/or deplete CCR8-expressing cells to which they are bound.
  • the proteins according to the present invention deplete CCR8-expressing cells to which they are bound.
  • the proteins according to the present invention deplete Tregs to which they are bound.
  • the proteins according to the present invention also deplete or reduce tumor infiltrating regulatory T cells to which they are bound.
  • the antibody or antigen-binding fragment thereof according to the present invention depletes CCR8-expressing cells to which it is bound. In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention depletes Tregs to which it is bound. In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention also depletes or reduces tumor infiltrating regulatory T cells to which it is bound.
  • the term “deplete” or “depleting”, with respect to CCR8-expressing cells or Tregs refers to the killing, elimination, lysis or induction of such killing, elimination or lysis, so as to negatively affect the number of CCR8 expressing cells present in a sample or in a subject.
  • the antibody or antigen binding fragment thereof according to the present invention allows targeting, blocking proliferation, and/or depleting CCR8-expressing cells or Treg cells.
  • the depletion is via ADCC.
  • the depletion is via ADCP. In one embodiment, the depletion is via CDC.
  • the antibody of the present invention leads, directly or indirectly, to the depletion of CCR8-expressing cells (e.g., leads to a 10%, 20%, 50%, 60%, 70%, 80%, 85% or greater elimination or decrease in number of CCR8 expressing cells).
  • the antibody or antigen-binding fragment thereof according to the present invention does not inhibit the binding of interleukin-2 (IL-2) to CCR8 and depletes Tregs to which they are bound.
  • IL-2 interleukin-2
  • the antibody or antigen-binding fragment thereof according to the present invention is a pegylated antibody or fragment thereof.
  • An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody.
  • the antibody, or fragment thereof typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment.
  • PEG polyethylene glycol
  • the pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
  • polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (DY12- DY120) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.
  • the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the present invention, such as, for example, as described in EP0154316 (Nishimura et al.) and EP0401384 (Ishikawa et al.).
  • the isolated antibody or antigen-binding fragment thereof as described hereinabove is for use as a medicament.
  • the present invention further relates to a nucleic acid encoding the isolated antibody or antigen-binding fragment thereof according to the invention.
  • the nucleic acid encoding the isolated antibody or antigenbinding fragment thereof of the invention may be expressed as a single nucleic acid molecule that encodes the isolated antibody or antigen-binding fragment thereof, or as multiple (e.g., two or more) nucleic acid molecules that are co-expressed.
  • Polypeptides encoded by nucleic acid molecules that are co-expressed may associate through, e.g., disulfide bonds or other means, to form the isolated antibody or antigen-binding fragment thereof as described hereinabove.
  • the nucleic acid molecule is DNA. In another embodiment, the nucleic acid molecule is RNA, for example, in the form of messenger RNA (mRNA). [0151] In one embodiment, the nucleic acid is linear. In another embodiment, the nucleic acid is circular.
  • the nucleic acid as described hereinabove is for use as a medicament.
  • Another object of the invention is a vector comprising a nucleic acid according the invention.
  • at least one nucleic acid molecule is comprised in the one vector.
  • the expression “at least one nucleic acid” is intended to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleic acid molecules.
  • the vector allows the controlled expression of at least one isolated antibody or antigen-binding fragment thereof.
  • controlled expression is intended to refer to an expression that is controlled in time and/or space.
  • the controlled expression of the isolated antibody or antigenbinding fragment thereof according to the invention may occur in a specific location of the body, such as, e.g., a specific organ, and/or for a specific time period.
  • the vector is a viral vector.
  • viral vectors include adenovirus, adeno-associated virus (AAV), alphavirus, herpesvirus, lentivirus, non-integrative lentivirus, retrovirus, vaccinia virus and baculovirus.
  • the vector is a non-viral vector.
  • non-viral vectors include inorganic particles (e.g., gold, calcium phosphate), lipidic emulsions, lipidic nanoparticles (e.g., liposomes), DNA-binding protein or peptide.
  • the vector as described hereinabove is for use as a medicament.
  • the present invention also relates to an immunotoxin fusion protein against a CCR8-expressing cell, such as a T reg cell or a cancer cell, comprising the isolated antibody or antigen-binding fragment thereof according to the invention and a toxin or fragment thereof.
  • An immunotoxin is a ligand combined with a toxin, which can be used to kill cells expressing receptors for the ligand. Immunotoxin treatment is also known as ligand- targeted therapeutics. Thus, the immunotoxins contain a targeting moiety (a ligand) for delivery and a toxic moiety for cytotoxicity.
  • the ligands currently used are monoclonal antibodies, cytokines/growth factors and soluble receptors.
  • the immunotoxin fusion protein comprises an antigenbinding fragment of the antibody of the invention.
  • the antigenbinding fragment comprises two single chain Fvs of the variable region of the antibody of the invention.
  • the antigen-binding fragment further comprises one or more linker(s).
  • the immunotoxin fusion protein comprises a truncated toxin.
  • the immunotoxin fusion protein can further comprise a modified amino terminal.
  • the immunotoxin fusion protein can further comprise a signal peptide at its amino terminal, following translation but prior to secretion.
  • the present invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof, nucleic acid or the vector according to the invention, and at least one additional pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable vehicle is selected in a group comprising or consisting of a solvent, a diluent, a carrier, an excipient, a dispersion medium, a coating, an antibacterial agent, an antifungal agent, an isotonic agent, an absorption delaying agent and any combinations thereof.
  • the carrier, diluent, solvent or excipient must be “acceptable” in the sense of being compatible with the isolated antibody or fragment thereof, nucleic acid or the vector according to the invention, or derivative thereof, and not be deleterious upon being administered to an individual.
  • the vehicle does not produce an adverse, allergic or other untoward reaction when administered to an individual, preferably a human individual.
  • the pharmaceutical compositions should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory offices, such as, for example, the Food and Drugs Administration (FDA) Office or the European Medicines Agency (EMA).
  • FDA Food and Drugs Administration
  • EMA European Medicines Agency
  • the pharmaceutical composition further comprises at least one additional therapeutic agent.
  • the at least one additional therapeutic agent is selected from the group comprising or consisting of anticancer agents and checkpoint inhibitors.
  • the at least one additional therapeutic agent is an anticancer agent.
  • Anticancer agents are known from the state of the art.
  • Non-limitative examples of anticancer agents include acalabrutinib, alectinib, alemtuzumab, anastrozole, avapritinib, avelumab, belinostat, bevacizumab, bleomycin, blinatumomab, bosutinib, brigatinib, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin copanlisib, cytarabine, daunorubicin, decitabine, dexamethasone, docetaxel, doxorubicin, encorafenib, erdafitinib, etoposide, everolimus, exemestane, fludarabine, 5-fluorouracil, gemcitabine, ifosfamide, imatinib Mesylate, leuprolide
  • the anticancer agent is to be administered in combination with, concomitantly or sequentially, the isolated antibody or antigen-binding fragment thereof, nucleic acid, the vector or the pharmaceutical composition according to the invention.
  • the at least one additional therapeutic agent is a checkpoint inhibitor therapy.
  • checkpoint inhibitor therapy is defined as the administration of at least one checkpoint inhibitor to the subject.
  • Checkpoint inhibitors that may also be referred to as immune checkpoint inhibitors or ICI
  • ICI immune checkpoint inhibitors
  • Checkpoint inhibitor therapy aims at preventing the activation of inhibitory receptors expressed on T cells by ligands expressed by the tumor cells.
  • Checkpoint inhibitor therapy thus aims at preventing the inhibition of T cells present in the tumor, i.e., tumor infiltrating T cells, and thus at enhancing the subject immune response towards the tumor cells.
  • checkpoint inhibitors include, without being limited to, inhibitors of the cell surface receptor PD-1 (programmed cell death protein 1), also known as CD279 (cluster differentiation 279); inhibitors of the ligand PD-L1 (programmed death-ligand 1), also known as CD274 (cluster of differentiation 274) or B7-H1 (B7 homolog 1); inhibitors of the cell surface receptor CTLA4 or CTLA-4 (cytotoxic T-lymphocyte- associated protein 4), also known as CD 152 (cluster of differentiation 152); inhibitors of LAG-3 (lymphocyte-activation gene 3), also known as CD223 (cluster differentiation 223); inhibitors of TIM-3 (T-cell immunoglobulin and mucin-domain containing-3), also known as HAVCR2 (hepatitis A virus cellular receptor 2) or CD366 (cluster differentiation 366); inhibitors of TIGIT (T cell immunoreceptor with Ig and ITIM domains), also known as VSIG9 (V-Set And Immun
  • the at least one checkpoint inhibitor is selected from the group comprising or consisting of inhibitors of PD-1, inhibitors of PD-L1, inhibitors of CTLA 4 and any mixtures thereof.
  • the therapeutic agent is a programmed cell death protein 1 (PD-1) inhibitor.
  • the PD-1 inhibitor is an isolated antibody or antigen-binding fragment thereof selective for PD-1, preferably a monoclonal antibody.
  • Non limitative examples of FDA approved anti -PD-1 therapeutic antibodies include pembrolizumab (Keytruda®, Merck), nivolumab (Opdivo®, Bristol Myers Squibb), cemiplimab (Libtayo®, Sanofi Genzyme) and dostarlimab (Jemperli®, GlaxoSmithKline).
  • the PD-1 inhibitor is a small molecule or a nucleic acid, e.g., an interfering RNA.
  • the antibody or antigen-binding fragment thereof, nucleic acid, expression vector, pharmaceutical composition or medicament according to the present invention is used in combination with a second antibody that induces, via ADCC, the death of a cell expressing an antigen to which the second antibody binds.
  • the second antibody e.g., of IgGl or IgG3 isotype
  • NK cells have an important role in inducing ADCC and increased reactivity of NK cells can be directed to target cells through use of such a second antibody.
  • the second antibody is specific for a cell surface antigen, e.g., membrane antigen.
  • the second antibody is specific for a tumor antigen (e.g., molecules specifically expressed by tumor cells), such as CD20, CD52, ErbB2 (or HER2/Neu), CD33, CD22, MUC-1, CEA, KDR, aVp3, etc., particularly lymphoma antigens (e.g., CD20).
  • a tumor antigen e.g., molecules specifically expressed by tumor cells
  • CD20 CD52, ErbB2 (or HER2/Neu)
  • the present invention further relates to the isolated antibody or antigen-binding fragment thereof, nucleic acid, vector or pharmaceutical composition according to the invention, for use as a medicament.
  • the present invention further relates to the isolated antibody or antigen-binding fragment thereof, nucleic acid, vector, pharmaceutical composition or medicament according to the invention, for use for treating and/or preventing a disease, preferably a CCR8-related disease in a subject in need thereof.
  • a disease preferably a CCR8-related disease in a subject in need thereof.
  • CCR8-related disease encompasses diseases wherein CCR8 is expressed or overexpressed by at least one cell type, and/or wherein CCR8- expressing cells are overrepresented and/or mislocalized.
  • Non-limitative examples of CCR8-related diseases include cancer, in particular CCR8-expressing T-cell lymphomas or leukaemia, or cancers where CCR8-expressing cells have infiltrated and promote the development of cancer; and diseases associated with or related to immune responses dysfunction.
  • the CCR8-related disease is cancer.
  • the cancer is selected from the group comprising or consisting of colon cancer, melanoma, breast cancer, urothelial cancer, adenoid cystic carcinoma, adrenal gland cancer, ataxia-telangiectasia, atypical mole syndrome, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, carcinoid tumor, cervical cancer, colorectal cancer, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, islet cell tumor, kidney cancer, laryngeal cancer, leukemia, liver cancer, lobular carcinoma, hepatocellular carcinoma, lung cancer, glioma, meningioma, nasopharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, peritoneal cancer, pituitary gland tumor, polycythemia vera, prostate cancer, renal cell carcinoma, retinoblastoma, salivary gland cancer, sarcoma
  • the cancer is selected from the group comprising or consisting of colon cancer, melanoma, breast cancer and urothelial cancer.
  • the cancer is colon cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is urothelial cancer.
  • the CCR8-related disease is a disease associated with or related to immune responses dysfunction.
  • the disease associated with or related to immune responses dysfunction is an inflammatory disease or a hypersensitivity disease.
  • inflammatory diseases according to the invention include cholangitis and arthritis.
  • the present invention further relates to a method for treating and/or preventing a disease, preferably a CCR8-related disease, more preferably a cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the isolated antibody or antigen-binding fragment thereof, nucleic acid, vector, pharmaceutical composition or medicament according to the invention.
  • a disease preferably a CCR8-related disease, more preferably a cancer
  • terapéuticaally effective amount it is meant a level or amount of isolated antibody or antigen-binding fragment thereof, nucleic acid, vector, pharmaceutical composition or medicament according to the invention, that is necessary and sufficient for depleting CCR8-expressing Treg cells infiltrating a tumor in an individual in need thereof, without causing significant negative or adverse side effects to the individual.
  • the term “therapeutically effective amount” further refers to the level or amount of isolated antibody or antigen-binding fragment thereof, nucleic acid, vector, pharmaceutical composition or medicament according to the invention, that is necessary and sufficient for slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of cancer, in particular solid cancer (tumor progression); or alleviating the symptoms of cancer in particular solid cancer; or curing cancer in particular solid cancer, without causing significant negative or adverse side effects to the individual.
  • the present invention further relates to the use of the isolated antibody or antigenbinding fragment thereof, nucleic acid, vector or pharmaceutical composition according to the invention, for the manufacture of a medicament, preferably for the treatment and/or prevention of a CCR8-related disease, more preferably a cancer.
  • the present invention further relates to the use of the isolated antibody or antigenbinding fragment thereof, nucleic acid, vector or pharmaceutical composition according to the invention, for depleting CCR8-expressing tumor Treg cells.
  • the Treg overexpress CCR8.
  • Another object of the present invention relates to the use of the antibody or antigen-binding fragment thereof with another therapeutic agent as described hereinabove, in the treatment of diseases in a subject in need thereof, wherein said antibody or antigen-binding fragment thereof is used as an adjuvant for the therapeutic agent.
  • the present invention thus relates to an antibody or antigen-binding fragment thereof as described herein (preferably in a pharmaceutical composition or medicament), for use as an adjuvant in a cancer therapy.
  • the present invention thus relates to an antibody or antigen-binding fragment thereof as described herein (preferably in a pharmaceutical composition or medicament), for use as an adjuvant in a therapy for an infectious disease.
  • the present invention relates to the use of the antibody or fragment thereof as described herein, for potentiating an immune response induced by a cancer therapy in a patient in need thereof.
  • the antibody or fragment thereof according to the present invention may be used as immunotherapeutic agent, particularly to treat a wide variety of cancers (e.g., cancers associated with immunosuppression and/or immune exhaustion).
  • cancers e.g., cancers associated with immunosuppression and/or immune exhaustion.
  • the antibody or fragment thereof according to the present invention may potentiate an immune response induced by a cancer therapy in a patient comprising administering said antibody or fragment thereof to a subject in an amount effective to potentiate an immune response induced by the cancer therapy in the patient.
  • the term “adjuvant” refers to a compound or a combination of compounds that potentiates at therapy, such as, for example, a cancer therapy.
  • Adjuvants may increase the effective immune response against low or non-immunogenic tumor cells.
  • the adjuvant is used with a well-known cancer therapeutic agent in the treatment of cancer and thus potentiates the immune response towards cancer cells.
  • an adjuvant may potentiate an immune response during a cancer therapy, decrease T cell exhaustion (without decreasing T cells activation), increase the survival of T cells, enhance NK cells cytotoxicity, decrease the tumor growth and/or the tumor size, and/or increase in survival, treats or prevents cancer metastasis.
  • potentiation of a cancer therapy in the presence of an adjuvant is defined by comparison with a cancer therapy administered alone.
  • the antibody or fragment thereof as described herein can increase or improve the immune response of a subject.
  • an “immune response” refers to a response by a cell of the immune system, such as a B cell, T cell (CD4 or CD8), regulatory T cell, antigen- presenting cell, dendritic cell, monocyte, macrophage, NKT cell, NK cell, basophil, eosinophil, or neutrophil, to a stimulus.
  • the response is specific for a particular antigen (an “antigen-specific response”), and refers to a response by a CD4 T cell, CD8 T cell, or B cell via their antigen-specific receptor.
  • an immune response is a T cell response, such as a CD4+ response or a CD8+ response.
  • T cell response such as a CD4+ response or a CD8+ response.
  • responses by these cells can include, for example, cytotoxicity, proliferation, cytokine or chemokine production, trafficking, or phagocytosis, and can be dependent on the nature of the immune cell undergoing the response.
  • immune potentiating agents may be useful in treating a wide variety of infectious diseases, particularly pathogenic agents which promote immunosuppression and/or immune exhaustion. Also, such immune potentiating agents may be useful in boosting the immunization efficacy of vaccines (e.g., infectious disease and cancer vaccines).
  • vaccines e.g., infectious disease and cancer vaccines
  • Another object of the present invention relates to the use of the antibody or antigen-binding fragment thereof according to the present invention to deplete CCR8 expressing Treg cells in a subject in need thereof, wherein a therapeutically effective amount of an antibody or fragment thereof of the present invention is to be administered to the subject.
  • the present invention thus further relates to a method for depleting CCR8 expressing Treg cells in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment thereof, a pharmaceutical composition, or a medicament as described herein.
  • the antibody or antigen-binding fragment thereof as described hereinabove (preferably in a composition, pharmaceutical composition or medicament as describe hereinabove), is for use to deplete CCR8 expressing Treg cells.
  • the CCR8 expressing Treg cells are tumor infiltrating Tregs.
  • Another obj ect of the present invention relates to a method for desensitizing cells to stimulations by CCL1 in a subject in need thereof, comprising administering to the subject the antibody or antigen-binding fragment thereof, a pharmaceutical composition, or a medicament according to the present invention.
  • the present invention thus further relates to the use of the antibody or antigenbinding fragment thereof or the immunotoxin fusion protein according to the invention for killing CCR8-expressing cells, such as T reg cells or cancer cells.
  • Still another object of the invention is the use of the antibody or antigen-binding fragment thereof according to the invention targeting both human and rabbit CCR8, in a rabbit model for pre-clinical studies.
  • Figure 1 is a chromatography and subsequent quantification showing the purity of the antibody mAb2.
  • the electrophoresis was performed in denaturating conditions (SDS-PAGE) on Bio-Rad gels 15% (Bio-Rad 4568084) and mAb2 was labelled.
  • Figure 2A-2B is a combination of graphs showing a flow cytometry analysis for the detection of CCL1. Represented herein are the positive control CCL1 (Fig. 2A) and negative control C3a (Fig. 2B).
  • Figure 3 is an histogram representing the percentage of CCL1 fixation on HuT- 78 using mAbl, mAb2, mAb3, mAb4 and mAb5, in the flow cytometry assay as exemplified in Fig. 2.
  • Figure 4 is a flow cytometry plot showing the detection of human Treg cells (cells expressing FoxP3) using the mAb as described hereinabove.
  • Figure 5 is a flow cytometry plot showing the detection of rabbit CD4-positive cells using the mAb as described hereinabove. The 1.5% corresponds to the expected proportion or peripheral Treg cells.
  • Mouse immunization and fusion Anti human CCR8 monoclonal antibodies (mAb) were obtained in immunizing BalB-C mouse with a syngenic cell line stably expressing hCCR8, according to the method previously described (WO 02/081523). After the immunization, the splenocytes were harvested (50* 10 6 cells) and fused by electrofusion with the mouse fusion cell line SP2O (20* 10 6 cells) to obtain producing mAbs hybridomas. The fusion product was diluted in 400 ml HAT containing medium (DMEM 10 % FCS) and dispensed in 20 96-well plates. One week after the fusion, the growing hybridomas supernatant were screened.
  • DMEM 10 % FCS medium
  • Hybridoma screening The hybridomas supernatants were screened by flow cytometry on the human HuT-78 cell line (ATCC TIB 161) that naturally express CCR8. 50 pl of HuT-78 cells diluted at 10 6 /ml in PBS were dispensed into a 96-well plate. 50 pl of the hybridomas supernatants were added. As negative control, 50 pl of culture medium was added. As positive control, 50 pl of mouse anti-hCCR8 (diluted at 2,5 pl/ml) (Biolegend L263G8) was added. After a 10 min incubation at 4°C, the plate was centrifuged (1200g during 10 min) and supernatant discarded.
  • the 5 hybridomas were developed in flasks (200 ml culture medium).
  • the mAbs were purified from the culture supernatants on rat anti-mouse columns.
  • the hybridomas 1,2, 3, 5 supernatant that are IgG2a were purified on LO-MG2a column (from Synabs).
  • the hybridoma 4 supernatant that is IgGl was purified on LO-MG1 column (from Synabs).
  • CCR8 presents 4 extracellular regions: one N-terminus tail, one C-terminus tail, one extracellular loop (ECL) and two intracellular loops (ICL).
  • pepscan was performed. This pepscan consisted in firstly coating an ELISA plate with several peptides corresponding to the whole extracellular regions of CCR8 and secondly adding the antibodies.
  • the peptides were coated overnight at 4°C at 5 pl/ml in borate buffer pH 9. After a washing step with PBS buffer, the different mAbs diluted at 5 pl/ml in PBS were added to the plate and incubated Ih at 37°C. After a washing step with PBS buffer, the goat anti mouse IgG Hrp (IcL GGHL-90P) 1000 X diluted was added to the plate and incubated 30 min at 37°C After a washing step in PBS, a TMB solution was added to the plate and incubated 10 min at room temperature. The colorimetric reaction was stopped in adding a Sulfuric acid solution at pH 2. The yellow coloration of the ELISA wells plate was red at 450 nm. Results
  • the 5 mAbs recognized the peptide 2 only.
  • the peptide 2 corresponds to the region AA 9-25 of the full length human CCR8 sequence.
  • VTTVTDYYYPDIFSSPC non-sulfonated peptide 2
  • VTTVTDY(SO 3 H)YY(SO 3 H)PDIFSSPC a comparative ELISA was performed in coating a non-sulfonated peptide 2 (VTTVTDY(SO 3 H)YY(SO 3 H)PDIFSSPC ).
  • the sulfonated peptide 2 was also purchased from Eurogentec.
  • the ELISA protocol was similar to this detailed for the pepcan (see Example 1).
  • Positive control 50 pl of HUT-78 suspended in PBS (10 6 ml) were incubated 10 min at 4°C with 50 pl PBS, then 50 pl of CCL1 Alexa Fluor647 (Almac cat: CAFO7) diluted 10 X was added for a further 10 min incubation at 4°C.
  • Negative control 50 pl of HUT-78 suspended in PBS (10 6 ml) were incubated 10 min at 4°C with 50 pl PBS, then 50 pl of C3a Alexa Fluor647 (Almac cat CAF91 Al) diluted 10 X was added for a further 10 min incubation at 4°C.
  • the ligand CCL1 When the ligand CCL1 binds to the GPCR, it first induces an intracellular activation cascade and then initiates the internalization of the ligand-GPCR complex. This phenomenon, well known as desensitization, renders the cell temporarily insensitive to any further stimulation by the same ligand. We show here that antibodies also cause internalization of the antibody-GPCR complex.
  • a volume of 200 pl of HUT-78 suspended in culture medium (10 6 ml) was dispensed in a 96-well plate and incubated at 37°C. At various periods of time (0 min, Ih, 2h, 3h, 3h30 and 3h55), the mAb 1, at a final 5 pg/ml concentration, was added. A negative control without mAbl was also tested.
  • Treg are characterized by the transcription factor FoxP3 expression. It was investigated if the 5 mAbs also recognize Treg.
  • Flow cytometry human: A flow cytometry test was performed on human PBMC (peripheral blood mononuclear cell) isolated from a fresh blood by a centrifugation on Ficoll. After a permeabilization treatment (FIX & PERM® Cell Permeabilization Kit Thermofisher GAS003) the isolated PBMC were doubly labelled with FOXP3 Alexa 488 (BD Biosciences) and with each mAbs Alexa Fluor 647.
  • PBMC peripheral blood mononuclear cell
  • Flow cytometry (rabbit): A double labelling of rabbit PBMC (isolated from fresh blood with a centrifugation on Ficoll) was performed with the mouse anti rabbit CD4 FITC mAb (Mybiosource cat: MBS 216606) and with one of the 5 mAbs Alexa Fluor 647.
  • VTTVTDYYYPDIFSSPC SEQ ID NO: 1
  • VTAVTDYYYPDIFSSPC SEQ ID NO: 2
  • a flow cytometry test was also performed to verify if the 5 mAbs are able to bind to a subpopulation of the peripheral rabbit CD4+ lymphocytes.
  • the clones 1, 3 and 5 recognized 1,5% of the CD4 positive cell that correspond to proportion of peripheral Treg

Abstract

The present invention relates to antibodies specific for the chemokine (C-C motif) receptor 8 (CCR8). The present invention further relates to the use of these antibodies for depleting CCR8-expressing tumor regulatory T cells, and to prevent and/or treat cancer.

Description

ANTI-CCR8 ANTIBODIES AND USES THEREOF
FIELD OF INVENTION
[0001] The present invention relates to antibodies specific for the chemokine (C-C motif) receptor 8 (CCR8) and their use for depleting or inhibiting regulatory T cells in tumors.
BACKGROUND OF INVENTION
[0002] Chemokine (C-C motif) receptor 8 (CCR8) is a G protein-coupled receptor (GPCR) chemokine receptor. In human, the CCR8 ligands are Chemokine (C-C motif) ligand 1, 16 and 18 (CCL1, CCL16 and CCL18).
[0003] CCR8 is mainly expressed by T reg cells and sometimes by T lymphomas or leukemias. When an immune response is triggered, cells involved in the immune response such as macrophages produce CCL1. The CCL1-CCR8 binding permits the Treg homing. It provokes activation of the cytoskeleton, which allows the T reg cells to migrate up the CCL1 gradient to the site of the immune reaction where T reg cells negatively regulate the immune reaction by secreting cytokines such as IL- 10.
[0004] In the particular case of an immune reaction directed against a tumor, it was shown that T reg cells infiltrate tumors and promote their progression by inhibiting the activity of immune cells, such as cytotoxic T lymphocytes.
[0005] Therefore, antibodies against CCR8 are useful for the treatment of cancer and more generally the treatment of diseases associated with an immune response dysfunction.
[0006] WO2021194942, WO2021194942 and WO2021163064 recently reported new clues regarding the use of anti-CCR8 antibody in treating cancer. [0007] WO2021194942 discloses an anti-CCR8 antibody exerting antibody-dependent cellular cytotoxicity (ADCC)-mediate depletion of CCR8-expressing cells (such as T reg cells) and use thereof in mouse model of colon cancer, alone or in combination with a mouse anti-mPD-1 Ab.
[0008] WO2021163064 discloses an anti-CCR8 antibody that may comprise one mutation/or modification (e.g., afucosylation) or may also be chimeric, that may favor depletion of Treg and reduces tumor growth in MC38 syngeneic model in mice. WO2021163064 discloses that this anti-CCR8 antibody may be used as monotherapy or in combination with an anticancer agent such as an immune checkpoint inhibitor (e.g., PD-1).
[0009] WO2021142002 discloses human or cyno antibodies that specifically bind the N- terminal extracellular domain of human CCR8 and inducing ADCC in 293T cells, and therapeutic use thereof.
[0010] Despite these advances made over the last few years, the current clinical benefits are still unsatisfying and there is still a need to improve this strategy in order to potentiate the response in patient in need. There is thus an unmet medical need to improve these anticancer therapies.
[0011] Here, the inventors provide a new antibody targeting CCR8, with specific CDRs sequences, having the capacity to inhibit the binding of CCL1 to CCR8 and to induce CCR8 internalization. This new anti-CCR8 antibody will target tumor-infiltrating immune cells, thereby reducing tumor progression and thus treat subjected afflicted with cancer.
SUMMARY
[0012] The present invention relates to an isolated antibody or antigen-binding fragment thereof that binds the chemokine (C-C motif) receptor 8 (CCR8).
[0013] In some embodiments, the isolated antibody or antigen-binding fragment thereof is monoclonal. [0014] In some embodiments, the isolated antibody or antigen-binding fragment thereof binds human CCR8 and rabbit CCR8.
[0015] In some embodiments, the isolated antibody or antigen-binding fragment thereof recognizes an epitope having or consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
[0016] In some embodiments, the isolated antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region which comprises at least one, preferably at least two, more preferably the three following complementary-determining regions (CDRs):
[0017] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a light chain variable region which comprises at least one, preferably at least two, more preferably the three following complementary- determining regions (CDRs):
[0018] In some embodiments, the epitope is post-transcriptionally modified.
[0019] In some embodiments, the epitope is sulfonated.
[0020] In some embodiments, the epitope is sulfonated at the amino acid position 7 and/or 9, wherein position numbering is with respect to SEQ ID NO: 1 or SEQ ID NO: 2.
[0021] In some embodiments, the isolated antibody or antigen-binding fragment thereof according to invention comprises a heavy chain variable region (VH) which comprises at least one, preferably at least two, more preferably the three following complementary- determining regions (CDRs):
VH-CDR1 : SEQ ID NO : 10,
VH-CDR2 : SEQ ID NO : 11 , and/or
VH-CDR3: SEQ ID NO: 12.
[0022] In some embodiments, the isolated antibody or antigen-binding fragment thereof according to invention comprises a light chain variable region (VL) which comprises at least one, preferably at least two, more preferably the three following complementary- determining regions (CDRs):
VL-CDR1 : SEQ ID NO: 13,
VL-CDR2: SEQ ID NO: 14, and/or
VL-CDR3 : SEQ ID NO : 15.
[0023] In some embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain variable region (VH) which comprises the three following complementary-determining regions (CDRs):
- VH-CDR1 : SEQ ID NO: 10,
- VH-CDR2 : SEQ ID NO : 11 , and
- VH-CDR3 : SEQ ID NO : 12, and further comprises a light chain variable region (VL) which comprises the three following complementary-determining regions (CDRs):
VL-CDR1 : SEQ ID NO: 13,
VL-CDR2: SEQ ID NO: 14, and/or VL-CDR3 : SEQ ID NO : 15.
[0024] In some embodiments, the isolated antibody or antigen-binding fragment thereof to CCR8 inhibits the binding of chemokine (C-C motif) ligand 1 (CCL1) and/or chemokine (C-C motif) ligand 18 (CCL18) to CCR8.
[0025] The present invention further relates to a nucleic acid encoding the isolated antibody or antigen-binding fragment thereof according to the invention.
[0026] The present invention further relates to a vector containing a nucleic acid according to the invention.
[0027] The present invention further relates to a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof, the nucleic acid or the vector according to the invention, and at least one pharmaceutically acceptable excipient. [0028] In some embodiments, the pharmaceutical composition further comprises at least on additional therapeutic agent.
[0029] In some embodiments, the additional therapeutic agent is a programmed cell death protein 1 (PD-1) inhibitor.
[0030] The present invention further relates to an isolated antibody or antigen-binding fragment thereof, a nucleic acid, a vector or a pharmaceutical composition according to the invention, for use as a medicament.
[0031] The present invention further relates to the isolated antibody or antigen-binding fragment thereof, nucleic acid, vector or pharmaceutical composition for use according to the invention, for treating and/or preventing a CCR8-related disease in a subject in need thereof.
[0032] In some embodiments, the disease is cancer.
[0033] In some embodiments, the cancer is caused by cells expressing CCR8, whether cancerous or non-cancerous cells.
[0034] The present invention further relates to a method for treating and/or preventing a disease, preferably a CCR8-related disease, comprising administering to a subject in need thereof a therapeutically effective amount of the isolated antibody or antigen-binding fragment thereof according to the invention.
[0035] The present invention further relates to the use of an isolated antibody or antigenbinding fragment thereof, a nucleic acid, a vector or a pharmaceutical composition according to the invention, for the manufacture of a medicament, preferably for the treatment and/or prevention of a CCR8-related disease. DEFINITIONS
[0036] In the present invention, the following terms have the following meanings:
[0037] The term “about”, preceding a figure encompasses plus or minus 10%, or less, of the value of said figure. It is to be understood that the value to which the term “about” refers is itself also specifically, and preferably, disclosed.
[0038] The term “affinity” and “avidity” are well-known in the art and are used to defined the strength of an antibody-antigen complex. Affinity measures the strength of interaction between an epitope and an antigen binding site on an antibody. It may be expressed by an affinity constant KA or by a dissociation constant KD. Avidity (or functional affinity) gives a measure of the overall strength of an antibody-antigen complex. It may depend on different parameters, including in particular the affinity of the antibody or antigen-binding fragment thereof for an epitope, (ii) the valency of both the antibody and the antigen and (iii) structural arrangement of the parts that interact.
[0039] The terms “antibody” and “immunoglobulin” may be used interchangeably and refer to a protein having a combination of two heavy and two light chains whether or not it possesses any relevant specific immunoreactivity. “Antibodies” refers to such assemblies which have significant known specific immunoreactive activity to an antigen of interest (e.g., human CCR8). The term “anti-hCCR8 antibodies” is used herein to refer to antibodies which exhibit immunological specificity for human CCR8 protein. As explained elsewhere herein, “specificity” for human CCR8 does not exclude crossreaction with species homologues of hCCR8, such as, for example, with simian CCR8. Antibodies and immunoglobulins comprise light and heavy chains, with or without an interchain covalent linkage between them. Basic immunoglobulin structures in vertebrate systems are relatively well understood. The generic term “immunoglobulin” comprises five distinct classes of antibody that can be distinguished biochemically. Although the following discussion will generally be directed to the IgG class of immunoglobulin molecules, all five classes of antibodies are within the scope of the present invention. With regard to IgG, immunoglobulins comprise two identical light polypeptide chains of molecular weight of about 23 kDa, and two identical heavy chains of molecular weight of about 53-70 kDa. The four chains are joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region. The light chains of an antibody are classified as either kappa (K) or lambda (X). Each heavy chain class may be bonded with either a K or X light chain. In general, the light and heavy chains are covalently bonded to each other, and the “tail” regions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. Those skilled in the art will appreciate that heavy chains are classified as gamma (y), mu (p), alpha (a), delta (□) or epsilon (a) with some subclasses among them (e.g., yl-y4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgD or IgE, respectively. The immunoglobulin subclasses or “isotypes” (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, etc.) are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the present invention. As indicated above, the variable region of an antibody allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the light chain variable domain (VL domain) and heavy chain variable domain (VH domain) of an antibody combine to form the variable region that defines a three-dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site presents at the end of each arm of the “Y”.
[0040] The term “antigen-binding fragment”, as used herein, refers to a part or region of the antibody according to the present invention, which comprises fewer amino acid residues than the whole antibody. An “antigen-binding fragment” binds antigen and/or competes with the whole antibody from which it was derived for antigen binding (e.g., specific binding to human CCR8). Antibody antigen-binding fragments encompasses, without any limitation, single chain antibodies, Fv, Fab, Fab', Fab'-SH, F(ab)’2, Fd, defucosylated antibodies, diabodies, triabodies and tetrabodies. [0041] The term “effector T cells” refers to a group of cells that includes several T cells types (e.g., CD4+ and CD8+ T cells). It includes helpers T cells (Th cells) that help other leukocytes in immunologic processes, including maturation of B cells into plasma cells and memory B cells and cytotoxic T cells (Tc cells, CTLs, T-killer cells, killer T cells) that destroy virus-infected cells and tumor cells, and are also implicated in transplant rejection.
[0042] The term “epitope” refers to a specific arrangement of amino acids located on a protein or proteins to which an antibody or antigen-binding fragment thereof or an antibody mimetic bind. Epitopes often consist of a chemically active surface grouping of molecules such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be linear (or sequential) or conformational, i.e., involving two or more sequences of amino acids in various regions of the antigen that may not necessarily be contiguous.
[0043] The terms “Fc domain”, “Fc portion”, and “Fc region” refer to a C-terminal fragment of an antibody heavy chain, e.g., from about amino acid (aa) 230 to about aa 450 of human gamma heavy chain or its counterpart sequence in other types of antibody heavy chains (e.g., a, 6, a and p for human antibodies), or a naturally occurring allotype thereof.
[0044] The term “Fv”, as used herein, refers to the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one HCVR and one LCVR in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the heavy and light chain) that contribute to antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
[0045] The term “heavy chain region” includes amino acid sequences derived from the constant domains of an immunoglobulin heavy chain. A protein comprising a heavy chain region comprises at least one of a CHI domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. In an embodiment, the antibody or antigen-binding fragment thereof according to the present invention may comprise the Fc region of an immunoglobulin heavy chain (e.g., a hinge portion, a CH2 domain, and a CH3 domain). In another embodiment, the antibody or antigen-binding fragment thereof according to the present invention lacks at least a region of a constant domain (e.g., all or part of a CH2 domain). In certain embodiments, at least one, and preferably all, of the constant domains are derived from a human immunoglobulin heavy chain. For example, in one preferred embodiment, the heavy chain region comprises a fully human hinge domain. In other preferred embodiments, the heavy chain region comprising a fully human Fc region (e.g., hinge, CH2 and CH3 domain sequences from a human immunoglobulin). In certain embodiments, the constituent constant domains of the heavy chain region are from different immunoglobulin molecules. For example, a heavy chain region of a protein may comprise a CH2 domain derived from an IgGl molecule and a hinge region derived from an IgG3 or IgG4 molecule. In other embodiments, the constant domains are chimeric domains comprising regions of different immunoglobulin molecules. For example, a hinge may comprise a first region from an IgGl molecule and a second region from an IgG3 or IgG4 molecule. As set forth above, it will be understood by one of ordinary skill in the art that the constant domains of the heavy chain region may be modified such that they vary in amino acid sequence from the naturally occurring (wild-type) immunoglobulin molecule. That is, the antibody or antigen-binding fragment thereof according to the present invention may comprise alterations or modifications to one or more of the heavy chain constant domains (CHI, hinge, CH2 or CH3) and/or to the light chain constant domain (CL). Exemplary modifications include additions, deletions or substitutions of one or more amino acids in one or more domains.
[0046] The term “hinge region” includes the region of a heavy chain molecule that joins the CHI domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., 1998. J Immunol. 161 (8):4083 -90). [0047] The term “identity” or “identical”, when used in a relationship between the sequences of two or more amino acid sequences, or of two or more nucleic acid sequences, refers to the degree of sequence relatedness between amino acid sequences or nucleic acid sequences, as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. “Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., “algorithms”). Identity of related amino acid sequences or nucleic acid sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Lesk A. M. (1988). Computational molecular biology: Sources and methods for sequence analysis. New York, NY: Oxford University Press; Smith D. W. (1993). Biocomputing: Informatics and genome projects. San Diego, CA: Academic Press; Griffin A. M. & Griffin H. G. (1994). Computer analysis of sequence data, Part 1. Totowa, NJ: Humana Press; von Heijne G. (1987). Sequence analysis in molecular biology: treasure trove or trivial pursuit. San Diego, CA: Academic press; Gribskov M. R. & Devereux J. (1991). Sequence analysis primer. New York, NY: Stockton Press; Carillo et al., 1988. SIAM J Appl Math. 48(5): 1073-82. Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Genetics Computer Group, University of Wisconsin, Madison, WI; Devereux et al., 1984. Nucleic Acids Res. 12(1 Pt 1):387-95), BLASTP, BLASTN, and FASTA (Altschul et al., 1990. J Mol Biol. 215(3):403-10). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894). The well-known Smith Waterman algorithm may also be used to determine identity.
[0048] The term "immunotoxin" refers to a bifunctional molecule comprising targeting moiety for delivery (a ligand) and a toxic moiety (toxin) for cytotoxicity. The immunotoxin can be used to kill cells expressing receptors for the ligand. [0049] The term “mammal” refers to any mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.
[0050] The term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprised in the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies or antigen-binding fragment thereof according to the present invention may be prepared by the hybridoma methodology first described by Kohler et al., 1975. Nature. 256(5517):495-7, or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (Patent US4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., 1991. Nature. 352(6336):624-8 and Marks et al., 1991. J Mol Biol. 222(3):581-97, for example.
[0051] “Nanobodies” are well-known in the art and refer to antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally-occurring heavy chain antibodies (Muyldermans, 2013. Annu Rev Biochem. 82:775-97). These heavy chain antibodies may contain a single variable domain (VHH) and two constant domains (CH2 and CH3).
[0052] The term “nucleic acid” or “polynucleotide” refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Nucleic acid” or “Polynucleotides” include, without limitation single-and doublestranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double- stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “Nucleic acid” or “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term “nucleic acid” or “polynucleotide” also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications has been made to DNA and RNA; thus, “nucleic acid” or “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.
[0053] The terms “prevent”, “preventing” and “prevention” refer to prophylactic and preventative measures, wherein the object is to reduce the chances that a subject will develop the pathologic condition or disorder over a given period of time. Such a reduction may be reflected, e.g., in a delayed onset of at least one symptom of the pathologic condition or disorder in the subject.
[0054] The term “regulatory T cell” or “Treg cell” refers to a specialized type of T cells, in particular of CD4+ T cell, that can suppress the responses of other T cells. Treg cells play a critical role in the induction and maintenance of peripheral self-tolerance to antigens, including those expressed by tumors.
[0055] The term “single-chain Fv”, also abbreviated as “sFv” or “scFv”, refers to antibody fragments that comprise the VH and VL antibody domains connected into a single amino acid chain. Preferably, the scFv amino acid sequence further comprises a peptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding (Pliickthun, 1994. Antibodies from Escherichia coli. In Rosenberg & Moore (Eds.), The pharmacology of monoclonal antibodies. Handbook of Experimental Pharmacology, 113:269-315. Springer: Berlin, Heidelberg). [0056] The term “subject” refers to a mammal, preferably a human. In one embodiment, a subject may be a “patient”, i.e., a warm-blooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of a disease. The term “mammal” refers here to any mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is a primate, more preferably a human.
[0057] The term “therapeutically effective amount” refers to the level or amount of an antibody as described herein that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of a disease, disorder, or condition; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of the disease, disorder, or condition; (3) bringing about ameliorations of the symptoms of the disease, disorder, or condition; (4) reducing the severity or incidence of the disease, disorder, or condition; or (5) curing the disease, disorder, or condition. A therapeutically effective amount may be administered prior to the onset of the disease, disorder, or condition, for a prophylactic or preventive action. Alternatively, or additionally, the therapeutically effective amount may be administered after initiation of the disease, disorder, or condition, for a therapeutic action.
[0058] The term “treating” or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully "treated" for a cancer or an infection if, after receiving a therapeutic amount of an antibody according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of cancer cells (or tumor size), or pathogenic cells; reduction in the percent of total cells that are cancerous or pathogenic; and/or relief to some extent, one or more of the symptoms associated with the specific disease or condition; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.
[0059] As used herein, the term “variable” refers to the fact that certain regions of the variable domains VH and VL differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its target antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called “hypervariable loops” in each of the VL domain and the VH domain which form part of the antigen binding site. The first, second and third hypervariable loops of the V light chain domain are referred to herein as LI (X), L2 (X) and L3 (X) and may be defined as comprising residues 24-33 (L1(X), consisting of 9, 10 or 11 amino acid residues), 49-53 L2 (X), consisting of 3 residues) and 90-96 (L3(X), consisting of 6 residues) in the VL domain (Morea et al., 2000. Methods. 20(3):267-79). The first, second and third hypervariable loops of the VK light chain domain are referred to herein as L1(K), L2(K) and L3(K) and may be defined as comprising residues 25-33 (L1(K), consisting of 6, 7, 8, 11, 12 or 13 residues), 49-53 (L2(K), consisting of 3 residues) and 90-97 (L3(K), consisting of 6 residues) in the VL domain (Morea et al., 2000. Methods. 20(3):267-79). The first, second and third hypervariable loops of the VH domain are referred to herein as Hl, H2 and H3 and may be defined as comprising residues 25-33 (Hl, consisting of 7, 8 or 9 residues), 52-56 (H2, consisting of 3 or 4 residues) and 91-105 (H3, highly variable in length) in the VH domain (Morea et al., 2000. Methods. 20(3):267-79). Unless otherwise indicated, the terms LI, L2 and L3 respectively refer to the first, second and third hypervariable loops of a VL domain, and encompass hypervariable loops obtained from both VK and V isotypes. The terms Hl, H2 and H3 respectively refer to the first, second and third hypervariable loops of the VH domain, and encompass hypervariable loops obtained from any of the known heavy chain isotypes, including gamma (y), mu (p), alpha (a), delta (5) or epsilon (a). DETAILED DESCRIPTION
[0060] This invention relates to an isolated antibody or antigen-binding fragment thereof that binds the chemokine (C-C motif) receptor 8 (CCR8).
[0061] In some embodiments, the isolated antibody or antigen-binding fragment thereof binds to CCR8 from any animal species. In some embodiments, the isolated antibody or antigen-binding fragment thereof binds to any mammalian CCR8. In some embodiments, the isolated antibody or antigen-binding fragment thereof binds to both human and rabbit CCR8. In some embodiments, the isolated antibody or antigen-binding fragment thereof binds human CCR8, wherein the amino acid sequence of human CCR8 corresponds to SEQ ID NO: 3. In some embodiments, the isolated antibody or antigen-binding fragment thereof binds rabbit CCR8, wherein the amino acid sequence of rabbit CCR8 corresponds to SEQ ID NO: 18. In some embodiments, the isolated antibody or antigen-binding fragment thereof binds mouse CCR8, wherein the amino acid sequence of mouse CCR8 corresponds to SEQ ID NO: 19. In some embodiments, the isolated antibody or antigenbinding fragment thereof binds rat CCR8, wherein the amino acid sequence of rat CCR8 corresponds to SEQ ID NO: 20.
[0062] In some embodiments, the isolated antibody or antigen-binding fragment thereof recognizes an epitope having at least 75% sequence identity with the amino acid sequence SEQ ID NO: 1 and/or SEQ ID NO: 2.
[0063] Within the scope of this invention, at least 75% sequence identity means 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9, 99.99, 99.999 or 100% sequence identity.
[0064] In some embodiments, the isolated antibody or antigen-binding fragment thereof recognizes an epitope having the amino acid sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2.
[0065] In some embodiments, the isolated antibody or antigen-binding fragment thereof recognizes an epitope consisting of the amino acid sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2. [0066] In some embodiments, the isolated antibody or antigen-binding fragment thereof recognizes an epitope having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the isolated antibody or antigen-binding fragment thereof recognizes an epitope having the amino acid sequence of SEQ ID NO: 2.
[0067] In some embodiments, the isolated antibody or antigen-binding fragment thereof recognizes an epitope consisting of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the isolated antibody or antigen-binding fragment thereof recognizes an epitope consisting of the amino acid sequence of SEQ ID NO: 2.
[0068] In some embodiments, the epitope is post-transitionally modified. In some embodiments, the epitope is sulfonated.
[0069] As used herein, the term “sulfonated epitope” refers to the addition of at least one sulfonic acid molecule (SO3H) on at least one moiety, e.g., an amine or a thiol, of the side chain of at least one amino acid, e.g., a tyrosine or a cysteine, within the amino acid sequence of the epitope.
[0070] In some embodiments, the epitope is sulfonated at the amino acid position 7 and/or 9, wherein position numbering is with respect to SEQ ID NO: 1 or SEQ ID NO: 2.
[0071] In some embodiments, the amino acid sequence of the sulfonated epitope is VTTVTDY(SO3H)YY(SO3H)PDIFSSPC or VTAVTDY(SO3H)YY(SO3H)PDIFSSPC.
[0072] In the following, and unless explicitly mentioned otherwise, CDR numbering and definitions are according to the IMGT® numbering system.
[0073] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region (abbreviated herein as HCVR or VH) which comprises at least one, preferably at least two, more preferably the three following complementary-determining regions (CDRs):
VH-CDR1 : EYTMH (SEQ ID NO: 10);
VH-CDR2: GINPKNGVTRYNQKFKG (SEQ ID NO: 11); and/or VH-CDR3: GYHGPFAY (SEQ ID NO: 12). [0074] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a HCVR which comprises the three following CDRs:
VH-CDR1 : EYTMH (SEQ ID NO: 10);
VH-CDR2: GINPKNGVTRYNQKFKG (SEQ ID NO: 11); and VH-CDR3: GYHGPFAY (SEQ ID NO: 12).
[0075] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a light chain variable region (abbreviated herein as LCVR or VL) which comprises at least one, preferably at least two, more preferably the three following complementary-determining regions (CDRs):
VL-CDR1 : RASQEISGYLS (SEQ ID NO: 13);
VL-CDR2: AASTLDS (SEQ ID NO: 14); and/or VL-CDR3: LHYANAPYT (SEQ ID NO: 15).
[0076] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a LCVR which comprises the three following CDRs:
VL-CDR1 : RASQEISGYLS (SEQ ID NO: 13);
VL-CDR2: AASTLDS (SEQ ID NO: 14); and
VL-CDR3: LHYANAPYT (SEQ ID NO: 15).
[0077] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises: a HCVR which comprises at least one, preferably at least two, more preferably the three following CDRs:
VH-CDR1 : EYTMH (SEQ ID NO: 10);
VH-CDR2: GINPKNGVTRYNQKFKG (SEQ ID NO: 11); and/or VH-CDR3: GYHGPFAY (SEQ ID NO: 12); and a LCVR which comprises at least one, preferably at least two, more preferably the three following CDRs:
VL-CDR1 : RASQEISGYLS (SEQ ID NO: 13);
VL-CDR2: AASTLDS (SEQ ID NO: 14); and/or
VL-CDR3: LHYANAPYT (SEQ ID NO: 15). [0078] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises: a HCVR which comprises the three following CDRs:
VH-CDR1 : EYTMH (SEQ ID NO: 10);
VH-CDR2: GINPKNGVTRYNQKFKG (SEQ ID NO: 11); and VH-CDR3: GYHGPFAY (SEQ ID NO: 12); and a LCVR which comprises the three following CDRs:
VL-CDR1 : RASQEISGYLS (SEQ ID NO: 13);
VL-CDR2: AASTLDS (SEQ ID NO: 14); and VL-CDR3: LHYANAPYT (SEQ ID NO: 15).
[0079] In one embodiment, any of CDR1, CDR2 and/or CDR3 of the HCVR with SEQ ID NOs 10-12 can be characterized as having 1, 2, 3 or more amino acids being substituted by a different amino acid.
[0080] In one embodiment, any of CDR1, CDR2 and/or CDR3 of the LCVR with SEQ ID NOs 13-15 can be characterized as having 1, 2, 3 or more amino acids being substituted by a different amino acid.
[0081] In one embodiment, any of CDR1, CDR2 and/or CDR3 of the HCVR with SEQ ID NOs 10-12 and/or of the LCVR with SEQ ID NOs 13-15 can be characterized as having 1, 2, 3 or more amino acids being substituted by a different amino acid.
[0082] As used herein, the phrase “characterized as having [...] amino acids being substituted by a different amino acid” in reference to a given sequence, refers to the occurrence, in said sequence, of conservative amino acid modifications.
[0083] As used herein, the expression “conservative amino acid modifications” refers to modifications that do not significantly affect or alter the binding characteristics of the antibody or antigen-binding fragment thereof containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antigen-binding fragment thereof by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
[0084] Conservative amino acid substitutions are typically those in which an amino acid residue is replaced with an amino acid residue having a side chain with similar physicochemical properties. Specified variable region and CDR sequences may comprise 1, 2, 3 or more amino acid insertions, deletions and/or substitutions. Where substitutions are made, preferred substitutions will be conservative modifications. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), [3- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDRs and/or variable regions of the antibody or antigen-binding fragment thereof according to the present invention can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., the properties set forth herein, such as, e.g., the binding to CCR8) using the assays described herein. In another embodiments, a string of amino acids within the CDRs and/or variable regions of the antibody or antigen-binding fragment thereof according to the present invention can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
[0085] In one embodiment, any of CDR1, CDR2 and/or CDR3 of the HCVR with SEQ ID Nos 10-12 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more of identity with the particular CDR or sets of CDRs listed in the corresponding SEQ ID NOs.
[0086] In one embodiment, any of CDR1, CDR2 and/or CDR3 of the LCVR with SEQ ID NOs 13-15 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more of identity with the particular CDR or sets of CDRs listed in the corresponding SEQ ID NOs. [0087] In one embodiment, any of CDR1, CDR2 and/or CDR3 of the HCVR with SEQ ID NOs 10-12 and/or of the LCVR with SEQ ID NOs 13-15 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more of identity with the particular CDR or sets of CDRs listed in the corresponding SEQ ID NOs.
[0088] In one embodiment, the LCVR and/or the HCVR further comprises a leader sequence, preferably located N terminally from the LCVR amino acid sequence or N terminally from the HCVR amino acid sequence respectively. Examples of leader sequences include, but are not limited to, SEQ ID NO: 16 and 17.
[0089] SEQ ID NO: 16
[0090] MGWSWIFLFLLSGTAGVLS
[0091] SEQ ID NO: 17
[0092] MDMRVPAHVFGFLLLWFPGTRC
[0093] In one embodiment, the LCVR comprises an amino acid sequence leader sequence SEQ ID NO: 16 located N terminally from the HCVR amino acid sequence.
[0094] In one embodiment, the LCVR comprises an amino acid leader sequence SEQ ID NO: 17 located N terminally from the LCVR amino acid sequence.
[0095] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a fully or substantially fully human heavy chain constant region (abbreviated herein as HCCR or CH) and/or light chain constant region (abbreviated herein as LCCR or CL).
[0096] In some embodiments, the binding of the isolated antibody or antigen-binding fragment thereof to CCR8 inhibits the binding of chemokine (C-C motif) ligand 1 (CCL1) and/or chemokine (C-C motif) ligand 18 (CCL18) to CCR8.
[0097] An “isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other proteins or antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds CCR8 is substantially free of proteins or antibodies that specifically bind antigens other than CCR8). An isolated antibody that specifically binds CCR8 may, however, have cross-reactivity to other antigens, such as CCR8 molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals, in particular those that would interfere with diagnostic or therapeutic uses of the protein or antibody, including without limitation, enzymes, hormones, and other proteinaceous or non-proteinaceous components.
[0098] An antibody or antigen-binding fragment thereof is said to be “specific for”, “immunospecific” or to “specifically bind” an antigen if it reacts at a detectable level with said antigen (e.g., CCR8), preferably with an affinity constant (KA) of greater than or equal to about 106 M'1, preferably greater than or equal to about 107 M'1, 108 M'1, 5* 108 M'1, 109 M'1, 5* 109 M'1 or more. Affinity of an antibody or antigen-binding fragment thereof for its cognate antigen is also commonly expressed as an equilibrium dissociation constant (KD). An antibody or antigen-binding fragment thereof is said to be “immunospecific”, “specific for” or to “specifically bind” an antigen if it reacts at a detectable level with said antigen (e.g., CCR8), preferably with a KD of less than or equal to 10'6 M, preferably less than or equal to 10'7 M, 5* 10'8 M, 10'8 M, 5* 10'9 M, 10'9 M or less.
[0099] Affinities of antibodies or antigen-binding fragment thereof can be readily determined using conventional techniques, for example, those described by Scatchard, 1949. Ann NY Acad Sci. 51 :660-672. Binding properties of an antibody or antigenbinding fragment thereof to antigens, cells or tissues may generally be determined and assessed using immunodetection methods including, for example, ELISA, immunofluorescence-based assays, such as immuno-histochemistry (IHC) and/or fluorescence-activated cell sorting (FACS) or by surface plasmon resonance (SPR, e.g., using BIAcore®).
[0100] In one embodiment, the isolated antibody or antigen-binding fragment thereof is purified. [0101] In one embodiment, the antibody or antigen-binding fragment thereof is purified to:
(1) greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% or more by weight of protein or of antibody or antigen-binding fragment thereof as determined by the Lowry method, and most preferably more than 96%, 97%, 98% or 99% by weight;
(2) a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or
(3) homogeneity as shown by SDS-PAGE under reducing or non-reducing conditions and using Coomassie blue or, preferably, silver staining.
[0102] In a preferred embodiment, the isolated antibody or antigen-binding fragment thereof is monoclonal. In another embodiment, the isolated antibody or antigen-binding fragment thereof is polyclonal.
[0103] In some embodiments, the isolated antibody is whole. In some embodiments, the isolated antibody is truncated. In certain embodiments, the truncated antibody is an antigen-binding fragment; in practice, an antigen-binding fragment retains the ability to bind an epitope on an antigen, but lacks parts of its amino acid sequence that are not involved in epitope binding, e.g., fragment crystallizable region (Fc).
[0104] In some embodiments, the isolated antibody or antigen-binding fragment thereof is a recombinant antibody. In some embodiments, the isolated antibody or antigenbinding fragment thereof is a modified antibody, e.g., an antibody o with optimized Fc inducing an increased engagement of Fc receptors and improved isolated antibodydependent cellular cytotoxicity (ADCC).
[0105] In some embodiments, the antibody or antigen-binding fragment thereof, or parts thereof, is comprised in a fusion protein.
[0106] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is a molecule selected from the group comprising or consisting of a whole antibody, a humanized antibody, a single chain antibody, a dimeric single chain antibody, a Fv, a Fab, a Fab', a Fab'-SH, a F(ab)’2, a Fd, a defucosylated antibody, a bispecific antibody, a diabody, a triabody and a tetrabody. [0107] Antigen-binding fragments of antibodies can be obtained using standard methods. For instance, Fab or F(ab')2 fragments may be produced by protease digestion of the isolated antibodies, according to conventional techniques.
[0108] It will also be appreciated that antibodies or antigen-binding fragments thereof according to the present invention can be modified using known methods. For example, to slow clearance in vivo and obtain a more desirable pharmacokinetic profile, the antibody or antigen-binding fragment thereof may be modified with polyethylene glycol (PEG). Methods for coupling and site-specifically conjugating PEG to an antibody or antigen-binding fragment thereof are described in, e.g., Leong et al., 2001. Cytokine. 16(3): 106-19; Delgado et al., 1996. Br J Cancer. 73(2): 175-82.
[0109] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is a molecule selected from the group comprising or consisting of a unibody, a domain antibody, and a nanobody.
[0110] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a fully or substantially fully human heavy chain constant region (abbreviated herein as HCCR or CH) and/or light chain constant region (abbreviated herein as LCCR or CL).
[0111] In one embodiment, the constant region is of human origin.
[0112] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a fully or substantially fully rabbit HCCR and/or LCCR.
[0113] In one embodiment, the constant region is of rabbit origin.
[0114] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is a rabbit antibody or fragment thereof.
[0115] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a fully or substantially fully murine HCCR and/or LCCR. [0116] In one embodiment, the constant region is of murine origin.
[0117] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is a murine antibody or fragment thereof.
[0118] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is a chimeric antibody or fragment thereof.
[0119] A “chimeric antibody”, as used herein, refers to an antibody or antigen-binding fragment thereof comprising a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked in nature. The amino acid sequences may normally exist in separate proteins that are brought together in the fusion protein or they may normally exist in the same protein but are placed in a new arrangement in the fusion protein. A chimeric protein may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. The term “chimeric antibody” encompasses herein antibodies and antigen-binding fragment thereof in which the constant region, or a portion thereof, is altered, replaced or exchanged so that the variable region is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region, or portion thereof, having a different or altered antigen specificity; or with corresponding sequences from another species or from another antibody class or subclass.
[0120] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is a humanized antibody or fragment thereof.
[0121] A “humanized antibody”, as used herein, refers to a chimeric antibody or antigenbinding fragment thereof which contains minimal sequence derived from a non-human immunoglobulin. It includes antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell, e.g., by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. Humanized antibodies or antigen-binding fragment thereof according to the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
[0122] A “humanized antibody” retains a similar antigenic specificity as the original antibody. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody may be increased.
[0123] Methods for humanizing the antibody or antigen-binding fragment thereof according to the present invention are well-known in the art. The choice of human variable domains, both light and heavy, to be used in making the humanized antibody or antigen-binding fragment thereof is very important to reduce antigenicity. According to the so-called “best-fit” method, the sequence of the variable domain of an antibody or antigen-binding fragment thereof according to the present invention is screened against the entire library of known human variable-domain sequences. The human sequence that is closest to the mouse sequence is then accepted as the human framework (FR) for the humanized antibody (Sims et al., 1993. J Immunol. 151(4):2296-308; Chothia & Lesk, 1987. J Mol Biol. 196(4):901-17).
[0124] Another method for humanizing the antibody or antigen-binding fragment thereof according to the present invention uses a particular framework from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., 1992. Proc Natl Acad Sci USA. 89(10):4285-9; Presta et al., 1993. J Immunol. 151(5):2623-32). It is further important that antibodies be humanized with retention of high affinity for hCCR8 and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies and antigen-binding fragments thereof are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its epitope.
[0125] Another method for humanizing the antibody or antigen-binding fragment thereof according to the present invention is to use a transgenic or transchromosomic animal carrying parts of the human immune system for immunization. As a host, these animals have had their immunoglobulin genes replaced by functional human immunoglobulin genes. Thus, antibodies produced by these animals or in hybridomas made from the B cells of these animals are already humanized. Examples of such transgenic or transchromosomic animal include, without limitation: the XenoMouse (Abgenix, Fremont, CA), described in Patents US5,939,598, US6,075,181, US6,114,598, US6, 150,584 and US6, 162,963; the HuMAb Mouse® (Medarex, Inc.), described in Lonberg et al., 1994. Nature. 368(6474):856-859; Lonberg & Huszar, 1995. Int Rev Immunol. 13(l):65-93; Harding & Lonberg, 1995. Ann N Y Acad Sci. 764:536-46; Taylor et al., 1992. Nucleic Acids Res. 20(23):6287-95; Chen et al., 1993. Int Immunol. 5(6):647-56; Tuaillon et al., 1993. Proc Natl Acad Sci USA. 90(8):3720-4; Choi et al.,
1993. Nat Genet. 4(2): 117-23; Chen et al., 1993. EMBO J. 12(3):821-30; Tuaillon et al.,
1994. J Immunol. 152(6):2912-20; Taylor et al., 1994. Int Immunol. 6(4):579-91; Fishwild et al., 1996. NatBiotechnol. 14(7):845-51 ; the KM Mouse®, described in Patent application W02002043478; the TC mice, described in Tomizuka et al., 2000. Proc Natl Acad Sci USA. 97(2):722-7; and the OmniRat™ (OMT, Inc.), described in Patent application W02008151081; Geurts et al., 2009. Science. 325(5939):433; Menoret et al., 2010. Eur J Immunol. 40(10):2932-41.
[0126] Humanized antibodies and antigen-binding fragments thereof may also be produced according to various other techniques, such as by using, for immunization, other transgenic animals that have been engineered to express a human antibody repertoire (Jakobovitz et al., 1993. Nature. 362(6417):255-8), or by selection of antibody repertoires using phage display methods. Such techniques are known to the skilled person and can be implemented starting from monoclonal antibodies or antigen-binding fragments thereof as disclosed in the present application.
[0127] In some embodiments, the antibody or antigen-binding fragment thereof according to the present invention is produced in a mammalian species. In some embodiments, the mammalian species is selected from the group comprising or consisting of mouse, rat, goat, horse, pig, dog, cat, non-human primates such as chimpanzee, and the like. In some embodiments, the mammalian species is the mouse (e.g., mus musculus and the like).
[0128] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is from the IgG class.
[0129] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is from the human IgGl subclass. In another embodiment, the antibody or antigen-binding fragment thereof according to the present invention is from the human lgG2 subclass.
[0130] The Fc region of IgG antibodies interacts with cellular Fey receptors (FcyR) to stimulate and regulate downstream effector mechanisms. There are five activating receptors, namely FcyRI (CD64), FcyRIIa (CD32a), FcyRIIc (CD32c), FcyRIIIa (CD 16a) and FcyRIIIb (CD 16b), and one inhibitory receptor FcyRIIb (CD32b). The communication of IgG antibodies with the immune system is controlled and mediated by FcyRs, which relay the information sensed and gathered by antibodies to the immune system, providing a link between the innate and adaptive immune systems, and particularly in the context of biotherapeutics (Hayes J et al., 2016. J Inflamm Res 9: 209- 219).
[0131] IgG subclasses vary in their ability to bind to FcyR and this differential binding determines their ability to elicit a range of functional responses. For example, in humans, FcyRIIIa is the major receptor involved in the activation of antibody-dependent cell-mediated cytotoxicity (ADCC) and IgG3 (followed closely by IgGl) display the highest affinities for this receptor, reflecting their ability to potently induce ADCC. IgG2 have been shown to have weak binding for this receptor.
[0132] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention binds FcyR with high affinity, preferably binds an activating receptor with high affinity.
[0133] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention binds FcyRI and/or FcyRIIa and/or FcyRIIc and/or FcyRIIIa and/or FcyRIIIb with high affinity.
[0134] In one embodiment, the IgGl antibody binds to at least one Fc activating receptor. For example, the antibody may bind to one or more receptor selected from FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa and FcyRIIIb. In one embodiment, the antibody is capable of binding to FcyRIIIa. In one embodiment, the antibody is capable of binding to FcyRIIa. In one embodiment, the antibody is capable of binding to FcyRIIIa, FcyRIIc and optionally FcyRI. In one embodiment, the antibody is capable of binding to FcyRIIIa, FcyRIIa and optionally FcyRI.
[0135] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention binds to at least one activating Fey receptor with a dissociation constant of less than about 10-6M, 10-7M, 10-8M, 10-9M or 10-10M.
[0136] In one embodiment, the IgGl antibody binds to FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and/or FcyRIIIb with a higher affinity than it binds to FcyRIIb, with low affinity.
[0137] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is an IgGl antibody, preferably a human IgGl antibody.
[0138] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention may comprise human heavy chain constant regions sequences and allow to target, block, and/or deplete CCR8-expressing cells to which they are bound. [0139] In one embodiment, the proteins according to the present invention deplete CCR8-expressing cells to which they are bound. In one embodiment, the proteins according to the present invention deplete Tregs to which they are bound. In one embodiment, the proteins according to the present invention also deplete or reduce tumor infiltrating regulatory T cells to which they are bound.
[0140] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention depletes CCR8-expressing cells to which it is bound. In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention depletes Tregs to which it is bound. In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention also depletes or reduces tumor infiltrating regulatory T cells to which it is bound.
[0141] The term “deplete” or “depleting”, with respect to CCR8-expressing cells or Tregs refers to the killing, elimination, lysis or induction of such killing, elimination or lysis, so as to negatively affect the number of CCR8 expressing cells present in a sample or in a subject. In one embodiment, the antibody or antigen binding fragment thereof according to the present invention allows targeting, blocking proliferation, and/or depleting CCR8-expressing cells or Treg cells. In one embodiment, the depletion is via ADCC.
[0142] In one embodiment, the depletion is via ADCP. In one embodiment, the depletion is via CDC.
[0143] Thus, in one embodiment, the antibody of the present invention leads, directly or indirectly, to the depletion of CCR8-expressing cells (e.g., leads to a 10%, 20%, 50%, 60%, 70%, 80%, 85% or greater elimination or decrease in number of CCR8 expressing cells).
[0144] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention does not inhibit the binding of interleukin-2 (IL-2) to CCR8 and depletes Tregs to which they are bound. [0145] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is a pegylated antibody or fragment thereof.
[0146] An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. The pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (DY12- DY120) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the present invention, such as, for example, as described in EP0154316 (Nishimura et al.) and EP0401384 (Ishikawa et al.).
[0147] In one embodiment, the isolated antibody or antigen-binding fragment thereof as described hereinabove is for use as a medicament.
[0148] The present invention further relates to a nucleic acid encoding the isolated antibody or antigen-binding fragment thereof according to the invention.
[0149] In one embodiment, the nucleic acid encoding the isolated antibody or antigenbinding fragment thereof of the invention may be expressed as a single nucleic acid molecule that encodes the isolated antibody or antigen-binding fragment thereof, or as multiple (e.g., two or more) nucleic acid molecules that are co-expressed. Polypeptides encoded by nucleic acid molecules that are co-expressed may associate through, e.g., disulfide bonds or other means, to form the isolated antibody or antigen-binding fragment thereof as described hereinabove.
[0150] In one embodiment, the nucleic acid molecule is DNA. In another embodiment, the nucleic acid molecule is RNA, for example, in the form of messenger RNA (mRNA). [0151] In one embodiment, the nucleic acid is linear. In another embodiment, the nucleic acid is circular.
[0152] In one embodiment, the nucleic acid as described hereinabove is for use as a medicament.
[0153] Another object of the invention is a vector comprising a nucleic acid according the invention. In some embodiments, at least one nucleic acid molecule is comprised in the one vector.
[0154] Within the scope of the instant invention, the expression “at least one nucleic acid” is intended to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleic acid molecules.
[0155] In some embodiments, the vector allows the controlled expression of at least one isolated antibody or antigen-binding fragment thereof. As used herein, the expression “controlled expression” is intended to refer to an expression that is controlled in time and/or space. In other words, the controlled expression of the isolated antibody or antigenbinding fragment thereof according to the invention may occur in a specific location of the body, such as, e.g., a specific organ, and/or for a specific time period.
[0156] In certain embodiments, the vector is a viral vector. Non limitative examples of viral vectors include adenovirus, adeno-associated virus (AAV), alphavirus, herpesvirus, lentivirus, non-integrative lentivirus, retrovirus, vaccinia virus and baculovirus.
[0157] In certain embodiments, the vector is a non-viral vector. Non limitative examples of non-viral vectors include inorganic particles (e.g., gold, calcium phosphate), lipidic emulsions, lipidic nanoparticles (e.g., liposomes), DNA-binding protein or peptide.
[0158] In one embodiment, the vector as described hereinabove is for use as a medicament.
[0159] The present invention also relates to an immunotoxin fusion protein against a CCR8-expressing cell, such as a T reg cell or a cancer cell, comprising the isolated antibody or antigen-binding fragment thereof according to the invention and a toxin or fragment thereof.
[0160] An immunotoxin is a ligand combined with a toxin, which can be used to kill cells expressing receptors for the ligand. Immunotoxin treatment is also known as ligand- targeted therapeutics. Thus, the immunotoxins contain a targeting moiety (a ligand) for delivery and a toxic moiety for cytotoxicity. The ligands currently used are monoclonal antibodies, cytokines/growth factors and soluble receptors.
[0161] In one embodiment, the immunotoxin fusion protein comprises an antigenbinding fragment of the antibody of the invention. In some embodiments, the antigenbinding fragment comprises two single chain Fvs of the variable region of the antibody of the invention. In some embodiments, the antigen-binding fragment further comprises one or more linker(s).
[0162] In some embodiments, the immunotoxin fusion protein comprises a truncated toxin.
[0163] Optionally, the immunotoxin fusion protein can further comprise a modified amino terminal. Optionally, the immunotoxin fusion protein can further comprise a signal peptide at its amino terminal, following translation but prior to secretion.
[0164] The present invention further relates to a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof, nucleic acid or the vector according to the invention, and at least one additional pharmaceutically acceptable excipient.
[0165] In some embodiments, the pharmaceutically acceptable vehicle is selected in a group comprising or consisting of a solvent, a diluent, a carrier, an excipient, a dispersion medium, a coating, an antibacterial agent, an antifungal agent, an isotonic agent, an absorption delaying agent and any combinations thereof. The carrier, diluent, solvent or excipient must be “acceptable” in the sense of being compatible with the isolated antibody or fragment thereof, nucleic acid or the vector according to the invention, or derivative thereof, and not be deleterious upon being administered to an individual. Typically, the vehicle does not produce an adverse, allergic or other untoward reaction when administered to an individual, preferably a human individual.
[0166] For the particular purpose of human administration, the pharmaceutical compositions should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory offices, such as, for example, the Food and Drugs Administration (FDA) Office or the European Medicines Agency (EMA).
[0167] In some embodiments, the pharmaceutical composition further comprises at least one additional therapeutic agent. In some embodiments, the at least one additional therapeutic agent is selected from the group comprising or consisting of anticancer agents and checkpoint inhibitors.
[0168] In some embodiments, the at least one additional therapeutic agent is an anticancer agent.
[0169] Anticancer agents are known from the state of the art. Non-limitative examples of anticancer agents include acalabrutinib, alectinib, alemtuzumab, anastrozole, avapritinib, avelumab, belinostat, bevacizumab, bleomycin, blinatumomab, bosutinib, brigatinib, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin copanlisib, cytarabine, daunorubicin, decitabine, dexamethasone, docetaxel, doxorubicin, encorafenib, erdafitinib, etoposide, everolimus, exemestane, fludarabine, 5-fluorouracil, gemcitabine, ifosfamide, imatinib Mesylate, leuprolide, lomustine, mechlorethamine, melphalan, methotrexate, mitomycin, nelarabine, paclitaxel, pamidronate, panobinostat, pralatrexate, prednisolone, ofatumumab, rituximab, temozolomide, topotecan, tositumomab, trastuzumab, vandetanib, vincristine, vorinostat, zanubrutinib, and the likes.
[0170] In certain embodiments, the anticancer agent is to be administered in combination with, concomitantly or sequentially, the isolated antibody or antigen-binding fragment thereof, nucleic acid, the vector or the pharmaceutical composition according to the invention. [0171] In some embodiments, the at least one additional therapeutic agent is a checkpoint inhibitor therapy.
[0172] As used herein, a “checkpoint inhibitor therapy” is defined as the administration of at least one checkpoint inhibitor to the subject.
[0173] Checkpoint inhibitors (CPI, that may also be referred to as immune checkpoint inhibitors or ICI) molecules, often antibodies, block the interactions between inhibitory receptors (IRs) expressed on T cells and their ligands. As a cancer treatment, checkpoint inhibitor therapy aims at preventing the activation of inhibitory receptors expressed on T cells by ligands expressed by the tumor cells. Checkpoint inhibitor therapy thus aims at preventing the inhibition of T cells present in the tumor, i.e., tumor infiltrating T cells, and thus at enhancing the subject immune response towards the tumor cells.
[0174] Examples of checkpoint inhibitors include, without being limited to, inhibitors of the cell surface receptor PD-1 (programmed cell death protein 1), also known as CD279 (cluster differentiation 279); inhibitors of the ligand PD-L1 (programmed death-ligand 1), also known as CD274 (cluster of differentiation 274) or B7-H1 (B7 homolog 1); inhibitors of the cell surface receptor CTLA4 or CTLA-4 (cytotoxic T-lymphocyte- associated protein 4), also known as CD 152 (cluster of differentiation 152); inhibitors of LAG-3 (lymphocyte-activation gene 3), also known as CD223 (cluster differentiation 223); inhibitors of TIM-3 (T-cell immunoglobulin and mucin-domain containing-3), also known as HAVCR2 (hepatitis A virus cellular receptor 2) or CD366 (cluster differentiation 366); inhibitors of TIGIT (T cell immunoreceptor with Ig and ITIM domains), also known as VSIG9 (V-Set And Immunoglobulin Domain-Containing Protein 9) or VSTM3 (V-Set And Transmembrane Domain-Containing Protein 3); inhibitors of BTLA (B and T lymphocyte attenuator), also known as CD272 (cluster differentiation 272); inhibitors of CEACAM-1 (carcinoembryonic antigen-related cell adhesion molecule 1) also known as CD66a (cluster differentiation 66a).
[0175] In one embodiment, the at least one checkpoint inhibitor is selected from the group comprising or consisting of inhibitors of PD-1, inhibitors of PD-L1, inhibitors of CTLA 4 and any mixtures thereof. [0176] In some embodiments, the therapeutic agent is a programmed cell death protein 1 (PD-1) inhibitor. In a preferred embodiment, the PD-1 inhibitor is an isolated antibody or antigen-binding fragment thereof selective for PD-1, preferably a monoclonal antibody. Non limitative examples of FDA approved anti -PD-1 therapeutic antibodies include pembrolizumab (Keytruda®, Merck), nivolumab (Opdivo®, Bristol Myers Squibb), cemiplimab (Libtayo®, Sanofi Genzyme) and dostarlimab (Jemperli®, GlaxoSmithKline). In another embodiment, the PD-1 inhibitor is a small molecule or a nucleic acid, e.g., an interfering RNA.
[0177] In some embodiments, the antibody or antigen-binding fragment thereof, nucleic acid, expression vector, pharmaceutical composition or medicament according to the present invention is used in combination with a second antibody that induces, via ADCC, the death of a cell expressing an antigen to which the second antibody binds. In one embodiment, the second antibody (e.g., of IgGl or IgG3 isotype) induces ADCC toward a cell to which the antibody binds. NK cells have an important role in inducing ADCC and increased reactivity of NK cells can be directed to target cells through use of such a second antibody. In one embodiment, the second antibody is specific for a cell surface antigen, e.g., membrane antigen. In some embodiments, the second antibody is specific for a tumor antigen (e.g., molecules specifically expressed by tumor cells), such as CD20, CD52, ErbB2 (or HER2/Neu), CD33, CD22, MUC-1, CEA, KDR, aVp3, etc., particularly lymphoma antigens (e.g., CD20). Accordingly, the present invention also provides methods to enhance the anti-tumor effect of monoclonal antibodies directed against tumor antigen(s).
[0178] The present invention further relates to the isolated antibody or antigen-binding fragment thereof, nucleic acid, vector or pharmaceutical composition according to the invention, for use as a medicament.
[0179] The present invention further relates to the isolated antibody or antigen-binding fragment thereof, nucleic acid, vector, pharmaceutical composition or medicament according to the invention, for use for treating and/or preventing a disease, preferably a CCR8-related disease in a subject in need thereof. [0180] As used herein, the term “CCR8-related disease” encompasses diseases wherein CCR8 is expressed or overexpressed by at least one cell type, and/or wherein CCR8- expressing cells are overrepresented and/or mislocalized. Non-limitative examples of CCR8-related diseases include cancer, in particular CCR8-expressing T-cell lymphomas or leukaemia, or cancers where CCR8-expressing cells have infiltrated and promote the development of cancer; and diseases associated with or related to immune responses dysfunction.
[0181] In some embodiments, the CCR8-related disease is cancer.
[0182] In some embodiments, the cancer is selected from the group comprising or consisting of colon cancer, melanoma, breast cancer, urothelial cancer, adenoid cystic carcinoma, adrenal gland cancer, ataxia-telangiectasia, atypical mole syndrome, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, carcinoid tumor, cervical cancer, colorectal cancer, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, islet cell tumor, kidney cancer, laryngeal cancer, leukemia, liver cancer, lobular carcinoma, hepatocellular carcinoma, lung cancer, glioma, meningioma, nasopharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, peritoneal cancer, pituitary gland tumor, polycythemia vera, prostate cancer, renal cell carcinoma, retinoblastoma, salivary gland cancer, sarcoma, small intestine cancer, stomach cancer, testicular cancer, thyroid cancer, uterine (endometrial) cancer and vaginal cancer.
[0183] In some embodiments, the cancer is selected from the group comprising or consisting of colon cancer, melanoma, breast cancer and urothelial cancer.
[0184] In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is urothelial cancer.
[0185] In some embodiments, the CCR8-related disease is a disease associated with or related to immune responses dysfunction.
[0186] In some embodiments, the disease associated with or related to immune responses dysfunction is an inflammatory disease or a hypersensitivity disease. Non- limited examples of inflammatory diseases according to the invention include cholangitis and arthritis.
[0187] The present invention further relates to a method for treating and/or preventing a disease, preferably a CCR8-related disease, more preferably a cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the isolated antibody or antigen-binding fragment thereof, nucleic acid, vector, pharmaceutical composition or medicament according to the invention.
[0188] By “therapeutically effective amount”, it is meant a level or amount of isolated antibody or antigen-binding fragment thereof, nucleic acid, vector, pharmaceutical composition or medicament according to the invention, that is necessary and sufficient for depleting CCR8-expressing Treg cells infiltrating a tumor in an individual in need thereof, without causing significant negative or adverse side effects to the individual. In particular, the term “therapeutically effective amount” further refers to the level or amount of isolated antibody or antigen-binding fragment thereof, nucleic acid, vector, pharmaceutical composition or medicament according to the invention, that is necessary and sufficient for slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of cancer, in particular solid cancer (tumor progression); or alleviating the symptoms of cancer in particular solid cancer; or curing cancer in particular solid cancer, without causing significant negative or adverse side effects to the individual.
[0189] The present invention further relates to the use of the isolated antibody or antigenbinding fragment thereof, nucleic acid, vector or pharmaceutical composition according to the invention, for the manufacture of a medicament, preferably for the treatment and/or prevention of a CCR8-related disease, more preferably a cancer.
[0190] The present invention further relates to the use of the isolated antibody or antigenbinding fragment thereof, nucleic acid, vector or pharmaceutical composition according to the invention, for depleting CCR8-expressing tumor Treg cells. In some embodiments, the Treg overexpress CCR8. [0191] Another object of the present invention relates to the use of the antibody or antigen-binding fragment thereof with another therapeutic agent as described hereinabove, in the treatment of diseases in a subject in need thereof, wherein said antibody or antigen-binding fragment thereof is used as an adjuvant for the therapeutic agent.
[0192] The present invention thus relates to an antibody or antigen-binding fragment thereof as described herein (preferably in a pharmaceutical composition or medicament), for use as an adjuvant in a cancer therapy. The present invention thus relates to an antibody or antigen-binding fragment thereof as described herein (preferably in a pharmaceutical composition or medicament), for use as an adjuvant in a therapy for an infectious disease.
[0193] In one embodiment, the present invention relates to the use of the antibody or fragment thereof as described herein, for potentiating an immune response induced by a cancer therapy in a patient in need thereof.
[0194] In one embodiment, the antibody or fragment thereof according to the present invention may be used as immunotherapeutic agent, particularly to treat a wide variety of cancers (e.g., cancers associated with immunosuppression and/or immune exhaustion).
[0195] In one embodiment, the antibody or fragment thereof according to the present invention may potentiate an immune response induced by a cancer therapy in a patient comprising administering said antibody or fragment thereof to a subject in an amount effective to potentiate an immune response induced by the cancer therapy in the patient.
[0196] As used herein, the term “adjuvant” refers to a compound or a combination of compounds that potentiates at therapy, such as, for example, a cancer therapy. Adjuvants may increase the effective immune response against low or non-immunogenic tumor cells. In one embodiment, the adjuvant is used with a well-known cancer therapeutic agent in the treatment of cancer and thus potentiates the immune response towards cancer cells. For example, an adjuvant may potentiate an immune response during a cancer therapy, decrease T cell exhaustion (without decreasing T cells activation), increase the survival of T cells, enhance NK cells cytotoxicity, decrease the tumor growth and/or the tumor size, and/or increase in survival, treats or prevents cancer metastasis. In one embodiment, potentiation of a cancer therapy in the presence of an adjuvant, is defined by comparison with a cancer therapy administered alone.
[0197] In another embodiment, the antibody or fragment thereof as described herein can increase or improve the immune response of a subject.
[0198] As used herein, an “immune response” refers to a response by a cell of the immune system, such as a B cell, T cell (CD4 or CD8), regulatory T cell, antigen- presenting cell, dendritic cell, monocyte, macrophage, NKT cell, NK cell, basophil, eosinophil, or neutrophil, to a stimulus. In some embodiments of any of the aspects, the response is specific for a particular antigen (an “antigen-specific response”), and refers to a response by a CD4 T cell, CD8 T cell, or B cell via their antigen-specific receptor. In some embodiments of any of the aspects, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. Such responses by these cells can include, for example, cytotoxicity, proliferation, cytokine or chemokine production, trafficking, or phagocytosis, and can be dependent on the nature of the immune cell undergoing the response.
[0199] As with other known immunotherapeutic agents, the ability of the antibody or fragment thereof according to the present invention, to potentiate an immune response in a patient may have broader therapeutic implications outside the cancer field. For example, it has been proposed that immune potentiating agents may be useful in treating a wide variety of infectious diseases, particularly pathogenic agents which promote immunosuppression and/or immune exhaustion. Also, such immune potentiating agents may be useful in boosting the immunization efficacy of vaccines (e.g., infectious disease and cancer vaccines).
[0200] Another object of the present invention relates to the use of the antibody or antigen-binding fragment thereof according to the present invention to deplete CCR8 expressing Treg cells in a subject in need thereof, wherein a therapeutically effective amount of an antibody or fragment thereof of the present invention is to be administered to the subject. [0201] The present invention thus further relates to a method for depleting CCR8 expressing Treg cells in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment thereof, a pharmaceutical composition, or a medicament as described herein.
[0202] In one embodiment, the antibody or antigen-binding fragment thereof as described hereinabove (preferably in a composition, pharmaceutical composition or medicament as describe hereinabove), is for use to deplete CCR8 expressing Treg cells.
[0203] In one embodiment, the CCR8 expressing Treg cells are tumor infiltrating Tregs.
[0204] Another obj ect of the present invention relates to a method for desensitizing cells to stimulations by CCL1 in a subject in need thereof, comprising administering to the subject the antibody or antigen-binding fragment thereof, a pharmaceutical composition, or a medicament according to the present invention.
[0205] The present invention thus further relates to the use of the antibody or antigenbinding fragment thereof or the immunotoxin fusion protein according to the invention for killing CCR8-expressing cells, such as T reg cells or cancer cells.
[0206] Still another object of the invention is the use of the antibody or antigen-binding fragment thereof according to the invention targeting both human and rabbit CCR8, in a rabbit model for pre-clinical studies.
BRIEF DESCRIPTION OF THE DRAWINGS
[0207] Figure 1 is a chromatography and subsequent quantification showing the purity of the antibody mAb2. The electrophoresis was performed in denaturating conditions (SDS-PAGE) on Bio-Rad gels 15% (Bio-Rad 4568084) and mAb2 was labelled.
[0208] Figure 2A-2B is a combination of graphs showing a flow cytometry analysis for the detection of CCL1. Represented herein are the positive control CCL1 (Fig. 2A) and negative control C3a (Fig. 2B). [0209] Figure 3 is an histogram representing the percentage of CCL1 fixation on HuT- 78 using mAbl, mAb2, mAb3, mAb4 and mAb5, in the flow cytometry assay as exemplified in Fig. 2.
[0210] Figure 4 is a flow cytometry plot showing the detection of human Treg cells (cells expressing FoxP3) using the mAb as described hereinabove.
[0211] Figure 5 is a flow cytometry plot showing the detection of rabbit CD4-positive cells using the mAb as described hereinabove. The 1.5% corresponds to the expected proportion or peripheral Treg cells.
EXAMPLES
[0212] The present invention is further illustrated by the following examples.
Figure imgf000042_0001
Materials and Methods
[0213] Mouse immunization and fusion: Anti human CCR8 monoclonal antibodies (mAb) were obtained in immunizing BalB-C mouse with a syngenic cell line stably expressing hCCR8, according to the method previously described (WO 02/081523). After the immunization, the splenocytes were harvested (50* 106 cells) and fused by electrofusion with the mouse fusion cell line SP2O (20* 106 cells) to obtain producing mAbs hybridomas. The fusion product was diluted in 400 ml HAT containing medium (DMEM 10 % FCS) and dispensed in 20 96-well plates. One week after the fusion, the growing hybridomas supernatant were screened.
[0214] Hybridoma screening: The hybridomas supernatants were screened by flow cytometry on the human HuT-78 cell line (ATCC TIB 161) that naturally express CCR8. 50 pl of HuT-78 cells diluted at 106/ml in PBS were dispensed into a 96-well plate. 50 pl of the hybridomas supernatants were added. As negative control, 50 pl of culture medium was added. As positive control, 50 pl of mouse anti-hCCR8 (diluted at 2,5 pl/ml) (Biolegend L263G8) was added. After a 10 min incubation at 4°C, the plate was centrifuged (1200g during 10 min) and supernatant discarded. For the revelation, 50 pl of a goat anti mouse IgG Alexa Fluor 488 diluted 100 X in PBS (Biolegend L263G8) is added. After a 5 min incubation at 4°C, the plate was centrifuged and supernatant discarded. The cell pellet was suspended in 100 pl PBS and analysed by flow cytometry on BD Accuri. The fluorescence (expressed in mean Fluorescence) was calculated by the software BD CSampler.
[0215] Isotyping of the antibodies: The mAbs were isotyped by flow cytometry using the protocol described above, except that the revelation antibody was the rat anti mouse IgGl FITC (L0-MG1.2 Synabs) or rat anti mouse IgG2a FITC (LO-MG2a7 Synabs) or rat anti mouse IgG2b FITC (LO-MG2b2 Synabs) or rat anti mouse IgG3 FITC (L0-MG3-
7 Synabs) or rat anti mouse IgM FITC (L0-MM9 Synabs).
Results
[0216] 41 positive hybridomas were obtained and classified according to their fluorescence intensity (Table 1).
Figure imgf000043_0001
Figure imgf000044_0001
Table 1: Fluorescence obtained with the antibodies produced by the hybridomas
[0217] A clear shift intensity was observed between the 5 first hybridoma and the last 37 ones. It was hypothesized that the five first mAb recognized a particularly exposed epitope. [0218] These 5 mAbs were isotyped by flow cytometry (Table 2).
Figure imgf000044_0002
Table 2: Isotyping results
The 5 hybridomas were developed in flasks (200 ml culture medium). The mAbs were purified from the culture supernatants on rat anti-mouse columns. The hybridomas 1,2, 3, 5 supernatant that are IgG2a were purified on LO-MG2a column (from Synabs). The hybridoma 4 supernatant that is IgGl was purified on LO-MG1 column (from Synabs).
The purified mAbs were suspended in PBS. The mAbs purity was then controlled by electrophoresis on polyacrylamide gel in denaturating and non-denaturating conditions. The purity of each mAbs was higher than 90 % (see an example of purity for mAb2 on Figure 1). Example 2: Epitope determination
[0219] CCR8 presents 4 extracellular regions: one N-terminus tail, one C-terminus tail, one extracellular loop (ECL) and two intracellular loops (ICL).
[0220] The corresponding sequence is highlighted in bold in the full-length hCCR8 sequence (genebank P51685): MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGKLLLAVFYCLLFVFSLLGN SLVILVLVVCKKLRSITDVYLLNLALSDLLFVFSFPFOTYYLLDQWVFGTVMC KVVSGFYYIGFYSSMFFITLMSVDRYLAVVHAVYALKVRTIRMGTTLCLAVWL TAIMATIPLLVFYOVASEDGVLQCYSFYNOOTLKWKIFTNFKMNILGLLIPFTI FMFCYIKILHQLKRCONHNKTKAIRLVLIVVIASLLFWVPFNVVLFLTSLHSMHI LDGCSISOQLTYATHVTEIISFTHCCVNPVIYAFVGEKFKKHLSEIFOKSCSOIF NYLGRQMPRESCEKSSSCQQHSSRSSSVDYIL (SEQ ID NO: 3)
Materials and Methods
[0221] To determine the epitopes recognized by the selected antibodies, a pepscan was performed. This pepscan consisted in firstly coating an ELISA plate with several peptides corresponding to the whole extracellular regions of CCR8 and secondly adding the antibodies.
[0222] The peptides (purchased from Eurogentec) used were:
- Peptl : MDYTLDLSVTTV (SEQ ID NO: 4)
- Pept2 : VTT VTD YYYPDIF S SPC (SEQ ID NO : 1 )
- Pept3 : FSSPCDAELIQTNGK (SEQ ID NO: 5)
- Pept4: YLLDQWVFGTVMC (SEQ ID NO: 6)
- Pept5 : YQVASEDGVLQC (SEQ ID NO: 7)
- Pept6: VLQCYSFYNQQTLK (SEQ ID NO: 8)
- Pept7 : QTLKWKIFTNFKM (SEQ ID NO : 9)
[0223] The peptides were coated overnight at 4°C at 5 pl/ml in borate buffer pH 9. After a washing step with PBS buffer, the different mAbs diluted at 5 pl/ml in PBS were added to the plate and incubated Ih at 37°C. After a washing step with PBS buffer, the goat anti mouse IgG Hrp (IcL GGHL-90P) 1000 X diluted was added to the plate and incubated 30 min at 37°C After a washing step in PBS, a TMB solution was added to the plate and incubated 10 min at room temperature. The colorimetric reaction was stopped in adding a Sulfuric acid solution at pH 2. The yellow coloration of the ELISA wells plate was red at 450 nm. Results
[0224] All the 5 mAbs recognized the peptide 2 only. The peptide 2 corresponds to the region AA 9-25 of the full length human CCR8 sequence.
[0225] The region corresponding to this epitope was previously described as a CCL1 binding region. It was also described that a sulfonation (i.e., a post-transcriptional SOsH group addition) on the tyrosines Y15 and Y17 may occurs (Yu, Hoffhiness et al., 2017. Nature Method. 4: 583).
[0226] To verify if the 5 mAbs are able to recognize the two forms sulfonated and nonsulfonated CCR8, a comparative ELISA was performed in coating a non-sulfonated peptide 2 (VTTVTDYYYPDIFSSPC) and a Y15, Y17 sulfonated peptide 2 (VTTVTDY(SO3H)YY(SO3H)PDIFSSPC ).
[0227] The sulfonated peptide 2 was also purchased from Eurogentec. The ELISA protocol was similar to this detailed for the pepcan (see Example 1).
[0228] The ELISA (Table 3) shows that the sulfonation only weakly reduce the mAbs binding of the 5 mAbs.
Figure imgf000046_0001
Table 3: 450 nm absorbance using mAbs against peptide 2 or sulfonated peptide 2
[0229] The 5 mAbs recognize the same epitope and 4 of them exhibit the same IgG2a isotype. This result could mean that some of the mAbs are identical and came from an original activated B lymphocyte that has divided a large number of times in the spleen. To verify this, the corresponding Heavy and Light variable chain mRNA of each mAb was sequenced. Example 3: Inhibition of CCL1 binding
[0230] Since the epitope recognized by the 5 mAbs was described to correspond to the CCR8 ligands binding, it was asked if some of these mAbs are able to block the CCL1 binding.
[0231] As described earlier (Liu, Doijen et al., 2021. Biochemical Pharmacology. 188: 114565), it is possible to evaluate the capacity of the mAbs to block the CCL1 binding in using CCL1 labelled with Alexa fluor 647 (Almac UK cat: CAF07).
Materials and Methods
[0232] Positive control: 50 pl of HUT-78 suspended in PBS (106 ml) were incubated 10 min at 4°C with 50 pl PBS, then 50 pl of CCL1 Alexa Fluor647 (Almac cat: CAFO7) diluted 10 X was added for a further 10 min incubation at 4°C.
[0233] Negative control: 50 pl of HUT-78 suspended in PBS (106 ml) were incubated 10 min at 4°C with 50 pl PBS, then 50 pl of C3a Alexa Fluor647 (Almac cat CAF91 Al) diluted 10 X was added for a further 10 min incubation at 4°C.
[0234] Essays: 50 pl of HUT-78 suspended in PBS (106 ml) were incubated 10 min at 4°C with 50 pl of each mAbs diluted in PBS at 5 pg/ml, then CCL1 Alexa Fluor647 (Almac cat: CAFO7) diluted 10 X was added for a further 10 min incubation at 4°C. After the incubation, the plates were washed 3 times with PBS and analyzed by flow cytometry.
[0235] Example of the flow cytometry analysis of mAb 2 is shown on Figures 2A-B.
Results
[0236] Results are shown in Table 4.
Figure imgf000047_0001
Table 4: Mean fluorescence (CCL1 binding)
[0237] Results expressed in % CCL1 fixation (mean fluo assay-mean fluo negative Ctl/ mean fluo positive ctl-mean fluo negative ctl) are shown in Figure 3.
[0238] The results shows that all the mAh compete completely (mAb2) or partially with CCL1.
Example 4: Antibodies internalization
[0239] When the ligand CCL1 binds to the GPCR, it first induces an intracellular activation cascade and then initiates the internalization of the ligand-GPCR complex. This phenomenon, well known as desensitization, renders the cell temporarily insensitive to any further stimulation by the same ligand. We show here that antibodies also cause internalization of the antibody-GPCR complex.
Materials and Methods
[0240] A volume of 200 pl of HUT-78 suspended in culture medium (106 ml) was dispensed in a 96-well plate and incubated at 37°C. At various periods of time (0 min, Ih, 2h, 3h, 3h30 and 3h55), the mAb 1, at a final 5 pg/ml concentration, was added. A negative control without mAbl was also tested.
After a 4 h incubation, the plate was centrifugated and the supernatant discarded. For the revelation, 50 pl of a goat anti mouse IgG Alexa Fluor 488 diluted 100 X in PBS (Biolegend L263G8) was added. After a 5 min incubation at 4°C, the plate was centrifuged and supernatant discarded. The cell pellet was suspended in 100 pl PBS and analyzed by flow cytometry on BD Accuri. The fluorescence (expressed in mean Fluorescence) was calculated by the software BD C Sampler.
Results
[0241] Results are shown in Table 5.
Figure imgf000048_0001
Figure imgf000049_0001
Table 5: Mean fluorescence
[0242] The results show that the mAbl, in mimicking the ligand binding, also induces mAb-CCR8 complex internalization, leading to a CCR8 down expression.
Example 5: Human and rabbit Tree recognition
[0243] Treg are characterized by the transcription factor FoxP3 expression. It was investigated if the 5 mAbs also recognize Treg.
Materials and Methods
[0244] Flow cytometry (human): A flow cytometry test was performed on human PBMC (peripheral blood mononuclear cell) isolated from a fresh blood by a centrifugation on Ficoll. After a permeabilization treatment (FIX & PERM® Cell Permeabilization Kit Thermofisher GAS003) the isolated PBMC were doubly labelled with FOXP3 Alexa 488 (BD Biosciences) and with each mAbs Alexa Fluor 647.
[0245] Flow cytometry (rabbit): A double labelling of rabbit PBMC (isolated from fresh blood with a centrifugation on Ficoll) was performed with the mouse anti rabbit CD4 FITC mAb (Mybiosource cat: MBS 216606) and with one of the 5 mAbs Alexa Fluor 647.
[0246] Remark: a double labelling with anti FOXP3 was not performed since no anti rabbit FOXP3 usable for flow cytometry are currently available.
[0247] ELISA: see Example 2. Results
[0248] All of the 5 tested mAbs recognized the FOXP3 positive cells. As an example, the cell surface analysis of the PBMC double labelled FOXP3-mAbl performed on a FaCScan flow cytometer (Becton Dickinson) is show in Figure 4. [0249] The human CCR8 epitope recognized the 5 mAbs is similar to the corresponding rabbit CCR8 (genebank G1T2B2), one AA excepted:
- Human epitope: VTTVTDYYYPDIFSSPC (SEQ ID NO: 1)
- Rabbit epitope: VTAVTDYYYPDIFSSPC (SEQ ID NO: 2)
[0250] To verify if the 5 mAbs also recognize the rabbit CCR8, a comparative ELISA on the human peptide 2 (VTTVTDYYYPDIFSSPC) and its corresponding rabbit peptide (VTAVTDYYYPDIFSSPC) was performed. Results are shown in Table 6.
Figure imgf000050_0001
Table 6: 450 nm absorbance using mAbs against human or rabbit peptide 2
[0251] Clones 1, 3 and 5 recognize the rabbit peptide.
[0252] A flow cytometry test was also performed to verify if the 5 mAbs are able to bind to a subpopulation of the peripheral rabbit CD4+ lymphocytes. The clones 1, 3 and 5 recognized 1,5% of the CD4 positive cell that correspond to proportion of peripheral Treg
(Figure 5). This result confirms the result obtained with the ELISA.

Claims

1. An isolated antibody or antigen-binding fragment thereof that binds the chemokine (C-C motif) receptor 8 (CCR8), wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) which comprises the three following complementary-determining regions (CDRs):
- VH-CDR1 : SEQ ID NO: 10,
- VH-CDR2 : SEQ ID NO : 11 , and
- VH-CDR3 : SEQ ID NO : 12, and further comprises a light chain variable region (VL) which comprises the three following complementary-determining regions (CDRs):
VL-CDR1 : SEQ ID NO: 13,
VL-CDR2: SEQ ID NO: 14, and/or
VL-CDR3 : SEQ ID NO : 15.
2. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein said isolated antibody or antigen-binding fragment thereof is monoclonal.
3. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein said isolated antibody or antigen-binding fragment thereof binds human and rabbit CCR8.
4. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein said isolated antibody or antigen-binding fragment thereof recognizes an epitope consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
5. The isolated antibody or antigen-binding fragment thereof according to claim 4, wherein said epitope is post-transcriptionally modified, in particular said epitope is sulfonated.
6. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein binding of said isolated antibody or antigen-binding fragment thereof to CCR8 inhibits the binding of chemokine (C-C motif) ligand 1 (CCL1) and/or chemokine (C-C motif) ligand 18 (CCL18) to CCR8.
7. A nucleic acid encoding the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.
8. A vector containing a nucleic acid according to claim 7.
9. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the nucleic acid according to claim 7 or the vector according to claim 8, and at least one pharmaceutically acceptable excipient.
10. The pharmaceutical composition according to claim 9, further comprising at least one additional therapeutic agent, preferably a programmed cell death protein 1 (PD- 1) inhibitor.
11. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, a nucleic acid according to claim 7, a vector according to claim 8 or a pharmaceutical composition according to any one of claims 9 or 10, for use as a medicament.
12. The isolated antibody or antigen-binding fragment thereof, nucleic acid, vector or pharmaceutical composition for use according to claim 11, for treating and/or preventing a CCR8-related disease in a subject in need thereof.
13. The isolated antibody or antigen-binding fragment thereof, nucleic acid, vector or pharmaceutical composition for use according to claim 12, wherein said disease is cancer.
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