CN116917320A

CN116917320A - Murine cross-reactive human CCR8 binding agents

Info

Publication number: CN116917320A
Application number: CN202180094415.8A
Authority: CN
Inventors: H·Q·卢; C·斯托特勒斯; N·范博克塞尔; S·范兹瓦姆; J·博鲁; B·东布雷克特; J·范金德拉克尔; P·梅谢尔斯; R·马丁
Original assignee: Vlaams Instituut voor Biotechnologie VIB; Universite Libre de Bruxelles ULB
Current assignee: Vlaams Instituut voor Biotechnologie VIB; Universite Libre de Bruxelles ULB
Priority date: 2020-12-24
Filing date: 2021-12-23
Publication date: 2023-10-20

Abstract

The present invention relates to human CCR8 (hCCR 8) binding agents, wherein the hCCR8 binding agent is cross-reactive with murine CCR 8. Such binding agents are particularly useful for intratumoral regulatory T cell depletion and general immunotherapy.

Description

Murine cross-reactive human CCR8 binding agents

Technical Field

The present invention relates to human CCR8 (hCCR 8) binding agents, wherein the hCCR8 binding agents have cross-reactivity with murine CCR 8. Such binding agents are particularly useful for intratumoral regulatory T cell depletion and general immunotherapy.

Background

Regulatory T (Treg) cells are one of the components of the adaptive immune system, helping to maintain tolerance to self-antigens and preventing autoimmune diseases. However, treg cells have also been found to be highly enriched in the tumor microenvironment of a variety of different cancers (colobo and Piconese,2007; nishikawa and Sakaguchi,2014; roychoudhuri et al, 2015). In the tumor microenvironment, treg cells help immune escape by reducing Tumor Associated Antigen (TAA) -specific T cell immunity, thereby preventing effective anti-tumor activity. Thus, the high tumor infiltration of tregs is often associated with invasive phenotypes and poor prognosis in cancer patients (Shang et al, 2015; plitas et al, 2016).

Because of the recognition of the importance of tumor-infiltrating Treg cells and their potential role in suppressing anti-tumor immunity, various strategies have been proposed to modulate Treg cells in the tumor microenvironment. Several studies have demonstrated that modulation of Treg can provide significant therapeutic benefits (Elpek et al, 2007).

However, one major challenge associated with Treg modulation is that systemic removal or suppression of Treg cells can elicit autoimmunity. Thus, in order to prevent autoimmunity, it is important to specifically eliminate tumor-infiltrating Treg cells while retaining tumor-reactive effector T cells and peripheral Treg cells (e.g., circulating blood Treg cells).

Wang et al (PloSONE 2012, e 30793) report increased expression of CCR8 on tumor-infiltrating foxp3+ T cells and suggest that blocking CCR8 may result in inhibition of Treg migration into tumors. Due to the high and relatively specific expression of CCR8 on tumor-infiltrating tregs, neutralizing monoclonal antibodies against CCR8 have been suggested for modulating and clearing this Treg population in cancer treatment (EP 3431105 A1 and WO2019/157098 A1). WO2018/181425 shows that in mice, neutralizing anti-CCR 8 mabs are able to clear Treg cells in tumor tissue by antibody-dependent cell-mediated cytotoxicity (ADCC), thereby enhancing tumor immunity. Through their neutralizing activity, these antibodies inhibit Treg migration into tumors, reverse the inhibitory function of Treg and eliminate intratumoral Treg (WO 2019/157098 A1). Recently, wang et al (Cancer Immunol Immonother 2020, https:// doi.org/10.1007/s 00262-020-02583-y) showed that CCR8 blockade may disrupt the stability of intratumoral Tregs, rendering them a fragile phenotype, accompanied by reactivation of anti-tumor immunity and increasing anti-PD-1 therapeutic benefit.

Unfortunately, the clinical development of CCR 8-based Treg clearance therapies is hampered by the lack of CCR8 binding agents that have cross-reactivity between human CCR8 and CCR8 in related animal models. Indeed, monoclonal antibodies against human CCR8 have been described in WO2007044756 A2, including the preferred antibody 433H, but the antibodies lack cross-reactivity against murine CCR 8. EP3616720 A1 shows that ADCC-dependent Treg clearance using anti-murine CCR8 antibodies enhanced tumor immunity in a mouse model. However, since this antibody does not bind to human CCR8, it cannot be developed for human cancer treatment. In contrast, WO2020138489 A1 from the same applicant provides neutralising antibodies with ADCC activity against human CCR8 for use in the treatment of cancer. Its humanized antibody binds to human CCR8 and neutralizes CCL 1-induced calcium influx (calcium infiux). This suggests that binding to the N-terminal region of human CCR8 is an important factor in exerting neutralizing activity. Unfortunately, the monoclonal antibodies of the latter application were unable to bind to murine CCR8 and could not be tested in a murine model. Thus, the prior art only allows the development of anti-hCCR 8 antibodies for human cancer therapies by relying on preclinical in vivo data generated with surrogate antibodies. This carries the risk that antibodies being developed for human therapy cannot be used to test important factors such as toxicity and efficacy in preclinical animal models. In particular, the development of human and mouse cross-reactive binders would be the best approach, as most Treg clearance models for cancer treatment have been established in mice.

Thus, there is a great need for cross-reactive binders that bind to human CCR8 as well as CCR8 in animal models, in particular cross-reactive binders that bind to human CCR8 as well as CCR8 from related animal models (e.g. mice or macaque).

Disclosure of Invention

The inventors have now surprisingly identified binders of human CCR8 (hCCR 8) having cross-reactivity with murine CCR8 as detailed in the claims, thus meeting the above need. While the prior art suggests that N-terminal CCR8 epitope binding is necessary to exert CCR8 neutralizing activity, it has been found that cross-reactive neutralizing binders can be generated by binding to the extracellular loop of CCR 8. In particular, extracellular loop 2 of CCR8 has been found to be a suitable target for the generation of cross-reactive CCR8 binders, which is particularly surprising since it is one of the regions of minor sequence identity between human and murine CCR 8. It is therefore an object of the present invention to provide hCCR8 binding agents which are cross-reactive with murine CCR 8. Thus, in a first embodiment, the invention provides an hCCR8 binding agent, wherein the hCCR8 binding agent is cross-reactive with murine CCR 8.

Preferably, the hCCR8 binding agent binds to the extracellular loop of hCCR 8. Preferably, hCCR8 binds to the extracellular loop 2 of hCCR 8.

In other embodiments, the hCCR8 binding agent comprises a single domain antibody moiety that binds hCCR 8.

In further embodiments, the single domain antibody portion comprises three Complementarity Determining Regions (CDRs), namely CDR1, CDR2, and CDR3, wherein CDR3 is selected from the group consisting of: (a) the amino acid sequence of NGRQTGWRTRVDY (SEQ ID NO: 7);

(b) NAAPYYWGAYRRQES (SEQ ID NO: 8); (c)

YAQDSYKIYKSRYTQDY (SEQ ID NO: 9); (d)

YAQQSYKIYKSRYTQDY (SEQ ID NO: 10); (e)

YAQDTYKIYKSRYTQDY (SEQ ID NO: 11); (f) An amino acid sequence having at least 80% amino acid sequence identity to SEQ ID No. 7, 8, 9, 10 or 11; and (e) an amino acid sequence having a 3, 2 or 1 amino acid difference from SEQ ID NO 7, 8, 9, 10 or 11.

Preferably, CDR1 is selected from: (a) the amino acid sequence of GGIRSIIP (SEQ ID NO: 1); (b)

The amino acid sequence of GSIFSLLD (SEQ ID NO: 2); (c) the amino acid sequence of GSIFSLRT (SEQ ID NO: 3); (d) An amino acid sequence having at least 80% amino acid identity to SEQ ID No. 1, 2 or 3; and (e) an amino acid sequence having a 3, 2, 1 amino acid difference from SEQ ID NO 1, 2 or 3; CDR2 is selected from: (a) the amino acid sequence of ISTAGSA (SEQ ID NO: 4); (b) the amino acid sequence of ITSGGST (SEQ ID NO: 5); (c) the amino acid sequence of ISAGGAT (SEQ ID NO: 6); (d) An amino acid sequence having at least 80% amino acid identity to SEQ ID No. 4, 5 or 6; and (e) an amino acid sequence having a 3, 2, 1 amino acid difference from SEQ ID NO. 4, 5 or 6.

In further embodiments, the single domain antibody portion further comprises four Framework Regions (FRs) having at least 50%, preferably at least 60%, more preferably at least 70%, still more preferably at least 80%, more preferably at least 85% sequence identity to SEQ ID NOS: 12 to 16.

In further embodiments, the single domain antibody portion comprises the amino acid sequence of SEQ ID NO. 17 or SEQ ID NO. 21.

In further embodiments, the hCCR8 binding agent comprises a single domain antibody moiety that binds to human CCR8, and further comprises at least one cytotoxic moiety.

Preferably, the cytotoxic moiety induces antibody-dependent cellular cytotoxicity (ADCC), induces antibody-dependent cellular phagocytosis (ADCP), induces complement-dependent cytotoxicity (CDC), binds and activates T cells, or comprises a cytotoxic payload.

It is a further object of the invention to provide nucleic acids encoding hCCR8 binding agents.

It is a further object of the invention to provide hCCR8 binding agents for use as a medicament.

It is a further object of the invention to provide hCCR8 binding agents for use in the treatment of tumors. Preferably, the tumor is selected from breast cancer, endometrial cancer, lung cancer, gastric cancer, head and neck cancer, squamous cell carcinoma, skin cancer, colorectal cancer, renal cancer and T-cell lymphoma.

Preferably, administration of hCCR8 binding agent results in clearance of tumor-infiltrating regulatory T cells (tregs).

In other embodiments, the treatment further comprises administering a checkpoint inhibitor. A checkpoint inhibitor is a compound that blocks the binding of checkpoint proteins to their chaperones, thereby activating immune system functions. Preferably, the checkpoint inhibitor blocks a protein selected from the group consisting of PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, VISTA, B7-1 and B7-2. More preferably, the checkpoint inhibitor blocks PD-1 or PD-L1.

Drawings

FIG. 1 shows the evaluation of binding of three VHHs (VHH-57, VHH-64 and VHH-67) derived from llamas immunized with human CCR8 to human CCR8 overexpressed in HEK293 cells by flow cytometry.

FIG. 2 shows the assessment of binding of three VHHs (VHH-57, VHH-64 and VHH-67) to cynomolgus CCR8 transiently expressed in HEK293T cells by flow cytometry.

FIG. 3 provides an overview of the binding properties of VHH-57 and VHH-67 in HEK293T cells transiently transfected with human CCR8 (hCR 8) or mouse CCR8 (mCCR 8) using flow cytometry as compared to mock transfected cells.

FIG. 4 shows an evaluation of the potential of VHH-57, VHH-64 and VHH-67 to functionally inhibit the effect of human CCL1 ligand on cAMP accumulation in CHO-K1 cells stably expressing recombinant CCR 8.

FIG. 5 shows evaluation of binding of 9 VHH-Fc fusions VHH-Fc-203 (SEQ ID NO: 49), VHH-Fc-204 (SEQ ID NO: 57), VHH-Fc-205 (SEQ ID NO: 65), VHH-Fc-209 (SEQ ID NO: 47), VHH-Fc-210 (SEQ ID NO: 55), VHH-Fc-211 (SEQ ID NO: 63), VHH-Fc-215 (SEQ ID NO: 51), VHH-Fc-216 (SEQ ID NO: 59) and VHH-Fc-217 (SEQ ID NO: 67) to stably transfected human CCR8 in HEK293 cells compared to two control anti-CCR 8 mAbs.

FIG. 6 shows an evaluation of the binding of VHH-Fc fusion VHH-Fc-203, VHH-Fc-204, VHH-Fc-205, VHH-Fc-209, VHH-Fc-210, VHH-Fc-211, VHH-Fc-215, VHH-Fc-216 and VHH-Fc-217 to transiently overexpressed cynomolgus CCR8 in HEK293 cells compared to two control anti-CCR 8 mAbs.

FIG. 7 shows an evaluation of the binding of three VHH-Fc fusions VHH-Fc-204, VHH-Fc-210 and VHH-Fc-216 to stable expressed cynomolgus CCR8 in HEK293T cells compared to two control anti-CCR 8 mAbs and a control without VHH.

FIG. 8 shows an evaluation of the binding of VHH-Fc fusion VHH-Fc-203, VHH-Fc-204, VHH-Fc-205, VHH-Fc-209, VHH-Fc-210, VHH-Fc-211, VHH-Fc-215, VHH-Fc-216 and VHH-Fc-217 to transiently overexpressed mouse CCR8 in HEK293 cells compared to two control anti-CCR 8 mAbs.

FIG. 9 shows an evaluation of binding of VHH-Fc fusion VHH-Fc-203, VHH-Fc-204, VHH-Fc-205, VHH-Fc-209, VHH-Fc-210, VHH-Fc-211, VHH-Fc-215, VHH-Fc-216 and VHH-Fc-217 to mouse CCR8 expressed in BW5147 cells.

FIG. 10 shows the amino acid sequences of VHH-64 (SEQ ID NO: 30), VHH-57 (SEQ ID NO: 31) and VHH-67 (SEQ ID NO: 32), which are murine cross-reactive hCR 8 binders. Complementarity Determining Regions (CDRs) identified by the IMGT method are underlined, while CDRs identified by the Kabat method are bolded. Asterisks indicate the amino acids mutated in the optimized cross-reactive hCR 8 binders VHH-84 (SEQ ID NO: 17), VHH-119 (SEQ ID NO: 18), VHH-120 (SEQ ID NO: 19), VHH-121 (SEQ ID NO: 20) and VHH-122 (SEQ ID NO: 21).

FIG. 11 shows the in vivo effects of VHH-Fc-205 (SEQ ID NO: 65) and VHH-Fc-215 (SEQ ID NO: 51) in MC38 tumors compared to isotypes.

FIG. 12 shows the evaluation of PBMC-mediated ADCC activity on HEK292 cells expressing hCR 8 in both Afucosylated (AF) and nonfucosylated (non-afucosylated) forms of VHH-Fc-256 (SEQ ID NO: 52) compared to isotype.

Detailed Description

The invention will be described with reference to specific embodiments and with reference to specific drawings, but the invention is not limited thereto.

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature and techniques employed in connection with the molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly employed in the art.

As previously described, the present invention provides a human CCR8 (hCCR 8) binding agent, wherein the hCCR8 binding agent is cross-reactive with murine CCR8 (CCR 8). Such compounds are particularly useful because they are capable of binding to human CCR8 expressed on cells, e.g., regulatory T cells, particularly intratumoral regulatory T cells, and clearing such cells by their cytotoxic activity. CCR8 is a member of the beta chemokine receptor family, which is predicted to resemble the G-coupled receptor seven transmembrane protein. The identified CCR8 ligands include their natural cognate ligand CCL1 (I-309). The entry number of human CCR8 in the UniProt knowledge base (UniProt Knowledgebase) is P51685, while the entry number of murine CCR8 in the UniProt knowledge base is P56484.

CCR8 binding agents

As used herein, the term "binding agent" for a specific antigen refers to a molecule that is capable of specifically binding to the antigen. In particular, as used herein, a human CCR8 binding agent refers to a molecule capable of specifically binding hCCR 8. Such binding agents are also referred to herein as "hCCR8 binding agents".

As used herein, a "cross-reactive binding agent" refers to a binding agent that specifically binds to a target molecule (e.g., CCR 8) in two different species. CCR8 binding agents that are cross-reactive with murine and human CCR8 specifically bind to human and murine forms of CCR8. As is known in the art, cross-reactive binders typically have slightly different affinities for target proteins in two species. Preferably, the ratio of dissociation constant of the cross-reactive binding agent to human CCR8 to that of CCR8 to other species ranges from 1000:1 to 1:1000, such as from 500:1 to 1:500, particularly from 200:1 to 1:200, more particularly from 100:1 to 1:100. Thus, in particular, the dissociation constant difference of human CCR8 from, for example, murine CCR8 is equal to or less than 100-fold.

"specific binding", "specifically binding" (bind specifically) and "specifically binding" (specifically bind) are understood in particular to mean the dissociation constant (K) of the binding agent for the antigen of interest _d ) Less than about 10 ^-6 M、10 ^-7 M、10 ^-8 M、10 ^-9 M、10 ^-10 M、10 ^-11 M、10 ^-12 M or 10 ^-13 M. In a preferred embodiment, the dissociation constant is less than 10 ^-8 M, e.g. in the range of 10 ^-9 M、10 ^-10 M、10 ^-11 M、10 ^-12 M or 10 ^-13 M. For example, binding agent affinity for membrane targets can be determined by using surface plasmon resonance-based assays of virus-like particles (e.g., BIAcore assay described in PCT application publication No. WO 2005/012359), cellular enzyme-linked immunosorbent assay (ELISA), and Fluorescence Activated Cell Sorting (FACS) readout. A preferred method for determining apparent Kd or EC50 values is to use hCR 8 overexpressing cells at 21℃by using FACS.

In a specific embodiment, the binding portion of the hCCR8 binding agent is a protein, more specifically an hCCR8 binding polypeptide. In other embodiments, the binding portion of hCCR8 binding agent is antibody-based or non-antibody-based, preferably antibody-based. Non-antibody based binding agents include, but are not limited to, affibody (affibody), kunitz domain peptide, monobody (adnectin), anticalin, engineered ankyrin repeat domain (DARPin), centyrin, fynomer, avimer; affilin; affitin, peptides, and the like. In a specific embodiment, the hCCR8 binding agents of the invention bind to the extracellular portion of hCCR8, particularly the extracellular portion of hCCR8 expressed on regulatory T cells. In a specific embodiment, the hCCR8 binding agent of the invention binds to the extracellular loop of hCCR8, in particular extracellular loop 2.

As used herein, the terms "antibody", "antibody fragment" and "active antibody fragment" refer to a protein comprising an immunoglobulin (Ig) domain or an antigen binding domain capable of specifically binding an antigen, in this case hCCR8 protein. An "antibody" may also be an intact immunoglobulin from natural or recombinant sources, and may be an immunoreactive portion of an intact immunoglobulin. The antibody may be a multimer of immunoglobulin molecules, such as a tetramer. In a preferred embodiment, the binding agent comprises an hCCR8 binding moiety, which hCCR8 binding moiety is an antibody or an active antibody fragment. In another aspect of the invention, the binding agent is an antibody. In another aspect of the invention, the antibody is monoclonal. The antibody may additionally or alternatively be humanized or human. In another aspect, the antibody is a human antibody, or in any case an antibody having a form and characteristics that allow for its use and administration in a human individual. Antibodies may be from any species including, but not limited to, mice, rats, chickens, rabbits, goats, cattle, non-human primates, humans, dromedaries, camels, llamas, alpacas, and sharks.

The term "antigen binding fragment" refers to the antigen binding portion of the intact polyclonal or monoclonal antibody that retains the ability to specifically bind to the target antigen or a single chain thereof, fusion proteins comprising the antibody, and any other modified configuration of immunoglobulin molecules comprising an antigen recognition site. Antigen binding fragments include, but are not limited to: fab; fab'; f (ab') ₂ The method comprises the steps of carrying out a first treatment on the surface of the An Fc fragment; single domain antibodies (sdabs or dAb fragments). These fragments are derived from the whole antibody by using methods conventional in the art, for example by proteolytic cleavage with enzymes such as papain to produce Fab fragments or pepsin to produce F (ab') ₂ Fragments. Antigen binding fragments as used herein also refer to fragments comprising heavy and/or light chainsFusion proteins of variable regions, such as single chain variable fragments (scfvs).

As used herein, the term "monoclonal antibody" refers to an antibody composition having a homogeneous population of antibodies. It will be appreciated that monoclonal antibodies are highly specific for a single antigenic site. Furthermore, in contrast to conventional antibody (polyclonal) preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The binding agent of the invention preferably comprises a monoclonal antibody moiety which binds hCCR 8.

In one aspect of the invention, the binding agent comprises an active antibody fragment. The term "active antibody fragment" refers to any antibody or portion of an antibody-like structure that itself has a high affinity for an epitope or epitope and contains one or more antigen binding sites, such as Complementarity Determining Regions (CDRs), thereby accounting for this specificity. Non-limiting examples include immunoglobulin domains, fab, F (ab') 2, scFv, heavy chain-light chain dimers, immunoglobulin single variable domains, single domain antibodies (sdAb or dAb),And single chain structures (e.g., an intact light chain or an intact heavy chain), and antibody constant domains that have been engineered to bind antigen. An additional requirement for the "activity" of the fragment according to the invention is that the fragment is capable of binding hCCR8. The term "immunoglobulin (Ig) domain" or more specifically "immunoglobulin variable domain" (abbreviated as "IVD") refers to an immunoglobulin domain consisting essentially of framework regions separated by complementarity determining regions. Typically, an immunoglobulin domain consists essentially of four "framework regions," which are referred to in the art and hereinafter as "framework region 1" or "FR1," respectively; "frame region 2" or "FR2"; "frame region 3" or "FR3"; "frame region 4" or "FR4"; these framework regions are separated by three "complementarity determining regions" or "CDRs", which are referred to in the art and below as "complementarity determining region 1" or "CDR1", respectively; "complementarity determining region 2" or "CDR2"; "complementarity determining region 3" or "CDR3". Thus (2) The general structure or sequence of an immunoglobulin variable domain can be as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin Variable Domains (IVD) confer specificity to an antigen by carrying an antigen binding site. Typically, in conventional immunoglobulins, the heavy chain variable region (VH) and the light chain variable region (VL) interact to form antigen binding sites. In this case, the Complementarity Determining Regions (CDRs) of VH and VL will contribute to the antigen binding site, i.e., a total of 6 CDRs will be involved in the formation of the antigen binding site. In view of the above definitions, the antigen binding domain or Fab fragment, F (ab') 2 fragment, fv fragment (e.g., disulfide linked Fv or scFv fragment) of a conventional 4-chain antibody (e.g., igG, igM, igA, igD or IgE molecule; known in the art), or the antigen binding domain of a diabody (diabody) derived from such a conventional 4-chain antibody (all known in the art), is bound to a corresponding epitope of an antigen by a pair of (associated) immunoglobulin domains, such as light and heavy chain variable domains, i.e., by VH-VL pairs of the immunoglobulin domains, which together bind to an epitope of the corresponding antigen. As used herein, a single domain antibody (sdAb) refers to a protein having an amino acid sequence comprising 4 Framework Regions (FRs) and 3 Complementarity Determining Regions (CDRs) according to the form FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The single domain antibodies of the invention are equivalent to an "immunoglobulin single variable domain" (abbreviated "ISVD") and refer to molecules in which an antigen binding site is present on and formed from a single immunoglobulin domain. This separates a single domain antibody from a "conventional" antibody or fragment thereof, in which two immunoglobulin domains, particularly two variable domains, interact to form an antigen binding site. The binding site of a single domain antibody is formed by a single VH/VHH or VL domain. Thus, the antigen binding site of a single domain antibody is formed from no more than 3 CDRs. Thus, a single domain may be a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof; so long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit consisting essentially of a single variable domain such that a single antigen binding structure The domain need not interact with another variable domain to form a functional antigen binding unit).

Thus, in one embodiment, the hCCR8 binding agent as detailed above comprises a single domain antibody moiety. In particular, the single domain antibody may be(as defined herein) or a suitable fragment thereof (note>And->Is a registered trademark of Ablynx corporation (a company of Sanofi). For->Reference is made to the following further description and description in the prior art, e.g. WO 2008/020079. "VHH domains", also known as VHH, VHH antibody fragments and VHH antibodies, were originally described as antigen-binding immunoglobulin (Ig) (variable) domains of "heavy chain antibodies" (i.e., "light chain-free antibodies"; see, e.g., hamers-Casterman et al, nature 363:446-8 (1993)). The term "VHH domain" is chosen to distinguish these variable domains from heavy chain variable domains (referred to herein as "VH domains") found in conventional 4-chain antibodies and light chain variable domains (referred to herein as "VL domains") found in conventional 4-chain antibodies. For VHH and->For further description, reference is made to the review article of Muyledermans (Reviews in Molecular Biotechnology 74:277-302,2001), and the following patent applications mentioned as general background: WO 94/04678, WO 95/04079 and WO 96/34103 at the university of Brussell freedom; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Union; WO 97/49505, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the institute of biotechnology (VIB); WO 03/050531 to Alganomics and Ablynx; WO 01/90190 of the national research Committee of Canada; WO 03/025020 (=ep 1433793) of the institute of antibodies (Institute of Antibodies); and further published patent applications by Ablynx company WO 04/041687, WO 04/041682, WO 04/041685, WO 04/041683, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825. As described in these documents, +.>(in particular VHH sequences and partially humanized +.>) May be characterized in particular as the presence of one or more "marker" residues in one or more framework sequences. For->Including humanization and/or camelization of nanobodies (nanobodies), as well as other modifications, portions or fragments, derivatives or "Nanobody fusions", multivalent or multispecific constructs (including some non-limiting examples of linker sequences) and for increasing->Various modifications of the half-life of their preparations can be found, for example, in WO 08/101985 and WO 08/142164. VHH and- >Is one of the smallest antigen binding fragments that fully retains the binding affinity and specificity of a full-length antibody (see, e.g., greenberg et al, nature 374:168-73 (1995);

Hassazadeh-Ghasssaboeh et al, nanomedicine (Lond), 8:1013-26 (2013)).

The binding agents of the invention may be monospecific, bispecific or multispecific. A "multispecific binding agent" may be specific for a different epitope of one target antigen or polypeptide, or may contain antigen binding domains specific for more than one target antigen or polypeptide (Kufer et al Trends Biotechnol 22:238-44 (2004)).

In one aspect of the invention, the binding agent is a monospecific binding agent. As discussed further below, in an alternative aspect, the binding agent is a bispecific binding agent.

As used herein, "bispecific binding agent" refers to a binding agent that is capable of binding to two different epitopes on a single antigen or polypeptide, or two different epitopes on two different antigens or polypeptides.

The bispecific binding agents of the invention as discussed herein can be produced by the following method: biological methods, such as somatic hybridization; or genetic methods, such as expression of a non-native DNA sequence encoding a desired binding agent structure in a cell line or organism; chemical means (e.g., by chemical coupling, gene fusion, non-covalent association, or otherwise binding to one or more molecular entities (e.g., another binding agent to a fragment thereof)); or a combination thereof.

Techniques and products that allow for the production of monospecific or bispecific binders are known in the art, as broadly reviewed in the literature, as well as alternative forms, binder-drug conjugates, binder design methods, in vitro screening methods, constant regions, post-translational and chemical modifications, improved features that trigger Cancer cell death (e.g., fc domain engineering) (Tiller K and Tessier P, annu Rev Biomed eng.17:191-216 (2015), speiss C et al Molecular Immunology67:95-106 (2015), weiner G, nat Rev Cancer,15:361-370 (2015), fan G et al, J Hematol Oncol8:130 (2015)).

The hCCR8 binding agent of the invention may be a blocking or non-blocking binding agent. In a specific embodiment, the hCCR8 binding agents of the invention are blocking binding agents, also described in the art as neutralizing binding agents. In a specific embodiment, the hCCR8 binding agents of the invention inhibit hCCL1 and/or other hCCR8 ligands from binding to hCCR 8.

As used herein, an "epitope" or "antigenic determinant" refers to a site on an antigen that binds to a binding agent (e.g., an antibody). Epitopes can be formed from contiguous amino acids (linear epitopes) or non-contiguous amino acids juxtaposed by tertiary folding of the protein (conformational epitopes), as is well known in the art. Epitopes formed by consecutive amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3, more typically at least 5 or 8 to 10 amino acids in a unique spatial conformation. Methods for determining epitope spatial conformation are well known in the art and include, for example, X-ray crystallography and 2D nuclear magnetic resonance. See, e.g., methods in Molecular Biology, volume 66, glenn E.Morris, et al (1996) epitope mapping protocol (Epitope Mapping Protocols).

As used herein, the term "sequence identity" refers to two polypeptide or polynucleotide sequences that are identical over a comparison window (i.e., on an amino acid-to-amino acid, or nucleotide-to-nucleotide basis, respectively). The "percent sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions in the two sequences at which the same amino acid or nucleic acid base (any related) occurs to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to yield the percent sequence identity.

As used herein, the term "substantially identical (substantially identical)" or "substantially identical (substantial identity)" refers to a characteristic of a polypeptide or polynucleotide sequence, wherein the polypeptide or polynucleotide comprises a sequence having at least 80% sequence identity, preferably at least 85% sequence identity, more preferably 90% sequence identity, still more preferably 95% sequence identity, still more preferably 99% sequence identity, as compared to a reference sequence, wherein the percent sequence identity is calculated by aligning the reference sequence to the polypeptide or polynucleotide sequence, and may include deletions or additions of 20% or less of the total amount of the reference sequence over a comparison window. The reference sequence may be a subset of the larger sequence. Optimal alignment of sequences can be performed by conventional software or methods known to those of ordinary skill in the art.

As used herein, the term "corresponds to" means that the polypeptide or polynucleotide sequence is the same as or similar to all or part of the reference polypeptide or polynucleotide sequence. In contrast, the term "complementary" as used herein in relation to a polypeptide or polynucleotide sequence refers to the complementary sequence being homologous to all or part of the reference polypeptide or polynucleotide sequence. For purposes of illustration, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA".

In one embodiment of the invention, a hCC binding agent as detailed above comprises a single domain antibody portion comprising at least one Complementarity Determining Region (CDR) of a single domain antibody portion as described herein, or an amino acid sequence having at least 80% amino acid identity to said CDR sequence, or an amino acid sequence having a 3, 2 or 1 amino acid sequence difference from said CDR sequence. It will be appreciated that the CDRs in the single domain antibody partial sequences and their positions can be readily identified by conventional methods known to those of ordinary skill in the art, such as, but not limited to, KABAT systems (KABAT), chothia, AHo, or international ImMunoGeneTics (IMGT) information systems (ImMunoGeneTics). A preferred method of determining CDR sequences is the IMGT method (Lefranc, M. -P.et al, 2009,Nucleic Acids Research,D1006-1012, http:// www.imgt.org).

As described in the examples below, specific and preferred hCR 8 binding agents according to the invention comprise a single domain antibody portion corresponding to SEQ ID NO 17-21 or 30-32. FIG. 10 shows a schematic representation of the amino acid sequence of SEQ ID NOS.30-32, in which CDRs are identified using the IMGT method (bold) or the Kabat method (underlined).

Thus, using IMGT methods, CDRs identified within a single domain antibody moiety as defined above correspond to:

SEQ ID NO:1(GGIRSIIP)

SEQ ID NO:2(GSIFSLLD)

SEQ ID NO:3(GSIFSLRT)

SEQ ID NO:4(ISTAGSA)

SEQ ID NO:5(ITSGGST)

SEQ ID NO:6(ISAGGAT)

SEQ ID NO:7(NGRQTGWRTRVDY)

SEQ ID NO:8(NAAPYYWGAYRRQES)

SEQ ID NO:9(YAQDSYKIYKSRYTQDY)

SEQ ID NO:10(YAQQSYKIYKSRYTQDY)

SEQ ID NO:11(YAQDTYKIYKSRYTQDY)

SEQ ID NO:22(GNIFSLLD)

alternatively, CDRs identified within a single domain antibody moiety as defined above correspond to:

SEQ ID NO:33(GGIRSIIPAG)

SEQ ID NO:34(GNIFSLLDMK)

SEQ ID NO:35(GSIFSLRTVG)

SEQ ID NO:36(AISTAGSADYADSVKG)

SEQ ID NO:37(DITSGGSTNYADPVKG)

SEQ ID NO:38(TISAGGATDYADSVKV)

SEQ ID NO:43(GSIFSLLDMK)

SEQ ID NO:44(DITSGGSTNYADSVKG)

SEQ ID NO:45(TISAGGATDYADSVKG)

SEQ ID NO:7(NGRQTGWRTRVDY)

SEQ ID NO:8(NAAPYYWGAYRRQES)

SEQ ID NO:9(YAQDSYKIYKSRYTQDY)

SEQ ID NO:10(YAQQSYKIYKSRYTQDY)

SEQ ID NO:11(YAQDTYKIYKSRYTQDY)

furthermore, the CDRs identified within the single domain antibody portion as defined above correspond to those identified using the Kabat method (Kabat, E.A. et al, 1991,Sequences of Proteins of Immunological Interest, fifth edition, NIH Press, no. 91-3242):

SEQ ID NO:75(IIPAG)

SEQ ID NO:76(LLDMK)

SEQ ID NO:77(LRTVG)

SEQ ID NO:36(AISTAGSADYADSVKG)

SEQ ID NO:37(DITSGGSTNYADPVKG)

SEQ ID NO:38(TISAGGATDYADSVKV)

SEQ ID NO:44(DITSGGSTNYADSVKG)

SEQ ID NO:45(TISAGGATDYADSVKG)

SEQ ID NO:78(RQTGWRTRVDY)

SEQ ID NO:79(APYYWGAYRRQES)

SEQ ID NO:80(QDSYKIYKSRYTQDY)

SEQ ID NO:81(QQSYKIYKSRYTQDY)

SEQ ID NO:82(YAQDTYKIYKSRYTQDY)

thus, the single domain antibody portion as detailed above comprises at least one, preferably at least two and most preferably three CDRs selected from SEQ ID NOs 1-11 and SEQ ID No. 22, or at least one, preferably at least two and most preferably three amino acid sequences having at least 80% amino acid identity with said CDR sequences, or at least one, preferably at least two and most preferably three amino acid sequences having a 3, 2 or 1 amino acid sequence difference with said CDR sequences.

Preferably, the single domain antibody portion as detailed above comprises a CDR3 selected from the group consisting of: (a) the amino acid sequence of NGRQTGWRTRVDY (SEQ ID NO: 7); (b) the amino acid sequence of NAAPYYWGAYRRQES (SEQ ID NO: 8); (c) YAQDSYKIYKSRYTQDY (SEQ ID NO: 9); (d)

YAQQSYKIYKSRYTQDY (SEQ ID NO: 10); (e)

YAQDTYKIYKSRYTQDY (SEQ ID NO: 11); (f) An amino acid sequence having at least 80% amino acid sequence identity to SEQ ID No. 7, 8, 9, 10 or 11; and (e) an amino acid sequence having a 3, 2 or 1 amino acid difference from SEQ ID NO 7, 8, 9, 10 or 11. More preferably, CDR3 corresponds to SEQ ID NO 7, 8, 9, 10 or 11.

In a preferred embodiment, CDR1 is selected from: (a) the amino acid sequence of GGIRSIIP (SEQ ID NO: 1); (b) the amino acid sequence of GSIFSLLD (SEQ ID NO: 2); (c) the amino acid sequence of GSIFSLRT (SEQ ID NO: 3); (d) the amino acid sequence of GNIFSLLD (SEQ ID NO: 22); (e) An amino acid sequence having at least 80% amino acid identity to SEQ ID No. 1, 2, 3 or 22; and (f) an amino acid sequence having a 3, 2, 1 amino acid difference from SEQ ID NO 1, 2, 3 or 22; and/or CDR2 is selected from: (a) the amino acid sequence of ISTAGSA (SEQ ID NO: 4); (b) the amino acid sequence of ITSGGST (SEQ ID NO: 5); (c) the amino acid sequence of ISAGGAT (SEQ ID NO: 6); (d) An amino acid sequence having at least 80% amino acid identity to SEQ ID No. 4, 5 or 6; and (e) an amino acid sequence having a 3, 2, 1 amino acid difference from SEQ ID NO. 4, 5 or 6.

In a further specific embodiment, the invention provides an hCCR8 binding agent comprising a combination of CDR1, CDR2 and CDR3 as described herein, including the permissible variations described for these CDR regions. In further specific embodiments, the binding agents of the invention comprise at least one CDR region of a single domain antibody moiety as described herein. In other embodiments, the binding agents of the invention comprise at least one CDR region of a single domain antibody portion having the amino acid sequence of SEQ ID NO. 30, 31 or 32. In other embodiments, the binding agents of the invention comprise three CDR regions of a single domain antibody portion having the amino acid sequence of SEQ ID NO. 30, 31 or 32.

In a more preferred embodiment, the single domain antibody portion as detailed above comprises three CDRs having the sequences SEQ ID NOs 1, 4 and 7, or comprises three CDRs having the sequences SEQ ID NOs 5, 8 and 22, or comprises a single domain antibody having the sequences SEQ ID NOs

Three CDRs of SEQ ID NOs 3, 6 and 9.

In further embodiments of the invention, the single domain antibody portion as detailed above further comprises a sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to at least one Framework Region (FR) of the single domain antibody portion described herein. In further embodiments of the invention, the single domain antibody portion as detailed above further comprises a sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to the four Framework Regions (FR) of the single domain antibody portion described herein. It will be appreciated that the method for determining the FR of the single domain antibody portion is the same as the method used to identify CDRs.

Thus, using IMGT methods, the FR identified within the single domain antibody portion as defined above corresponds to:

SEQ ID NO:12(DVQLVESGGGLVQPGGSLRLSCTVS)

SEQ ID NO:13(VGWYRQAPGKQRELVAT)

SEQ ID NO:14(MKWYRQAPGKQRELVAD)

SEQ ID NO:15(DYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYC)

SEQ ID NO:16(WGQGTLVTVSS)

SEQ ID NO:23(EVQLVESGGGLVQPGGSLRLSCAVS)

SEQ ID NO:24(EVQLVESGGGLVQPGGSLRLSCTAS)

SEQ ID NO:25(EVQLVESGGGLVQPGGSLRLSCTVS)

SEQ ID NO:26(AGWYRQVPGKQRELVAA)

SEQ ID NO:27(DYADSVKGRFTISRDNTKNTAYLQMNSLRPEDTAVYYC)

SEQ ID NO:28(NYADPVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYC)

SEQ ID NO:29(DYADSVKVRFTVSRDNGKNTVYLQMNSLRPEDTAVYYC)

SEQ ID NO:39(DVQLVESGGGLVQPGGSLRLSCAVS)

SEQ ID NO:40(DVQLVESGGGLVQPGGSLRLSCTAS)

SEQ ID NO:41(AGWYRQAPGKQRELVAA)

SEQ ID NO:42(NYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYC)

thus, a single domain antibody moiety as described above comprises at least one, preferably at least two, more preferably at least three and most preferably four amino acid sequences having at least 85%, preferably 90%, more preferably 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 12-16, 23-29 and 39-42.

Preferably, the single domain antibody portion as detailed above comprises four Framework Regions (FRs) according to the form FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 has at least 85%, preferably 90%, more preferably 95% sequence identity to the sequence of SEQ ID NO:12, 23-25 or 39-40, FR2 has at least 85%, preferably 90%, more preferably 95% sequence identity to the sequence of SEQ ID NO:13-14, 26 or 41, FR3 has at least 85%, preferably 90%, more preferably 95% sequence identity to the sequence of SEQ ID NO:15, 27-29 or 42, and FR4 has at least 85%, preferably 90%, more preferably 95% sequence identity to the sequence of SEQ ID NO: 16.

In further specific embodiments, the binding agents of the invention comprise an antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID No. 30, 31 or 32, or an amino acid sequence having 85%, 90% or 95% sequence identity thereto; wherein the binding agent comprises CDR1 of SEQ ID NO 1, 3 or 22, CDR2 of SEQ ID NO 4, 5 or 6 and CDR3 of SEQ ID NO 7, 8 or 9.

In a specific embodiment, the binding agent of the invention comprises an amino acid sequence corresponding to SEQ ID NO. 30. In a further specific embodiment, the binding agent of the invention comprises an amino acid sequence corresponding to SEQ ID NO. 31. In a further specific embodiment, the binding agent of the invention comprises an amino acid sequence corresponding to SEQ ID NO. 32.

Sequence optimized murine cross-reactive hCCR8 binding agent

As used herein, the term "humanized binding agent" refers to a binding agent that is produced by molecular modeling techniques to determine the optimal combination of human and non-human (e.g., mouse or rabbit) binding agent sequences, i.e., a combination in which the human component of the binding agent is maximized while causing little or no loss of binding affinity due to the variable region of the non-human antibody. For example, humanized antibodies, also known as chimeric antibodies, comprise amino acid sequences of a human framework region and constant regions from a human antibody to "humanize" or render Complementary Determining Regions (CDRs) from a non-human antibody non-immunogenic.

As used herein, the term "human binding agent" refers to a binding agent having an amino acid sequence that corresponds to an amino acid sequence of a binding agent that can be produced by a human and/or prepared using any technique known to those skilled in the art or disclosed herein for preparing human antibodies. It is also understood that the term "human antibody" includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising a murine light chain and a human heavy chain polypeptide.

For antibodies of full size, a single variable structureDomains (e.g., VHH and) Sequence optimization may be performed, for example, humanization (i.e., increasing the degree of sequence identity to the closest human germline sequence) and other optimization techniques (e.g., improving the physicochemical or other properties of the binding agent). In particular, humanized immunoglobulin single variable domains, (e.g., VHH and +.>) May be a single domain antibody in which at least one single amino acid residue (particularly at least one framework residue) is present, which amino acid residue is and/or corresponds to a humanized substitution (as further defined herein). />

Humanized single domain antibodies, in particular VHH andthere may be several advantages, such as reduced immunogenicity, compared to the corresponding naturally occurring VHH domain. Humanization refers to mutation such that immunogenicity is less or absent when administered to a human patient. The humanized substitutions are selected such that the resulting humanized amino acid sequence and/or VHH still retain the advantageous properties of the VHH, such as antigen binding ability.

In a specific embodiment, the cross-reactive hCCR8 binding agent as described above is an optimized cross-reactive hCCR8 binding agent.

Preferably, the optimised cross-reactive hCCR8 binding agent comprises a single domain antibody moiety as described above. More preferably, the single domain antibody is optimized by introducing a mutation, in particular a substitution, for example at any of positions 1, 40, 74 and/or 78 of SEQ ID NO. 30, or at any of positions 1, 27 and/or 62 of SEQ ID NO. 31, or at any of positions 1, 65, 69, 74, 99 and/or 100 of SEQ ID NO. 32.

These residues are highlighted in figure 10. These sequence-optimizing mutations were found to have substantially no effect on target binding parameters. The E1D mutation in all SEQ ID NOS: 30-32 was found to eliminate pyroglutamic acid formation. Furthermore, the mutation D99Q or S100T in SEQ ID NO. 32 was found to reduce aspartate isomerisation. Interestingly, mutations T74A and A78V in SEQ ID NO. 30 were found to improve the thermostability of the binding agent. As shown in the examples below, it was also found that substitution of asparagine residue (N) with serine (S) at position 27 of SEQ ID NO. 31 avoids deamidation when the binding agent is stored at 40 ℃. In addition, it was found that substitution of proline (P) with serine (S) at position 62 of SEQ ID NO:31 increases the thermostability of the binding agent. Similarly, substitution of valine (V) with glycine (G) at position 65 of SEQ ID NO:32 also increases the thermostability of the binding agent. The additional amino acid substitutions listed above are primarily used to further humanize the binding agent.

Thus, in a specific embodiment, the invention provides a binding agent comprising the amino acid sequence of SEQ ID NO. 30, optionally comprising one or more of substitutions E1D, T74A, A V and V40A. In other embodiments, the binding agent comprising the amino acid sequence of SEQ ID NO. 31 optionally comprises a substitution of one or more of E1D, N S and P62S. In a specific embodiment, the binding agent comprising the amino acid sequence of SEQ ID NO. 31 comprises the substitution N27S and optionally one or more further substitutions, such as E1D or P62S. In other embodiments, the binding agent comprising the amino acid sequence of SEQ ID NO. 32 optionally comprises one or more of substitutions E1D, V65G, V69I and G74A. In other specific embodiments, the binding agent comprising the amino acid sequence of SEQ ID NO. 32 comprises the substitution D99Q and optionally one or more additional substitutions, such as those selected from E1D, V65G, V I and G74A. In further embodiments, the binding agent comprising the amino acid sequence of SEQ ID NO. 32 optionally comprises one or more of substitutions E1D, V65G, V69I, G A and D99Q. In further embodiments, the binding agent comprising the amino acid sequence of SEQ ID NO. 32 optionally comprises one or more of substitutions E1D, V65G, V69I, G A and S100T.

In a further embodiment, the binding agent of the invention comprises at least one CDR region of a single domain antibody portion having the amino acid sequence of SEQ ID NO. 17. In other embodiments, the binding agents of the invention comprise three CDR regions of a single domain antibody portion having the amino acid sequence of SEQ ID NO. 17. In a further embodiment, the binding agent of the invention comprises at least one CDR region of a single domain antibody portion having the amino acid sequence of SEQ ID NO. 18. In other embodiments, the binding agents of the invention comprise three CDR regions of a single domain antibody portion having the amino acid sequence of SEQ ID NO. 18. In a further embodiment, the binding agent of the invention comprises at least one CDR region of a single domain antibody portion having the amino acid sequence of SEQ ID NO. 19. In other embodiments, the binding agents of the invention comprise three CDR regions of a single domain antibody portion having the amino acid sequence of SEQ ID NO. 19. In a further embodiment, the binding agent of the invention comprises at least one CDR region of a single domain antibody portion having the amino acid sequence of SEQ ID NO. 20. In other embodiments, the binding agents of the invention comprise three CDR regions of a single domain antibody portion having the amino acid sequence of SEQ ID NO. 20. In a further embodiment, the binding agent of the invention comprises at least one CDR region of a single domain antibody portion having the amino acid sequence of SEQ ID NO. 21. In other embodiments, the binding agents of the invention comprise three CDR regions of a single domain antibody portion having the amino acid sequence of SEQ ID NO. 21.

However, the amino acid sequences and/or the single domain antibodies of the invention may be suitably optimized at any position, in particular at any framework residue, e.g. at one or more tag residues (as defined above) or at one or more other framework residues (i.e. non-tag residues) or any suitable combination thereof. Such deletions and/or substitutions may also be designed in a manner that removes one or more sites of post-translational modification (e.g., one or more glycosylation sites), depending on the host organism used to express the amino acid sequences, single domain antibodies, or polypeptides of the invention, as is well within the ability of those skilled in the art. Alternatively, substitutions or insertions may be designed to introduce one or more sites for attachment of functional groups (as described herein), for example to allow site-specific pegylation.

In one embodiment of the invention, an optimized cross-reactive hCC binding agent as described above comprises a single domain antibody moiety comprising at least one Complementarity Determining Region (CDR) of a single domain antibody moiety as described herein, or an amino acid sequence having at least 80% amino acid identity to said CDR sequence, or an amino acid sequence having a 3, 2 or 1 amino acid sequence difference from said CDR sequence. It will be appreciated that the CDRs in the single domain antibody partial sequences and their positions can be readily identified by conventional methods known to those of ordinary skill in the art, such as, but not limited to, KABAT systems (KABAT), chothia, AHo, or international ImMunoGeneTics (IMGT) information systems (ImMunoGeneTics). A preferred method of determining CDR sequences is the IMGT method (Lefranc, M. -P.et al, 2009,Nucleic Acids Research,D1006-1012, http:// www.imgt.org).

Five specifically optimized hCCR8 binding agents of the invention comprise a single domain antibody portion corresponding to SEQ ID NOs 17, 18, 19, 20 or 21, as described in the examples below.

SEQ ID NO:1(GGIRSIIP)

SEQ ID NO:2(GSIFSLLD)

SEQ ID NO:3(GSIFSLRT)

SEQ ID NO:4(ISTAGSA)

SEQ ID NO:5(ITSGGST)

SEQ ID NO:6(ISAGGAT)

SEQ ID NO:7(NGRQTGWRTRVDY)

SEQ ID NO:8(NAAPYYWGAYRRQES)

SEQ ID NO:9(YAQDSYKIYKSRYTQDY)

SEQ ID NO:10(YAQQSYKIYKSRYTQDY)

SEQ ID NO:11(YAQDTYKIYKSRYTQDY)

SEQ ID NO:33(GGIRSIIPAG)

SEQ ID NO:35(GSIFSLRTVG)

SEQ ID NO:36(AISTAGSADYADSVKG)

SEQ ID NO:43(GSIFSLLDMK)

SEQ ID NO:44(DITSGGSTNYADSVKG)

SEQ ID NO:45(TISAGGATDYADSVKG)

SEQ ID NO:7(NGRQTGWRTRVDY)

SEQ ID NO:8(NAAPYYWGAYRRQES)

SEQ ID NO:9(YAQDSYKIYKSRYTQDY)

SEQ ID NO:10(YAQQSYKIYKSRYTQDY)

SEQ ID NO:11(YAQDTYKIYKSRYTQDY)

furthermore, using the Kabat method, CDRs identified within the single domain antibody moiety as defined above correspond to:

SEQ ID NO:75(IIPAG)

SEQ ID NO:76(LLDMK)

SEQ ID NO:77(LRTVG)

SEQ ID NO:36(AISTAGSADYADSVKG)

SEQ ID NO:44(DITSGGSTNYADSVKG)

SEQ ID NO:45(TISAGGATDYADSVKG)

SEQ ID NO:78(RQTGWRTRVDY)

SEQ ID NO:79(APYYWGAYRRQES)

SEQ ID NO:80(QDSYKIYKSRYTQDY)

SEQ ID NO:81(QQSYKIYKSRYTQDY)

SEQ ID NO:82(YAQDTYKIYKSRYTQDY)

thus, the single domain antibody portion as detailed above comprises at least one, preferably at least two and most preferably three CDRs selected from the group consisting of SEQ ID NOS: 1-11, or at least one, preferably at least two and most preferably three amino acid sequences having at least 80% amino acid identity to said CDR sequences, or at least one, preferably at least two and most preferably three amino acid sequences having a 3, 2 or 1 amino acid sequence difference from said CDR sequences.

Preferably, the single domain antibody portion as detailed above comprises a CDR3 selected from the group consisting of: (a) the amino acid sequence of NGRQTGWRTRVDY (SEQ ID NO: 7); (b) the amino acid sequence of NAAPYYWGAYRRQES (SEQ ID NO: 8); (c) YAQDSYKIYKSRYTQDY (SEQ ID NO: 9); (d) YAQQSYKIYKSRYTQDY (SEQ ID NO: 10); (e) YAQDTYKIYKSRYTQDY (SEQ ID NO: 11); (f) An amino acid sequence having at least 80% amino acid sequence identity to SEQ ID No. 7, 8, 9, 10 or 11; and (e) an amino acid sequence having a 3, 2 or 1 amino acid difference from SEQ ID NO 7, 8, 9, 10 or 11. More preferably, CDR3 corresponds to SEQ ID NO 7, 8, 9, 10 or 11.

In a preferred embodiment, CDR1 is selected from: (a) GGIRSIIP (amino acid sequence of SEQ ID NO: 1); (b) the amino acid sequence of GSIFSLLD (SEQ ID NO: 2); (c) the amino acid sequence of GSIFSLRT (SEQ ID NO: 3); (d) An amino acid sequence having at least 80% amino acid identity to SEQ ID No. 1, 2 or 3; and (e) an amino acid sequence having a 3, 2, 1 amino acid difference from SEQ ID NO 1, 2 or 3; CDR2 is selected from: (a) the amino acid sequence of ISTAGSA (SEQ ID NO: 4); (b) the amino acid sequence of ITSGGST (SEQ ID NO: 5); (c) the amino acid sequence of ISAGGAT (SEQ ID NO: 6); (d) An amino acid sequence having at least 80% amino acid identity to SEQ ID No. 4, 5 or 6; and (e) an amino acid sequence having a 3, 2, 1 amino acid difference from SEQ ID NO. 4, 5 or 6.

In a more preferred embodiment, the single domain antibody portion as detailed above comprises three CDRs having the sequences of SEQ ID NOS: 1, 4 and 7, or having the sequences of SEQ ID NOS: 2, 5 and 8, or having the sequences of SEQ ID NOS: 3, 6 and 9, or having the sequences of SEQ ID NOS: 3, 6 and 10, or having the sequences of SEQ ID NOS: 3, 6 and 11.

In further embodiments of the invention, the single domain antibody portion as detailed above further comprises a sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to at least one Framework Region (FR) of the single domain antibody portion described herein. It will be appreciated that the method for determining the FR of the single domain antibody portion is the same as the method used to identify CDRs.

SEQ ID NO:12(DVQLVESGGGLVQPGGSLRLSCTVS)

SEQ ID NO:13(VGWYRQAPGKQRELVAT)

SEQ ID NO:14(MKWYRQAPGKQRELVAD)

SEQ ID NO:15(DYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYC)

SEQ ID NO:16(WGQGTLVTVSS)

SEQ ID NO:39(DVQLVESGGGLVQPGGSLRLSCAVS)

SEQ ID NO:40(DVQLVESGGGLVQPGGSLRLSCTAS)

SEQ ID NO:41(AGWYRQAPGKQRELVAA)

SEQ ID NO:42(NYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYC)

thus, the single domain antibody portion as detailed above comprises at least one, preferably at least two, more preferably at least three and most preferably four amino acid sequences having at least 85%, preferably 90%, more preferably 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 12-16 and 39-42.

Preferably, the single domain antibody portion as detailed above comprises four Framework Regions (FRs) according to the form FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 has at least 85%, preferably 90%, more preferably 95% sequence identity to the sequence of SEQ ID NO:12, 39 or 40, FR2 has at least 85%, preferably 90%, more preferably 95% sequence identity to the sequence of SEQ ID NO:13, 14 or 41, FR3 has at least 85%, preferably 90%, more preferably 95% sequence identity to the sequence of SEQ ID NO:15 or 42, and FR4 has at least 85%, preferably 90%, more preferably 95% sequence identity to the sequence of SEQ ID NO: 16.

More preferably, the single domain antibody portion as detailed above comprises an amino acid sequence corresponding to SEQ ID NO. 17, 18, 19, 20 or 21. In a specific embodiment, the binding agent of the invention comprises the amino acid sequence of SEQ ID NO. 17. In a further specific embodiment, the binding agent of the invention comprises the amino acid sequence of SEQ ID NO. 18. In a further specific embodiment, the binding agent of the invention comprises the amino acid sequence of SEQ ID NO. 19. In a further specific embodiment, the binding agent of the invention comprises the amino acid sequence of SEQ ID NO. 20. In a further specific embodiment, the binding agent of the invention comprises the amino acid sequence of SEQ ID NO. 21.

In further specific embodiments, the binding agents of the invention comprise an antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID No. 17 or an amino acid sequence having 85%, 90% or 95% sequence identity thereto, wherein the binding agent comprises CDR1 of SEQ ID No. 1, CDR2 of SEQ ID No. 4 and CDR3 of SEQ ID No. 7.

In further specific embodiments, the binding agents of the invention comprise an antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO. 18 or an amino acid sequence having 85%, 90% or 95% sequence identity thereto, wherein the binding agent comprises CDR1 of SEQ ID NO. 2, CDR2 of SEQ ID NO. 5 and CDR3 of SEQ ID NO. 8.

In further specific embodiments, the binding agents of the invention comprise an antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO. 19 or an amino acid sequence having 85%, 90% or 95% sequence identity thereto, wherein the binding agent comprises CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 6 and CDR3 of SEQ ID NO. 9.

In further specific embodiments, the binding agents of the invention comprise an antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO. 20 or an amino acid sequence having 85%, 90% or 95% sequence identity thereto, wherein the binding agent comprises CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 6 and CDR3 of SEQ ID NO. 10.

In further specific embodiments, the binding agents of the invention comprise an antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO. 21 or an amino acid sequence having 85%, 90% or 95% sequence identity thereto, wherein the binding agent comprises CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 6 and CDR3 of SEQ ID NO. 11.

In a further aspect, the invention provides a cross-reactive binding agent, e.g. an antibody or antigen binding fragment thereof, which competes for specific binding to hCCR8 with a binding agent described herein, in particular with a hCCR8 single domain antibody moiety having the amino acid sequence of SEQ ID NO 17, 18, 19, 20 or 21. Thus, in a particular embodiment, the invention provides a cross-reactive CCR8 binding agent which competes for specific binding to hCCR8 with a single domain antibody having the amino acid sequence of SEQ ID No. 17, 18, 19, 20 or 21. In a specific embodiment, the cross-reactive binding agent competes for specific binding to hCCR8 and murine CCR 8. Whether a binding agent competes with the binding agent described herein for specific binding to hCCR8 can be readily determined using conventional methods known in the art. For example, to determine whether detecting binding agents compete, the binding agents of the invention are allowed to bind CCR8 protein under saturated conditions. Next, the ability to detect binding agents was evaluated. If the detection binding agent is unable to bind to the CCR8 protein, it can be inferred that the detection antibody competes with the binding agent of the invention for specific binding to CCR 8.

Cytotoxicity of cells

Another aspect of the invention is to provide a human CCR8 binding agent with cytotoxic activity. As used herein, "cytotoxic" or "cytotoxic activity" refers to the ability of a binding agent to be toxic to the cells to which it binds. As will be clear to the skilled person from the description of the invention, any type of cytotoxicity may be used in the context of the present invention. Cytotoxicity may be direct cytotoxicity, where the binding agent itself directly damages the cells (e.g., because it contains a chemotherapy payload), or it may be indirect, where the binding agent induces extracellular mechanisms that result in cell damage (e.g., antibodies that induce antibody-dependent cellular activity). More specifically, the binding agents of the invention may signal the immune system to destroy or eliminate the cells to which they bind, or the binding agents may carry a cytotoxic payload to destroy the cells to which they bind. In particular, the cytotoxic activity is due to the presence of cytotoxic moieties. Examples of such cytotoxic moieties include moieties that induce antibody-dependent cellular Activity (ADCC), induce antibody-dependent cellular phagocytosis (ADCP), induce complement-dependent cytotoxicity (CDC), bind and activate T cells, or contain a cytotoxic payload. Most preferably, the cytotoxic moiety induces antibody dependent cellular Activity (ADCC).

Antibody-dependent cellular cytotoxicity (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors recognize binding agents on target cells and subsequently cause lysis of the target cells. Examples of nonspecific cytotoxic cells that express Fc receptors include natural killer cells, neutrophils, monocytes and macrophages.

Complement Dependent Cytotoxicity (CDC) refers to the cleavage of a target in the presence of complement. The complement activation pathway is initiated by binding of the first component of the complement system (C1 q) to a binding agent complexed to a cognate antigen.

Antibody-dependent cellular phagocytosis (ADCP) refers to a cell-mediated response in which phagocytes (e.g., macrophages) expressing Fc receptors recognize a binding agent on a target cell, resulting in phagocytosis of the target cell.

CDC, ADCC, and ADCP can be measured using assays known in the art (Vafa et al, methods2014.1.1;65 (1): 114-26 (2013)).

Binding to and activating T cells refers to binding to a T cell marker different from hCCR8 and activation of said T cells thereby produced. Activation of T cells induces cytotoxic activity of T cells against cells to which the binding agents of the invention bind. Thus, in a specific embodiment, the binding agent of the invention binds hCCR8 and binds and activates T cells. For example, the cytotoxic moiety may bind hCD3. In other embodiments, the cytotoxic moiety comprises an antibody or antigen binding fragment thereof that binds hCD3. Thus, the binding agents of the invention bind hCCR8 and hCD3. Such binders bind to intratumoral tregs and direct the cytotoxic activity of T cells to these tregs, thereby clearing them from the tumor environment. In a specific embodiment, the binding agent of the invention comprises a moiety that binds hCCR8 and a moiety that binds hCD3, wherein at least one moiety is antibody-based, in particular wherein both moieties are antibody-based. Thus, in a particular embodiment, the invention provides a bispecific construct comprising an antibody or antigen-binding fragment thereof that specifically binds hCCR8 and an antibody or antigen-binding fragment thereof that specifically binds hCD3.

A cytotoxic payload refers to any molecular entity that causes direct damage to cells in contact with the cytotoxic payload. Cytotoxic payloads are known to those skilled in the art. In a specific embodiment, the cytotoxic payload is a chemical entity. Specific examples of such cytotoxic payloads include toxins, chemotherapeutic agents, and radioisotopes or radionuclides. In other embodiments, the cytotoxic payload comprises an agent selected from the group consisting of: alkylating agents, anthracyclines, cytoskeletal disrupting agents, epothilones, histone deacetylase inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, kinase inhibitors, nucleotide analogs and precursor analogs, peptide antibiotics, platinum-based agents, retinoids, vinca alkaloids and derivatives, peptide or small molecule toxins, and radioisotopes. The chemical entity may be coupled to a proteinaceous inhibitor, such as an antibody or antigen-binding fragment, using techniques known in the art. Such couplings may be covalent or non-covalent, and the coupling may be labile or reversible.

As is well known in the art, the Fc region of IgG antibodies interacts with several cellular fcγ receptors (fcγrs) to stimulate and regulate downstream effector mechanisms. There are five activating receptors, fcyri (CD 64), fcyriia (CD 32 a), fcyriic (CD 32 c), fcyriiia (CD 16 a) and fcyriiib (CD 16 b), and one inhibitory receptor fcyriib (CD 32 b). Communication of IgG antibodies with the immune system is controlled and mediated by fcγr, which conveys information sensed and collected by antibodies to the immune system, providing a link between the innate and adaptive immune systems, particularly in the case of biotherapy (Hayes J et al, 2016.J Inflamm Res 9:209-219).

IgG subclasses vary in their ability to bind fcγr, and this differential binding determines their ability to elicit a range of functional responses. For example, in humans fcγriiia is the primary receptor involved in the activation of antibody dependent cell mediated cytotoxicity (ADCC), and IgG3 and the immediately following IgG1 show the highest affinity for this receptor, reflecting their ability to effectively induce ADCC. While IgG2 has been shown to bind poorly to this receptor, binding agents with human IgG2 isotypes have also been found to be effective in eliminating tregs.

In a preferred embodiment of the invention, the binding agent of the invention induces antibody effector function, in particular in humans. In a specific embodiment, the binding agents of the invention bind fcγr with high affinity, preferably binding to the activating receptor with high affinity. Preferably, the binding agent binds fcyri and/or fcyriia and/or fcyriiia with high affinity. Particularly preferably, the binding agent binds fcγriiia. In particular embodiments, the binding agent is present in an amount of less than about 10 ^-6 M、10 ^-7 M、10 ^-8 M、10 ^-9 M、10 ^-10 M、10 ^-11 M、10 ^-12 M or 10 ^-13 The dissociation constant of M binds to at least one activated fcγ receptor. Fcγr binding can be achieved in several ways. For example, the cytotoxic moiety may comprise a crystallizable fragment (Fc) region portion or it may comprise a binding moiety, such as an antibody or antigen binding portion thereof that specifically binds fcγr.

In a specific embodiment, the cytotoxic moiety comprises a crystallizable fragment (Fc) region portion. Preferably, the Fc region portion is an IgG Fc domain derived from IgG1, igG2, igG3 and IgG4 antibodies. More preferably, the Fc region portion is an IgG Fc domain derived from a human IgG1 antibody. More preferably, the Fc region portion is an IgG Fc domain derived from a short hinge variant of a human IgG1 antibody. In specific embodiments, the Fc region portion comprises the amino acid sequence of SEQ ID NO:71 or an amino acid sequence having at least 80% sequence identity (e.g., at least 85% sequence identity or 90% sequence identity). In other embodiments, the Fc region portion comprises the amino acid sequence of SEQ ID NO. 71 or an amino acid sequence having at least 95%, 96%, 97% sequence identity, particularly at least 98% sequence identity, more particularly at least 99% sequence identity.

In one embodiment, the Fc region portion has been engineered to increase ADCC, ADCP and/or CDC activity.

ADCC may be increased by methods of reducing or eliminating the fucose portion of an Fc portion glycan and/or by introducing specific mutations (e.g., S298A/E333/K334A, S239D/I332E/a330L or G236A/S239D/a 330L/I332E) in the Fc region of an immunoglobulin (e.g., igG 1) (Lazar et al Proc Natl Acad Sci USA 103:2005-2010 (2006); smith et al Proc Natl Acad Sci USA 209:6181-6 (2012)). ADCP may also be increased by introducing specific mutations in the Fc portion of human IgG (Richards et al Mol Cancer Ther 7:2517-27 (2008)). Methods for engineering binders to increase ADCC, CDC and ADCP activity are described in Saunders (Frontiers in Immunology 2019,1296) and Wang et al (Protein Cell2019, 9:63-73).

In a specific embodiment of the invention, the binding agent comprising the Fc region portion is optimized to elicit an ADCC response, i.e. the ADCC response is enhanced, increased or improved relative to other hCCR8 binding agents comprising the Fc region portion, including those having cross-reactivity with murine CCR8 and e.g. unmodified anti-CCR 8 monoclonal antibodies. In a preferred embodiment, the hCCR8 binding agent has been engineered to elicit an enhanced ADCC response.

In a preferred embodiment of the invention, the binding agent comprising the Fc region portion is optimised to elicit an ADCP response, i.e. the ADCP response is enhanced, increased or improved relative to other hCCR8 binding agents comprising the Fc region portion, including those having cross-reactivity with murine CCR8 and, for example, unmodified anti-hCCR 8 monoclonal antibodies.

In further embodiments, the cytotoxic moiety comprises a moiety that binds to an fcγ receptor. More particularly fcγr, in particular activating a receptor, such as fcγri and/or fcγriia and/or fcγriiia, in particular fcγriiia. The moiety that binds to fcγr may be antibody-based or non-antibody-based, as described above. If based on antibodies, the moiety may bind fcγr via its variable region.

In other embodiments of the invention, the hCCR8 binding agent as detailed above comprises at least one Fc region portion and a single domain antibody portion that binds hCCR8 as detailed above.

In one embodiment of the invention, the hCCR8 binding agent is a genetically engineered polypeptide comprising at least one Fc region portion and a single domain antibody portion that binds hCCR8, linked together by a direct bond.

In further embodiments, hCCR8 bindingAn agent is a genetically engineered polypeptide comprising at least one Fc region portion and a single domain antibody portion that binds hCCR8, linked together by a direct bond or linker. Preferably, the linker is a peptide linker. Preferably, the linker is a flexible linker having an amino acid sequence consisting essentially of an extension of glycine (G) and serine (S) residues (i.e., a so-called "GS" or "GlySer" linker). In other embodiments, at least 80%, particularly at least 85%, more particularly at least 90% of the amino acid residues in the peptide linker are selected from glycine and serine. In a preferred embodiment, at least 95% of the amino acid residues in the peptide linker are selected from glycine and serine. In further specific embodiments, the peptide linker comprises 1 to 50 amino acids, e.g. 1 to 40, especially 1 to 30. In a specific embodiment, 5 to 25 amino acids, preferably 8 to 22 amino acids, e.g. 10 to 20 amino acids. A preferred example of such a GS linker comprises the sequence of GGGGS (SEQ ID NO: 46). In such linkers, the sequence of SEQ ID NO:46 may be repeated "n" times to optimize the length of the GS linker to achieve the appropriate properties of the binding agent so that the sequence of the linker will be (SEQ ID NO: 46) _n Is a sequence of (a). Typically the copy number "n" ranges from 1 to 10, or from 2 to 4. The amino acid sequences of the Fc region portion and/or the single domain antibody portion regions may be humanized to reduce immunogenicity to humans.

Thus, in a particular embodiment, the hCCR8 binding agent of the invention has the formula B-L-C; wherein B refers to an hCCR8 binding moiety as described herein, L refers to a linker as described herein, and C refers to a cytotoxic moiety as described herein. As will be appreciated from the disclosure herein, preferably B comprises a single domain antibody moiety that binds hCR 8, L is a direct bond or has a sequence (SEQ ID NO: 46) _n Wherein n is an integer from 1 to 10 and C is the Fc region portion.

In one embodiment, the hCR 8 binding agent of the invention has the formula B-L-C, wherein B is a single domain antibody moiety corresponding to SEQ ID NO:17-21 or 30-32 and L is a single domain antibody moiety corresponding to (SEQ ID NO: 46) _n Wherein "n" ranges from 1 to 10 and C is the Fc region portion.

Preferably, the hCR 8 junction of the inventionThe cocktail has the formula B-L-C, wherein B is a single domain antibody moiety corresponding to SEQ ID NO:17-21 or 30-32 and L is a single domain antibody moiety corresponding to (SEQ ID NO: 46) _n Wherein "n" is 2 or 4 and C is an IgG Fc domain derived from a short hinge variant of a human IgG1 antibody.

More preferably, the hCCR8 binding agent as detailed above comprises an amino acid sequence corresponding to any one of SEQ ID NOs 55 to 70.

In other embodiments, the hCR 8 binding agent of the invention has the formula B-C, wherein B is a single domain antibody moiety corresponding to SEQ ID NO:17-21 or 30-32, and C is an Fc region moiety.

Preferably, the hCR 8 binding agent of the invention has the formula B-C, wherein B is a single domain antibody moiety corresponding to SEQ ID NO:17-21 or 30-32, and C is an IgG Fc domain derived from a short hinge variant of a human IgG1 antibody.

More preferably, the hCCR8 binding agent as detailed above comprises an amino acid sequence corresponding to any one of SEQ ID NOs 47 to 54.

In a specific embodiment, as previously described, and C comprises the sequence of SEQ ID NO: 71.

In other embodiments, the invention provides nucleic acid molecules encoding hCCR8 binding agents as defined herein. In some embodiments, such provided nucleic acid molecules may contain codon optimized nucleic acid sequences. In further embodiments, the nucleic acid is included in an expression cassette within a suitable nucleic acid vector for expression in a host cell (e.g., bacterial, yeast, insect, fish, murine, simian, or human cell). In some embodiments, the invention provides host cells comprising a heterologous nucleic acid molecule (e.g., a DNA vector) that expresses a desired binding agent.

In particular embodiments, the binding agents of the invention are administered as therapeutic nucleic acids. The term "therapeutic nucleic acid" as used herein refers to any nucleic acid molecule that has a therapeutic effect when introduced into a eukaryotic organism (e.g., a mammal such as a human) and includes DNA and RNA molecules encoding the binding agents of the invention. As known to those of skill in the art, a nucleic acid may comprise an element that induces transcription and/or translation of the nucleic acid or increases the in vitro and/or in vivo stability of the nucleic acid.

In some embodiments, the invention provides a method of preparing an isolated hCCR8 binding agent as defined above. In some embodiments, such methods may comprise culturing a host cell comprising a nucleic acid (e.g., a heterologous nucleic acid that may comprise and/or be delivered to the host cell by a vector). Preferably, the host cells (and/or heterologous nucleic acid sequences) are arranged and constructed such that the binding agent is secreted from the host cells and isolated from the cell culture supernatant.

Treatment of

hCCR8 binding agents having the characteristics described herein represent a further object of the invention. hCCR8 binding agents are useful as pharmaceuticals. In other embodiments, the invention provides a method of treating a disease in an individual comprising administering an hCCR8 binding agent having cytotoxic activity, the binding agent being cross-reactive with murine CCR 8. Preferably, the disease is cancer, in particular a solid tumor.

In a preferred embodiment of the invention, the subject of aspects of the invention described herein is a mammal, preferably a cat, dog, horse, donkey, sheep, pig, goat, cow, hamster, mouse, rat, rabbit or guinea pig, but most preferably the subject is a human. Thus, in all aspects of the invention described herein, the individual is preferably a human.

As used herein, the terms "cancer," "cancerous," or "malignant" refer to or describe the physiological condition of a mammal that is typically characterized by unregulated cell growth.

As used herein, the term "tumor" when applied to an individual diagnosed with or suspected of having cancer refers to malignant or potentially malignant tumor or tissue mass of any size, and includes primary and secondary tumors. The terms "cancer," "malignancy," "neoplasm," "tumor," and "carcinoma" are also used interchangeably herein to refer to tumors and tumor cells that exhibit an abnormal growth phenotype characterized by a significant loss of control of cell proliferation. Generally, cells of interest for treatment include pre-cancerous (e.g., benign), malignant, pre-metastatic, and non-metastatic cells. The teachings of the present invention can be associated with any and all tumors.

Examples of tumors include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma, and sarcoma. More specific examples of such cancers include squamous cell carcinoma, myeloma, small-cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (HCC), hodgkin's lymphoma, non-hodgkin's lymphoma, acute Myelogenous Leukemia (AML), anaplastic Large Cell Lymphoma (ALCL), cutaneous T-cell lymphoma (CTCL), adult T-cell leukemia/lymphoma (ATLL), multiple myeloma, gastrointestinal (gastrointestinal) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, colorectal cancer, endometrial cancer, renal cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon cancer, and head and neck cancer.

In one aspect, the tumor is a solid tumor. Examples of solid tumors are sarcomas (including cancers produced by transformed cells of mesenchymal origin in tissue (e.g., cancellous bone, cartilage, fat, muscle, blood vessels, hematopoietic or fibrous connective tissue), carcinomas (including tumors produced by epithelial cells), mesotheliomas, neuroblastomas, retinoblastomas, and the like. Tumors that involve solid tumors include, but are not limited to, brain, lung, stomach, duodenum, esophagus, breast, colorectal, kidney, bladder, kidney, pancreas, prostate, ovary, melanoma, oral, sarcoma, eye, thyroid, urethra, vagina, neck, lymphoma, and the like.

In further specific embodiments, the tumor is selected from the group consisting of breast invasive cancer, colon adenocarcinoma, head and neck squamous cell carcinoma, gastric adenocarcinoma, lung adenocarcinoma (NSCLC), lung squamous cell carcinoma (NSCLC), renal clear cell carcinoma, skin melanoma, esophageal cancer, cervical cancer, hepatocellular carcinoma, merck cell carcinoma (Merkel cell carcinoma), small Cell Lung Carcinoma (SCLC), classical hodgkin lymphoma (cHL), urothelial carcinoma, microsatellite highly unstable (MSI-H) carcinoma, and mismatch repair deficient (dMMR) carcinoma.

In other embodiments, the tumor is selected from the group consisting of breast cancer, endometrial cancer, lung cancer, gastric cancer, head and neck squamous cell carcinoma, skin cancer, colorectal cancer, and renal cancer. In other embodiments, the tumor is selected from the group consisting of breast invasive cancer, colon adenocarcinoma, head and neck squamous cell carcinoma, gastric adenocarcinoma, lung adenocarcinoma (NSCLC), lung squamous cell carcinoma (NSCLC), renal clear cell carcinoma, and skin melanoma. In one aspect, the cancer involves CCR8 expressing tumors including, but not limited to, breast, endometrial, lung, gastric, head and neck squamous cell carcinoma, skin, colorectal and renal cancers. In specific embodiments, the tumor is selected from breast cancer, colon adenocarcinoma, and lung cancer.

In a specific embodiment, the tumor is a T cell lymphoma, particularly CCR8 expressing T cell lymphoma, including but not limited to adult T cell leukemia/lymphoma (ATLL), cutaneous T Cell Lymphoma (CTCL), and Anaplastic Large Cell Lymphoma (ALCL).

In other embodiments, the tumor is a tumor that carries a recurrent chromosomal rearrangement involving the DUSP22-IRF4 locus on 6p25.3 (i.e., a so-called DUSP22 rearrangement). Preferably, the tumor is a lymphoma bearing a DUSP22 rearrangement.

As used herein, the term "administration" refers to the act of administering a drug, prodrug, antibody, or other agent or therapeutic treatment to a physiological system (e.g., an individual or in vivo, in vitro, or ex vivo cells, tissues, and organs). Exemplary routes of administration to the human body may be through the mouth (oral), skin (transdermal), oral mucosa (buccal), ear, by injection (e.g., intravenous, subcutaneous, intratumoral, intraperitoneal, etc.), and the like. The term administration of the binding agents of the invention includes direct administration of the binding agent as well as indirect administration by administration of nucleic acid encoding the binding agent such that the binding agent is produced by the nucleic acid in the individual. Thus, administration of the binding agent includes DNA and RNA therapeutic methods that result in the production of the binding agent in vivo.

As used herein, "treating (and the various parts of speech thereof)" a tumor is defined as achieving at least one therapeutic effect, such as a reduction in the number of tumor cells, a reduction in the size of the tumor, a reduction in the rate of infiltration of cancer cells into surrounding organs, or a reduction in the rate of tumor metastasis or tumor growth. As used herein, the term "modulate" refers to the activity of a compound in affecting (e.g., promoting or treating) a cellular function, including but not limited to cell growth, proliferation, invasion, angiogenesis, apoptosis, and the like.

The positive therapeutic effect of cancer can be measured in a variety of ways (e.g., weber (2009) J nucleic Med 50,1S-10S). For example, T/C.ltoreq.42% is the lowest level of anti-tumor activity relative to tumor growth inhibition, according to the National Cancer Institute (NCI) standard. T/C <10% is considered to be a high level of anti-tumor activity, where T/C (%) = median tumor volume treated/median tumor volume control x 100. In some embodiments, the treatment achieved by the therapeutically effective amount is any one of Progression Free Survival (PFS), disease Free Survival (DFS), or total survival (OS). PFS is also known as "time to tumor progression (Time to Tumour Progression)", which refers to the length of time that cancer does not grow during and after treatment, and includes the amount of time that a patient experiences a complete response or a partial response, as well as the amount of time that a patient experiences stable disease. DFS refers to the length of time a patient remains disease free during and after treatment. OS refers to an increase in life expectancy compared to untreated (naive) or untreated (untreated) individuals or patients.

As used herein, "prevention" refers to delaying or preventing the onset of symptoms of cancer. Prevention may be absolute (such that no disease occurs) or may be effective in only some individuals or for a limited period of time.

In a preferred aspect of the invention, the individual has an established tumor, i.e. the individual has already had a tumor, e.g. classified as a solid tumor. Thus, the invention described herein can be used when an individual has had a tumor (e.g., a solid tumor). Thus, the present invention provides a treatment option that can be used to treat existing tumors. In one aspect of the invention, the individual has an existing solid tumor. The invention may be used for the prevention or preferably treatment of individuals suffering from solid tumors. In one aspect, the invention is not used for prophylaxis or prevention.

In one aspect, the use of the invention described herein can enhance tumor regression, can reduce or decrease tumor growth, and/or can increase survival time, e.g., as compared to other cancer treatments (e.g., standard of care treatments for a given cancer).

In one aspect of the invention, the methods of treating or preventing a tumor described herein further comprise the step of identifying an individual having a tumor, preferably identifying an individual having a solid tumor.

The dosage regimen of the therapies described herein that is effective to treat a patient having a tumor can vary depending upon factors such as the disease state, age and weight of the patient, and the ability of the therapy to elicit an anti-cancer response in the individual. The selection of an appropriate dosage will be within the ability of those skilled in the art. For example 0.01, 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50mg/kg. In some embodiments, such amounts are unit doses (or whole portions thereof) suitable for administration according to a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to the relevant population (i.e., using a therapeutic dosing regimen).

The binding agent or nucleic acid encoding the same according to any aspect of the invention as described herein may be in the form of a pharmaceutical composition, which additionally comprises a pharmaceutically acceptable carrier, diluent or excipient. As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity. The pharmaceutically acceptable carrier enhances or stabilizes the composition or may be used to facilitate the preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible as known to those skilled in the art (see, e.g., remington's Pharmaceutical Sciences, 18 th edition, mack printing company, 1990, pages 1289-1329; remington: the Science and Practice of Pharmacy, 21 st edition, uk medical press 2011; and subsequent versions thereof). Non-limiting examples of the pharmaceutically acceptable carrier include any standard pharmaceutical carrier, e.g., phosphate buffered saline solution, water, emulsions such as oil/water emulsions, and various types of wetting agents. Thus, the invention also provides the use of the binding agent of the invention in the manufacture of a medicament for the treatment of a tumour.

Such compositions include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, or liposomes. In some embodiments, the preferred form may depend on the intended mode of administration and/or therapeutic application. Pharmaceutical compositions containing the binding agents or nucleic acids of the invention may be administered by any suitable method known in the art, including but not limited to oral, mucosal, inhalation, topical, buccal, nasal, rectal, or parenteral (e.g., intravenous, infusion, intratumoral, intranodular, subcutaneous, intraperitoneal, intramuscular, intradermal, transdermal, or other types of administration involving physical disruption of the individual's tissues and administration of the pharmaceutical composition by disruption in the tissues). Such formulations may be in the form of injectable or infusible solutions, for example, suitable for intradermal, intratumoral or subcutaneous administration or intravenous infusion. In particular embodiments, the binding agent or nucleic acid is administered intravenously. Administration may include intermittent administration. Alternatively, administration may include continuous administration (e.g., infusion) for at least a selected period of time, concurrently with other compounds, or between administration of other compounds.

The formulations of the present invention generally comprise a therapeutically effective amount of a binding agent as in the present invention. "therapeutic level", "therapeutically effective amount" or "therapeutic amount" refers to the amount or concentration of an active agent that is suitable for safely treating a disorder to reduce or prevent symptoms of the disorder.

In some embodiments, the binding agent may be prepared with a carrier that protects it from rapid release and/or degradation (e.g., controlled release formulations such as implants, transdermal patches, and microencapsulated delivery systems). Biodegradable, biocompatible polymers may be used.

Those skilled in the art will appreciate that, for example, the route of delivery (e.g., oral, intravenous, subcutaneous, intratumoral, etc.) may affect the dose and/or the desired dose may affect the route of delivery. For example, focused delivery (e.g., intratumoral delivery in this example) may be desirable and/or useful when focusing on a particular point or location (e.g., intratumoral) of a particularly high concentration of agent. Other factors to be considered when optimizing a route and/or dosing regimen for a given treatment regimen may include, for example, the particular cancer being treated (e.g., type, stage, location, etc.), the clinical condition of the individual (e.g., age, general health, etc.), the presence or absence of combination therapies, and other factors known to the practitioner.

The pharmaceutical composition should generally be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, dispersions, liposomes or other ordered structures suitable for high drug concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of the binding agent in the appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained release or biodegradable formulations as discussed herein. Sterile injectable preparations may be prepared using non-toxic parenterally acceptable diluents or solvents. Each of the pharmaceutical compositions for use according to the present invention may include a pharmaceutically acceptable dispersing agent, wetting agent, suspending agent, isotonic agent, coating, antibacterial and antifungal agent, carrier, excipient, salt or stabilizer which is non-toxic to the individual at the dosage and concentration used. Preferably, such compositions may also comprise a pharmaceutically acceptable carrier or excipient for treating cancer that is compatible with a given method and/or site of administration, e.g., for parenteral (e.g., subcutaneous, intradermal, or intravenous injection), intratumoral, or peritumoral administration.

Although the embodiment of the therapeutic method or composition for use according to the invention is not necessarily effective to achieve a positive therapeutic effect in each individual, it should be achieved in the use of pharmaceutical compositions and dosing regimens, which are in accordance with good medical practice and have been tested by any statistical test known in the art, such as StCurrent t test, X ² Test, U test according to Mann and Whitney, kruskal-Wallis test (H test), jonckheere-Terpstra test and Wilcoxon test.

When a tumor, neoplastic disease, cancer or cancer is mentioned hereinabove and later, metastasis in the original organ or tissue and/or any other location is also alternatively or additionally implied, regardless of the location of the tumor and/or metastasis.

As discussed herein, the present invention relates to the clearance of regulatory T cells (tregs). Thus, in one aspect of the invention, treatment with hCCR8 binding agents having cytotoxic activity eliminates or reduces regulatory T cells, particularly tumor infiltrating regulatory T cells. In one aspect, the clearing is via ADCC. In another aspect, purging occurs via CDC. In another aspect, the purging is via ADCP.

Accordingly, the present invention provides a method for clearing regulatory T cells in a tumor of an individual comprising administering to said individual an hCCR8 binding agent having cytotoxic activity. In a preferred embodiment, tregs are cleared in solid tumors. By "clearance" is meant a decrease in the number, proportion or percentage of tregs relative to when hCCR8 binding agent with cytotoxic activity is not administered. In particular embodiments of the invention described herein, more than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99% of tumor-infiltrating regulatory T cells are cleared.

As used herein, "regulatory T cells" ("Treg", "Treg cells" or "Tregs") refer to cd4+ T lymphocyte lineages that specifically control autoimmunity, allergy and infection. Typically, they modulate the activity of T cell populations, but they can also affect certain innate immune system cell types. Tregs are generally identified by expression of the biomarkers CD3, CD4, CD25 and CD127 or Foxp 3. Naturally occurring Treg cells typically constitute about 5-10% of peripheral cd4+ T lymphocytes. However, in tumor microenvironments (i.e., tumor-infiltrating Treg cells), they may account for up to 20-30% of the total cd4+ T lymphocyte population.

Activated human Treg cells can kill target cells, such as effector T cells and APCs, directly through perforin or granzyme B-dependent pathways; cytotoxic T lymphocyte-associated antigen 4 (ctla4+) Treg cells induce indoleamine 2, 3-dioxygenase (IDO) expression by APCs, which inhibit T cell activation by reducing tryptophan; treg cells can release interleukin 10 (IL-10) and transforming growth factor (TGF beta) in vivo, thereby directly inhibiting T cell activation and inhibiting APC function by inhibiting the expression of MHC molecules CD80, CD86 and IL-12. Treg cells can also suppress immunity by expressing high levels of CTLA4, CTLA4 can bind CD80 and CD86 on antigen presenting cells and prevent proper activation of effector T cells. Furthermore, treg cells are known to express high levels of CD25, thereby competing with IL2 for binding to CD8 and reducing CD 8-induced proliferation and survival.

In a preferred embodiment of the invention, the ratio of effector T cells to regulatory T cells in a solid tumor is increased after administration of the binding agent of the invention. In some embodiments, the ratio of effector T cells to regulatory T cells in a solid tumor is increased to 5, 10, 15, 20, 40, or 80 or more.

Immune effector cells refer to immune cells that are involved in the effector phase of an immune response. Exemplary immune cells include bone marrow or lymphoid derived cells, such as lymphocytes (e.g., B cells and T cells, including cytolytic T Cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils.

Immune effector cells involved in the effector phase of the immune response express specific Fc receptors and perform specific immune functions. Effector cells may induce antibody-dependent cell-mediated cytotoxicity (ADCC), such as neutrophils capable of inducing ADCC. For example, fcaR-expressing monocytes, macrophages, neutrophils, eosinophils and lymphocytes are involved in specific killing of target cells and presentation of antigens to other components of the immune system, or in binding to antigen-presenting cells. Effector cells may also phagocytose target antigens, target cells or microorganisms. As described herein, the antibodies of the invention may be optimized for the ability to induce ADCC.

In preferred embodiments, the methods and compositions for clearing tregs are specific for tregs, with no effect on other T cells. In other embodiments, the methods and compositions of the invention eliminate tumor-infiltrating tregs to a greater extent than other tregs. In other embodiments, the methods and compositions of the invention eliminate tumor-infiltrating tregs to a greater extent than circulating tregs. In further embodiments, the methods and compositions of the invention eliminate tumor-invasive tregs to a greater extent than normal tissue-invasive tregs (e.g., intestinal tregs). The extent of clearance of a cell population is preferably compared by comparing the percent reduction of untreated and treated cell populations, such as shown in the examples.

In other specific embodiments, the methods and compositions of the invention reduce the ratio of Treg to T cells, particularly in tumors, more particularly in human tumors. In other embodiments, the methods and compositions of the invention reduce the ratio of Treg to T cells in a tumor to a greater extent than the ratio of Treg to T cells outside the tumor. In further embodiments, the methods and compositions of the invention reduce the ratio of Treg to T cells in a tumor to a greater extent than the ratio of Treg to T cells in normal tissue (particularly intestinal tissue).

In another aspect of the invention, treatment with an hCCR8 binding agent having cytotoxic activity or a nucleic acid encoding an hCCR8 binding agent as described herein can clear or reduce any type of hCCR8 expressing cell. Preferably, the cell is a tumor cell expressing hCCR 8. Accordingly, the present invention provides a method for clearing cells (preferably tumour cells) in an individual comprising administering to the individual an hCCR8 binding agent having cytotoxic activity or a nucleic acid encoding an hCCR8 binding agent as described herein.

In some embodiments, different anti-cancer agents may be administered in combination with the binding agents of the invention by the same or different delivery routes and/or according to different schedules. Alternatively or additionally, in some embodiments, one or more doses of the first active agent are administered substantially simultaneously with, and in some embodiments via a common route and/or as part of a single composition with, one or more other active agents. Those skilled in the art will also appreciate that some embodiments of the combination therapies provided according to the invention achieve synergistic effects; in some such embodiments, when the agents are used in different treatment regimens (e.g., as monotherapy and/or as part of different combination therapies), the dose of one or more of the agents used in the combination may be substantially different (e.g., lower) than the standard, preferred, or necessary dose and/or may be delivered by an alternative route.

In some embodiments, when two or more active agents are used in accordance with the present invention, such active agents may be administered simultaneously or sequentially. In some embodiments, the administration of one agent is specifically timed relative to the administration of another agent. For example, in some embodiments, the first agent is administered such that a particular effect is observed (or expected to be observed, e.g., based on a population study showing a correlation between a given dosing regimen and a particular effect of interest). In some embodiments, the desired relative dosing regimen of the agents administered in combination may be empirically assessed or determined, for example using an ex vivo, in vivo, and/or in vitro model; in some embodiments, such assessment or empirical determination is made in vivo, in a patient population (e.g., to establish a correlation), or alternatively in a particular patient of interest.

In another aspect of the invention, the cross-reactive hCCR8 binding agent has an improved therapeutic effect when combined with an immune checkpoint inhibitor. Combination therapy with cross-reactive hCCR8 binding agents and immune checkpoint inhibitors may have a synergistic effect in the treatment of established tumors. Thus, the interaction between the PD-1 receptor and the PD-L1 ligand may be blocked, resulting in a "PD-1 blocking". In one aspect, the combination can result in enhanced tumor regression, enhanced injury to tumor growth, or reduced or increased survival time, e.g., as compared to administration of a checkpoint inhibitor alone, using the invention as described herein. Thus, in a particular aspect of the invention, the invention provides an hCCR8 binding agent of the invention for use in the treatment of a tumour, wherein the treatment further comprises administration of an immune checkpoint inhibitor.

As used herein, "immune checkpoint" or "immune checkpoint protein" refers to a protein that belongs to an inhibitory pathway in the immune system, particularly for modulating T cell responses. Under normal physiological conditions, immune checkpoints are critical for preventing autoimmunity, especially during responses to pathogens. Cancer cells can alter modulation of immune checkpoint protein expression to avoid immune surveillance.

Examples of immune checkpoint proteins include, but are not limited to, PD-1, CTLA-4, BTLA, KIR, CD, B7H4, VISTA, and TIM3, as well as OX40, GITR, 4-1BB, and HVEM. An immune checkpoint protein may also refer to a protein that binds to other immune checkpoint proteins. Such proteins include PD-L1, PD-L2, CD80, CD86, HVEM, LLT1 and GAL9.

An "immune checkpoint protein inhibitor", "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to any molecule that can interfere with signal transduction and/or protein-protein interactions mediated by an immune checkpoint protein. In one aspect of the invention, the immune checkpoint protein is PD-1 or PD-L1. In a preferred aspect of the invention described herein, the immune checkpoint inhibitor interferes with PD-1/PD-L1 interactions via an anti-PD-1 or anti-PD-L1 antibody.

In further specific embodiments, the immune checkpoint is CTLA-4 (also known as CTLA4, cytotoxic T lymphocyte-associated protein 4, or CD 152), and the immune checkpoint inhibitor is an inhibitor of CTLA-4. In a specific embodiment, the binding agents of the invention are used to treat tumors, wherein the treatment further comprises administration of a CTLA-4 inhibitor, particularly an anti-CTLA-4 antibody, particularly a blocking anti-CTLA-4 antibody. The anti-CTLA-4 antibodies of the invention can bind to an epitope on human CTLA-4 in order to inhibit the interaction of CTLA-4 with human B7 counter-receptor. Because the interaction of human CTLA-4 with human B7 transduces a signal that results in inactivation of T cells carrying the human CTLA-4 receptor, antagonizing the interaction effectively induces, enhances, or prolongs activation of T cells carrying the human CTLA-4 receptor, thereby prolonging or enhancing the immune response. anti-CTLA-4 antibodies are described in U.S. patent No. 5,811,097;5,855,887;6,051,227; PCT application publication Nos. WO 01/14424 and WO 00/37504; in U.S. patent publication No. 2002/0039581. For the purpose of describing anti-CTLA-4 antibodies, each of these references is specifically incorporated herein by reference. An exemplary clinical anti-CTLA-4 antibody is human monoclonal antibody 10D1, as described in WO 01/14424 and U.S. patent application Ser. No. 09/644,668; antibody 10D1 was administered in single and multiple doses alone or in combination with vaccine, chemotherapy or interleukin 2 to more than 500 patients diagnosed with metastatic melanoma, prostate cancer, lymphoma, renal cell carcinoma, breast cancer, ovarian cancer and HIV. Other anti-CTLA-4 antibodies encompassed by the methods of the invention include, for example, those disclosed in the following documents: WO 98/42752; WO 00/37504; U.S. patent No. 6,207,156; hurwitz et al, (1998) Proc. Natl. Acad. Sci. USA 95 (17): 10067-10071; camahho et al, (2004) J.Clin.Oncology 22 (145): abstract number 2505 (antibody CP-675206); and Mokyr et al, (1998) Cancer Res.58:5301-5304. In certain embodiments, the methods of the invention comprise the use of an anti-CTLA-4 antibody, which is a human sequence antibody, preferably a monoclonal antibody, and in further embodiments is monoclonal antibody 10D1. In further specific embodiments, the CTLA-4 inhibitor is ipilimumab (ipilimumab) or tremelimumab (tremelimumab).

PD-1 (programmed cell death protein 1), also known as CD279, is a cell surface receptor expressed on activated T cells and B cells. Interaction with its ligand has been shown to attenuate T cell responses in vitro and in vivo. PD-1 binds to two ligands, PD-L1 and PD-L2.PD-1 belongs to the immunoglobulin superfamily. PD-1 signaling requires binding to a PD-1 ligand in close proximity to the peptide antigen presented by the Major Histocompatibility Complex (MHC) (Freeman, proc Natl Acad Sci USA 105,10275-6 (2008)). Thus, proteins, antibodies or small molecules that prevent the co-ligation of PD-1 and TCR on the T cell membrane are useful PD-1 antagonists.

In one embodiment, the PD-1 receptor antagonist is an anti-PD-1 antibody or antigen-binding fragment thereof that specifically binds to PD-1 and blocks the binding of PD-L1 to PD-1. The anti-PD-1 antibody may be a monoclonal antibody. The anti-PD-1 antibody may be a human or humanized antibody. An anti-PD-1 antibody is an antibody capable of specifically binding to the PD-1 receptor. anti-PD-1 antibodies known in the art and suitable for use in the present invention include nivolumab, pembrolizumab (pembrolizumab), pidotizumab (pidilizumab), BMS-936559, and torsemimab Li Shan.

The PD-1 antagonists of the invention also include compounds or agents that bind to and/or block PD-1 ligands to interfere with or inhibit ligand binding to PD-1 receptor, or that bind directly to and block PD-1 receptor without inducing inhibitory signal transduction through PD-1 receptor. In particular, PD-1 antagonists include small molecule inhibitors of the PD-1/PD-L1 signaling pathway. Alternatively, the PD-1 receptor antagonist may bind directly to the PD-1 receptor without triggering inhibitory signal transduction, and also bind to a ligand of the PD-1 receptor to reduce or inhibit ligand triggering signal transduction through the PD-1 receptor. By reducing the number and/or amount of ligands that bind to the PD-1 receptor and trigger inhibitory signal transduction, fewer cells are attenuated by the negative signal delivered by PD-1 signal transduction and a more robust immune response can be achieved.

In one embodiment, the PD-1 receptor antagonist is an anti-PD-L1 antibody or antigen-binding fragment thereof that specifically binds to PD-L1 and blocks the binding of PD-L1 to PD-1. The anti-PD-L1 antibody may be a monoclonal antibody. The anti-PD-L1 antibody may be a human antibody or a humanized antibody, such as atilizumab (atezolizumab) (MPDL 3280A) or aviumab (avelumab).

Any aspect of the invention described herein may be performed in combination with additional therapeutic agents, particularly additional cancer therapies. In particular, hCCR8 binding agents and optionally immune checkpoint inhibitors according to the invention may be administered in combination with co-stimulatory antibodies, chemotherapy and/or radiotherapy (by application of radiation to the outside of the body or by administration of a radioconjugated compound), cytokine-based therapies, targeted therapies, monoclonal antibody therapies or any combination thereof.

As used herein, a chemotherapeutic entity for combination therapy refers to an entity that is destructive to cells, i.e., an entity that reduces cell viability. The chemotherapeutic entity may be a cytotoxic drug. Contemplated chemotherapeutic agents include, but are not limited to, alkylating agents, anthracyclines, epothilones, nitrosoureas, ethyleneimine/methyl melamine, alkyl sulfonates, alkylating agents, antimetabolites, pyrimidine analogs, epipodophyllotoxins, enzymes (e.g., L-asparaginase); biological response modifiers, such as IFN-gamma, IL-2, IL-12 and G-CSF; platinum coordination complexes (e.g., cisplatin, oxaliplatin, and carboplatin), anthracenediones, substituted ureas (e.g., hydroxyurea), methylhydrazine derivatives (including N-Methylhydrazine (MIH) and procarbazine), adrenocortical inhibitors (e.g., mitotane (o, p' -DDD) and aminoglutethimide; hormones and antagonists including adrenocortical hormone antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens such as tamoxifen; androgens including propionic acid and fluorometholone/equivalents; antiandrogens such as flutamide, gonadotrophin releasing hormone analogs and leuprorelin; and non-steroidal antiandrogens such as flutamide.

Additional cancer therapies may be other antibodies or small molecule agents that reduce immunomodulation within the peripheral and tumor microenvironment, such as molecules targeting the tgfβ pathway, IDO (indoleamine deoxygenase), arginase, and/or CSF 1R.

"administering another therapy in combination" or a treatment comprising administering another therapy may refer to administering the additional therapy prior to, concurrently with, or after administration of any aspect according to the invention. Thus, the combination therapies may be administered simultaneously, separately or sequentially.

In a further embodiment, the invention provides a kit comprising any of the above-described binding agents. In some embodiments, the kit further comprises a pharmaceutically acceptable carrier or excipient. In other related embodiments, any of the components of the above combinations in the kit are present in unit doses, particularly as described herein. In still other embodiments, the kit includes instructions for administering any component or combination of the above to the individual. In specific embodiments, the kit comprises an hCCR8 binding agent as described herein and an immune checkpoint inhibitor, e.g., a PD-1 or PD-L1 inhibitor. The hCCR8 binding agent and immune checkpoint inhibitor may be present in the same or different compositions.

In a specific embodiment, the invention provides a package comprising a binding agent as described herein, wherein the package further comprises instructions for administration of the binding agent to a tumor patient concurrently receiving treatment with an immune checkpoint inhibitor.

Diagnosis of

hCCR8 binding agents described herein may also be used to predict, diagnose, prognose, and/or monitor a disease or condition in an individual. In a specific embodiment, the invention provides a method for monitoring a population of cells expressing hCCR8, the method comprising contacting the population of cells with an hCCR8 binding agent of the invention. In other embodiments, the invention provides the use of an hCCR8 binding agent as described herein as a chaperone diagnostic in a method for treating a disease in an individual, wherein the treatment comprises administration of Treg clearance therapy. More particularly, wherein the treatment comprises administration of a cytotoxic hCCR8 binding agent, such as an anti-hCCR 8 antibody having ADCC activity, in particular an anti-hCCR 8 antibody having ADCC activity and binding to the N-terminal region of hCCR 8. By its binding to the extracellular loop of hCCR8, the antibodies of the invention can be used to monitor treatment with hCCR8 binding agents (e.g., to monitor Treg clearance), which hCCR8 binding agents bind to the N-terminal portion of hCCR 8.

As used herein, the term "diagnosis" and its various forms of part of speech (diagnosis) generally refers to a process or behavior that identifies, determines, or infers a disease or disorder in an individual based on symptoms and signs and/or by the results of various diagnostic procedures (e.g., by knowing the presence, absence, and/or amount of one or more biomarker features of the disease or disorder being diagnosed). As used herein, "diagnosis of a disease" in an individual may particularly mean that the individual suffers from the disease, and is therefore diagnosed as suffering from the disease. As taught herein, an individual may be diagnosed as not suffering from the disease, although one or more of its reminded conventional symptoms or signs are displayed.

As used herein, the term "prognosis" and its various forms of parts of speech (prognostics) "generally refers to the expectation of the progression and recovery (e.g., probability, duration, and/or extent) of a disease or disorder. A good prognosis of a disease may generally include an expectation of satisfactory partial or complete recovery from the disease, preferably within an acceptable period of time. A good prognosis of the disease may more generally include an expectation that the disorder is preferably not further worsened or aggravated over a given period of time. Poor prognosis of a disease may generally include an expected sub-standard recovery and/or unsatisfactory slow recovery, or substantially no recovery or even further exacerbation of the disease.

In one aspect, the present invention relates to hCCR8 binding agents according to the invention for use in a method of diagnosis, prognosis and/or prognosis of a disease associated with altered expression and/or activity of human CCR 8. In other words, the present invention provides an (in vitro) method for diagnosing, predicting and/or prognosticating a disease associated with a change in expression and/or activity of CCR8 in an individual, wherein the method comprises measuring the amount of CCR8 in a sample from the individual.

In a further aspect, the present invention relates to an hCCR8 binding agent according to the invention for use in a method of diagnosis, prognosis and/or prognosis of a disease associated with altered expression and/or activity of human CCL 1. In other words, the present invention provides an (in vitro) method for diagnosing, predicting and/or prognosticating a disease associated with a change in expression and/or activity of CLL1 in an individual, wherein the method comprises measuring the amount of CCR8 in a sample from the individual.

According to a further aspect, the present invention relates to a kit for the diagnosis, prognosis and/or prognosis of a disease associated with altered expression and/or activity of CCR8 and/or CCL1, the kit comprising a means for measuring the amount of hCCR8 by using a hCCR8 binding agent as described herein. According to a preferred embodiment, the kit comprises a reference control obtained from an individual not suffering from the disease or from an individual having a known diagnosis, prognosis and/or prognosis of the disease.

In one embodiment, the hCCR8 binding agent as described above may advantageously be immobilized on a solid phase or carrier.

The kit may also comprise hCCR8 and/or fragments thereof in known amounts or concentrations, for example for use as controls, standards and/or calibrators. It may also comprise means for collecting a sample from the individual.

An advantage of the binding agents of the invention, in particular the binding agents lacking cytotoxic activity described herein, is that they are suitable for in vivo diagnostic use. Because they bind to the extracellular loop of CCR8, and in the absence of cytotoxic moieties, the binding agents of the invention can be administered to an individual without affecting therapeutic treatment with non-competitive CCR8 binding agents (e.g., prior art N-terminal binding agents). For example, a single domain antibody moiety as described herein, e.g., a VHH molecule as defined above and in the examples, can be administered, e.g., for imaging purposes, when a patient is subjected to treatment with, e.g., an anti-cancer drug (e.g., treg-clearance therapy). Non-cytotoxic binders can be used for imaging purposes, for example for monitoring Treg clearance for efficient CCR8 expression. Thus, in a specific embodiment, the invention provides a CCR8 binding agent comprising a CCR8 binding moiety as described herein and a detectable label. Detectable labels can be detected using, for example, radioactive, optical, magnetic resonance, and ultrasonic methods. In a specific embodiment, the detectable label is a fluorescent label. In particular embodiments, CCR8 binding agents of the invention (preferably lacking a cytotoxic moiety) are used to monitor non-competitive CCR8 binding agent therapies. In further embodiments, the CCR8 binding agents of the invention (preferably lacking a cytotoxic moiety) are used to monitor therapy of anti-CCR 8 antibodies that bind to the N-terminal region of hCCR 8. In particular, an anti-CCR 8 antibody that binds to the N-terminal region of hCCR8 is one of the antibodies disclosed in WO2020138489 A1, more particularly an anti-CCR 8 antibody comprising a light chain variable region comprising SEQ ID No. 59 and a heavy chain variable region comprising SEQ ID No. 41 in WO2020138489 A1. In a further embodiment is an anti-CCR 8 antibody comprising a light chain constant region comprising SEQ ID NO:52 and a heavy chain constant region comprising SEQ ID NO:53 in WO 2020138489A 1.

Examples

The following examples are provided to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

Two anti-human CCR8 blocking monoclonal antibodies (ONCC 8 and ONCC 10) were used as controls for the experiments described below. The sequence of ONCC8 was obtained by cloning the sequences of the light chain variable region and the heavy chain variable region from WO2020/0138489 A1 (SEQ ID NO:59 and SEQ ID NO:41 corresponding to WO2020/0138489 A1, respectively) into a human IgG1 backbone, whereas the sequence of ONCC10 was obtained by cloning the heavy chain variable region sequence and the light chain variable region sequence of mAb 433H from WO2007/044756 A1 into a human IgG1 backbone. The production of these antibodies was performed in HEK293 cells of Icosagen (Icosagen, edanita Tarl) or in CHO cells of Evitria (Evitria, zurich, switzerland).

Example 1.Generation of hCR 8-targeting single domain antibody portions

hCR 8 DNA immunization

Immunization of llamas and alpacas with human CCR8 DNA is essentially carried out as disclosed in Pardon E.et al (A general protocol for the generation of Nanobodies for structural Biology, nature Protocols,2014,9 (3), 674-693) and Henry K.A. and MacKenzie C.R. editions (Single-Domain Antibodies: biology, engineering and Emerging applications.Lausane: front Media). Briefly, animals were immunized four times at two week intervals with 2mg of DNA encoding human CCR8 inserted into expression vector pVAX1 (ThermoFisher Scientific company, V26020) and blood samples were collected. Three months later, all animals received three injections of 2mg of the same DNA, and then blood samples were collected.

Phage display library preparation

Phage display libraries derived from Peripheral Blood Mononuclear Cells (PBMC) were prepared and used as described in Pardon E.et al (A general protocol for the generation of Nanobodies for structural Biology, nature Protocols,2014,9 (3), 674-693) and in the company Henry K.A. and MacKenzie C.R. editors (Single-Domain Antibodies: biology, engineering and Emerging applications. Lausane: front Media). The VHH fragment was inserted into an M13 phagemid vector containing MYC and His6 tags. Coli TG1[ (F's) grown by infectious index'

traD36 proAB laclqZΔM15) supE thi-1Δ (lac-proAB) Δ (mcrB-hsdSM) 5 (rK-mK-) ] cells, followed by repeated infection (surifection) with VCSM13 helper phage to preserve the library.

Phage display library was selected in two rounds on HEK293T cells transiently transfected with human CCR8 inserted into pcDNA3.1 (ThermoFisher Scientific company, V79020) and then on CHO-K1 cells transiently transfected with human CCR8 inserted into pcDNA3.1. Polyclonal phagemid DNA was prepared with escherichia coli TG1 cells infected with eluted phage from the second round of selection. VHH fragments were amplified from these samples by PCR and subcloned into an e.coli expression vector with an in-frame N-terminal PelB signal peptide and C-terminal FLAG3 and His6 tags. The resulting VHH expression plasmid ligation mixture was used to transform electrocompetent E.coli TG1 cells and individual colonies were grown in 96-deep well plates. Expression of monoclonal VHH was essentially as described by Pardon E et al (A general protocol for the generation of Nanobodies for structural biology, nature Protocols,2014,9 (3), 674-693). Crude periplasmic extracts containing VHH were prepared by freezing bacterial pellet overnight, then re-suspending in PBS and centrifuging to remove cell debris.

Example 2.Production of stable hCR 8 cell lines

At 37℃with 5% CO ₂ The human embryonic kidney cell line HEK293 (ATTC number CRL-1573) was cultured in Dulbecco's modified Eagle's medium (DMDM, gibco) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) and 100U/ml penicillin and streptomycin (Gibco). Cells were incubated at 7.5X10 before transfection ⁵ The individual cell/well densities were seeded in 6-well plates (Greiner) and cultured overnight. When approximately 40% confluency was reached, cells were transfected with linearized pcdna3.1 encoding human CCR8 using FUGENEHD transfection reagent (Promega). After 6 hours, the cell supernatant was carefully removed and replaced with fresh complete DMEM. 48 smallAt this time, the medium was replaced with a medium containing 500. Mu.g/ml G-418 (ThermoFisher Scientific company) to select gentamicin resistant transfectants carrying the expression cassette. The medium was changed every 2-3 days. After 3 weeks, from each well 10 ³ Individual cells were started and subjected to limiting 1:2 dilutions to obtain monoclonal lines. Mouse anti-hCR 8/CD198 IgG2a (Biolegend, clone No. L263G8,360603) using phycoerythrin label 10 was obtained in a flow cytometer (Attune NxT, thermoFisher Scientific Co.) ⁴ Individual cells, thereby identifying a monoclonal line expressing hCCR 8.

Example 3.Screening of hCR 8 selective output

Recombinant cells expressing hCCR8 were recovered using a cell dissociated non-enzymatic solution (Sigma Aldrich, C5914-100 mL) and resuspended to 1.0 x 10 in FACS buffer ⁶ Final concentration of individual cells/ml. Dilutions of crude periplasmic extracts containing VHH (1:5 FACS buffer) were incubated with 5 μg/ml FACS buffer of mouse anti-FLAG biotinylated antibody (Sigma Aldrich, F9291-1 MG) for 30 min and shaken at room temperature. The cell suspension was dispensed into 96-well v-plates and incubated with the VHH/antibody mixture on ice with shaking for 1 hour. Binding of VHH to cells was detected with streptavidin R-PE (Invitrogen, SA 10044) diluted 1:400 (0.18. Mu.g/ml) in FACS buffer, shaken on ice and incubated for 30 min in the absence of light. Surface expression of human CCR8 on transiently transfected cell lines was confirmed by 2 μg/ml PE anti-human CCR8 (Biolegend, 360603) antibodies.

Example 4.Purification and evaluation of monovalent VHHs

The synthetic DNA fragment encoding hCCR 8-bound VHH was subcloned into an e.coli expression vector under the control of IPTG-inducible lac promoter, which has an N-terminal PelB signal peptide in-frame for periplasmic compartment targeting and C-terminal FLAG3 and His6 tags. The electrocompetent E.coli TG1 cells were transformed and the resulting clones were sequenced. VHH protein was purified from these clones by IMAC chromatography followed by desalting essentially as described in Pardon E.et al (A general protocol for the generation of Nanobodies for structural biology, nature Protocols,2014,9 (3), 674-693).

Eight purified VHHs obtained from human CCR8 immunization campaigns were selected and evaluated for their binding to hCCR8 by flow cytometry. All purified VHHs showed efficient binding to human CCR 8. FIG. 1 shows the binding of the three selected VHHs (i.e., VHH-57, VHH-64 and VHH-67).

Example 5.In vitro cross-reactivity assessment of monovalent VHH

Eight purified VHH obtained from human CCR8 immunization campaigns were further evaluated with mice by flow cytometry

Binding of CCR8 (SEQ ID NO: 74) to cynomolgus CCR 8. Three purified VHHs (VHH-57, VHH-64 and VHH-67, see below) showed potent binding to cynomolgus CCR8 (FIG. 2) and murine CCR8 (FIG. 3).

Example 6.Epitope mapping

In comparison to mock transfected control cells, VHH clones generated by human CCR8 immunization and selection activities were screened by flow cytometry for binding to human CCR8 (SEQ ID NO: 72) on stably transfected HEK293 cells, or HEK293 cells previously transfected with plasmid DNA encoding three different forms of human-mouse chimera CCR8, wherein one of the extracellular loops (ECLs) was replaced with the mouse CCR8 counterpart, and for binding to N-terminally deleted human CCR8 (. Delta.18-3 XHA), SEQ ID NO:73, see below. Comparing the binding (median fluorescence intensity) signal of a given VHH clone in seven cell lines can classify the clone as either an N-terminal human CCR8 binding agent (i.e., binding to hCCR8 cells but not to human CCR8 (delta 18-3 XHA) or control cells) or an extracellular loop hCCR8 binding agent (i.e., binding to hCCR8 cells and human CCR8 (delta 18-3 XHA) but not to control cells). Comparison of binding to cell lines expressing human-mouse chimera CCR8 allows for further classification of extracellular hCCR8 binding agents as ECL1, ECL2 or ECL3 binding agents.

These experiments classified VHH-57 and VHH-67 as extracellular loop 2 (ECL 2) human CCR8 binding agents, while VHH-64 showed competitive binding to CCR8 with VHH-57 and VHH-67.

Example 7.Binding and functional characterization of monovalent VHH

The potential of monovalent VHHs VHH-57, VHH-64 and VHH-67 to functionally inhibit human CCL1 signaling on CHO-K1 cells displaying human CCR8 was evaluated in cAMP accumulation experiments.

Prior to testing, CHO-K1 cells stably expressing recombinant human CCR8 were grown in antibiotic-free medium and isolated by washing with PBS-EDTA (5 mM EDTA), recovered by centrifugation and resuspended in KHR buffer (5mM KCl,1.25mM MgSO ₄ 124mM NaCl,25mM HEPES,13.3mM glucose, 1.25mM KH ₂ PO ₄ ，1.45mM CaCl ₂ 0.5g/l BSA, supplemented with 1mM IBMX). 12 microliter of cells were mixed in triplicate with 6 microliter of VHH (final concentration: 1. Mu.M) and incubated for 30 minutes. Thereafter, 6 μl of forskolin and human CCL1 (R&DSsystems, 845-TC or 272-I) at a final concentration corresponding to its EC80 value. Plates were then incubated for 30 minutes at room temperature. After addition of lysis buffer and incubation for 1 hour, fluorescence ratios were measured using HTRF kit (Cisbio, 62AM9 PE) according to the manufacturer's instructions.

All VHHs tested gave effective functional inhibition in the assay, pIC50 values ranging from 6.48±0.02M to 8.93±0.01M (n=2) (mean±standard deviation), indicating that these are blocking CCR8 binders (see fig. 4).

Example 8.Synthesis and purification of cross-reactive VHH-Fc fusions

To evaluate the binding properties of the VHH-Fc fusion of hCR 8 binders, nine VHH-Fc constructs (VHH-Fc-203, VHH-Fc-204, VHH-Fc-205, VHH-Fc-209, VHH-Fc-210, VHH-Fc-216) were produced by direct fusion (VHH-Fc-203, VHH-Fc-209 and VHH-Fc-218) or by flexible GlySer linker 10GS (VHH-Fc-205, VHH-Fc-211 and VHH-Fc-217) (20 GS refers to four repeats of SEQ ID NO:46, thus having a length of 20 amino acids) by combining anti-CCR 8 VHH with the human short hinge and the IgG1 Fc domain (SEQ ID NO: 30). Constructs VHH-Fc-203, VHH-Fc-204 and VHH-Fc-205 contain a VHH-57CCR8 binding moiety, VHH-Fc-209, VHH-Fc-210 and VHH-Fc-211 contain a VHH-64CCR8 binding moiety, and VHH-Fc-215, VHH-Fc-216 and VHH-Fc-217 contain a VHH-67CCR8 binding moiety.

The construct was cloned into a pQMCF mammalian expression vector with an in-frame secretion signal peptide to direct the expressed recombinant protein to the extracellular environment. Cloning, cell transfected protein production and protein a purification in choe bnalt854 1E9 cells were performed by icosangen (Icosagen Cell Factory, isannia tarr diagram).

Example 9.Demonstration of hCR 8 binding by VHH-Fc fusion

Nine multivalent VHH-Fc fusions were evaluated for their ability to bind human CCR8 on stably transfected HEK293 cells by flow cytometry experiments (fig. 5). Cells were incubated with multivalent VHH-Fc fusions at different concentrations for 30 min at 4℃and then washed twice with FACS buffer and then with R-phycoerythrin AffiniPure F (ab') ₂ The fragment goat anti-human IgG (Jackson ImmunoResearch, cat# 109-116-098) was incubated at 4℃for 30 minutes, followed by two washing steps. Dead cells were stained using TOPRO3 (Thermo Fisher Scientific, T3605).

All nine VHH-Fc fusions were highly comparable to human CCR8, with pEC50 values ranging from 8.19 to 9.04M.

Example 10.In vitro cross-reactivity assessment of VHH-Fc fusions

Cynomolgus CCR8 binding

All nine multivalent VHH-Fc fusions were evaluated by flow cytometry experiments for their ability to bind cynomolgus CCR8 on transiently transfected cells (fig. 6) and on stably expressed HEK293 cells (fig. 7). Cells were incubated with different concentrations of multivalent VHH-Fc fusion for 30 min at 4 ℃, then washed twice with FACS buffer, then with AF488 goat anti-mouse IgG (Life Technologies, a 11029) or AF488 donkey anti-rat IgG (Life Technologies, a 21208) for 30 min at 4 ℃, then two washing steps were performed. Dead cells were stained using TOPRO3 (Thermo Fisher Scientific, T3605).

All VHH-Fc fusions were found to bind highly to cynomolgus CCR8 with pEC50 values ranging from 7.97-8.67M, confirming that all 12 VHH-Fc species were cross-reactive with cynomolgus CCR8 and that the EC values were comparable to human CCR 8.

Mouse CCR8 binding

All nine multivalent VHH-Fc fusions were evaluated by flow cytometry experiments for their ability to bind to mouse CCR8 on transiently transfected cells (fig. 8) and to mouse CCR8 expressed endogenously on BW5147 cells (fig. 9). Cells were incubated with different concentrations of multivalent VHH-Fc fusion for 30 min at 4 ℃, then washed twice with FACS buffer, then with AF488 goat anti-mouse IgG (Life Technologies, a 11029) or AF488 donkey anti-rat IgG (Life Technologies, a 21208) for 30 min at 4 ℃, then two washing steps were performed. Dead cells were stained using TOPRO3 (Thermo Fisher Scientific, T3605).

All VHH-Fc fusions were found to bind highly to mouse CCR8 with pEC50 values ranging from 6.02 to 8.57M, confirming that all 12 VHH-Fc species were cross-reactive with mouse CCR 8.

Example 11.ADCC efficacy of VHH-Fc fusions

ADCC reporter assay

All nine VHH-Fc fusions were tested for their ability to activate human fcyriiia in an ADCC reporter gene assay kit (Promega, G7010, G7018) using the human CCR8 HEK293 cell line as target cells.

Engineered Jurkat cells stably transfected with V158 fcγriiia receptor and NFAT (nuclear factor of activated T cells) responsive firefly luciferase reporter as effector cells were used in this assay. HEK293 cells overexpressing human CCR8 were used as target cells. ADCC activity was quantified by the luciferase luminescence signal generated by NFAT pathway activation when VHH-Fc fusions were incubated with target cells and effector cells at a 2.5:1 effector cell to target cell ratio according to manufacturer's recommendations.

All nine VHH-Fc fusions were found to activate human fcyriiia, with pEC50 values ranging from about 7.74-9.29M based on 4 dilutions, which is the same range as the two control anti-human CCR8 antibodies (ONCC 8 and ONCC 10).

ADCC assay using human PBMC

Four VHH-Fc fusions (VHH-Fc-210, VHH-Fc-215, VHH-Fc-216 and VHH-Fc-217) as well as control monoclonal antibodies ONCC8 and isotype controls were tested in a ratio of 40:1 effector cells to target cells in an ADCC assay using human PBMC from three independent healthy donors. Briefly, HEK293 cells expressing human CCR8 were labeled with DiO and 5X 10 per well ³ Individual cells were seeded in 96-well round bottom plates. Binding agents were titrated in duplicate at 8-spots. The labeled target cells were conditioned with titrating binders and then incubated with effector cells for 3 hours. Specific lysis on target cells was monitored by PI live/dead staining (PI live/dead stain). Samples were collected on a NxT flow cytometer (Attune).

Based on the average of three independent experiments using human PBMCs from different healthy donors, all four VHH-Fc fusions showed potent ADCC activity with pEC50 values ranging from about 28.8-43.8M, which is the same range as ONCC8 controls.

Example 12.Effect of cross-reactive VHH-Fc fusions on tumor growth in MC38 syngeneic mouse models

The mouse MC38 model was used to demonstrate the efficacy of cross-reactive VHH-Fc fusions that bind to human and mouse CCR8 for tumor treatment.

Will be 5X 10 ⁵ Individual MC38 cells (100 μl) were subcutaneously injected into female C57BL/6J mice (7-9 weeks). On day 7 (average tumor size=132 mm) ³ ) Mice were divided into different groups. The different cohorts consisted of 10 mice for each disorder, and each group of mice was injected intraperitoneally once every two weeks with 200 μg of mouse IgG2a (control), VHH-Fc-205, or VHH-Fc-215 for 3 weeks. Body weight and tumor size were measured every two weeks.

Tumors were measured in two dimensions with calipers to monitor growth. Tumor size (in mm) was calculated using the following formula ³ )：

Tumor size= (w ² ×l)×0.52

Where w=width of tumor, l=length of tumor, in mm.

Tumor size median (in mm for all different queues ³ ) Depicted in fig. 11. From the slaveBeginning on day 18, the groups treated with VHH-Fc-205 and VHH-Fc-215 showed significantly reduced tumor size compared to isotype control. These data indicate that VHH-fc_205 and VHH-Fc-215 show efficacy in the MC38 syngeneic mouse model.

Example 12.Sequence optimisation of monovalent VHH

Sequence optimization was performed on VHH-57, VHH-64 and VHH-67 in an attempt to maximize improvement against human IGHV3 (SEQ ID NO:83, evqlvesgglvqpggslrlrlscaasgfssyamhwvrpgkglewvsvsvsvsssdgtyyadsvkgrft isdnskntlyllqmnslraedtavycar) and JH (SEQ ID NO:84, wgqgtlvtvss) germline consensus sequences, as well as sequences in terms of chemical and biophysical stability, while minimizing loss of target binding.

Evaluation of production, purification and binding Properties

Cloning into E.coli expression vectors with a C-terminal His6 (but without FLAG 3) tag standard E.coli expression and Immobilized Metal Affinity Chromatography (IMAC) procedures were performed as described in example 4.

A plurality of His6 tagged VHH variants were thereby generated and evaluated by flow cytometry for their ability to compete with their FLAG3-His6 tagged parent counterparts (VHH-57, VHH-64 or VHH-67) for binding to hCR 8. Cells were first incubated with different concentrations of monovalent sequence optimized variants that did not carry a FLAG3 tag at 4 ℃ for 30 min, then with fixed concentrations of FLAG 3-tagged parent counterparts (VHH-57, VHH-64 or VHH-67) at 4 ℃ for 30 min, then washed, and subsequently passed through the mouse M2 anti-FLAG mAb (Sigma Aldrich, cat#f-1804) and then through the R-phycoerythrin AffiniPure F (ab') ₂ The fragment goat anti-mouse IgG (Jackson ImmunoResearch, cat# 115-116-071) was tested for anti-FLAG. Similarly, variants were evaluated for their ability to compete with their parent VHH-Fc fusion counterparts (VHH-Fc-204, VHH-Fc-210 and VHH-Fc-216) for binding mCCR 8; the assay was performed as described in example 9.

Variants that retain binding capacity were selected for further biophysical and chemical stability analysis.

Biophysical stability

All VHH and VHH-Fc variants were subjected to thermal stability and aggregation assays to understand their melting and aggregation temperatures.

Intrinsic tryptophan fluorescence upon temperature-induced protein unfolding was monitored in a Unlce instrument (Unchained Labs, prisendon, calif., U.S.A.). 10 microliters of sample was added to the sample cuvette at 1mg/ml and the temperature was raised linearly from 25 ℃ to 95 ℃ at a rate of 0.5 ℃/min, pre-run for 180 seconds. The center of gravity average (BCM) and static light scattering (SLS at 266nm and 473 nm) signals were plotted against temperature to obtain melting temperatures (T) _m ) And aggregation temperature (T) _agg )。

When temperature-induced unfolding was performed on VHH samples, it was noted that the melting temperature (T _m ) This is measured by static light scattering at 266nm (smaller aggregate) and 473nm (larger aggregate) (T _agg ) Can be well verified.

Dynamic light scattering was performed using a Uncle instrument by adding 10. Mu.l of sample to a sample cuvette at 1 mg/ml. The laser and attenuator control was set to automatic with 10 acquisitions per data point run, each acquisition time being 10 seconds.

Size Exclusion Chromatography (SEC) coupled with multi-angle laser light scattering (MALLS) was performed by adding 120. Mu.l of a 1mg/ml sample to a Superdex 200 column (GE healthcare) on an Agilent HPLC system. The outlet of the column is coupled to a UV detector, then to a Refractive Index (RI) detector, and finally to a MALLS detector.

The SEC-MALLS data of VHH variants and their Fc-fusion counterparts are highly correlated. For most VHH-Fc fusions containing VHH entities, storage at 40℃for one week does not result in the presence of any soluble (insoluble) (SEC-MALLS) aggregates that might be observed.

Chemical stability

Purified VHH obtained from sequence optimization activities were selected for chemical stability assessment.

The following binders were selected:

VHH-84: VHH-64 with mutation E1D, V40A, T74A, A78V

VHH-119: VHH-57 with mutation E1D, N27S, P62S

VHH-120: VHH-67 with mutation E1D, V65G, V69I, G A

VHH-121: VHH-67 with E1D, V65G, V69I, G74A, D99Q

VHH-122: VHH-67 with E1D, V65G, V69I, G74A, S T

The samples were stored at 40℃for 4 weeks, while the reference samples were stored at-80 ℃. The forcibly oxidized samples (1 mg/ml) were supplemented with hydrogen peroxide to a final concentration of 10mM, then incubated at 37℃for 3 hours, and finally buffer exchanged for Phosphate Buffered Saline (PBS) using a PD MiDiTrap G-25 column (GE-Healthcare, chicago, ill.) according to the manufacturer's instructions. Samples were stored at-80 ℃ until the mass spectrum peptide was mapped (chromatography institute, belgium, cretinic). Peptide mapping involved treating 100 μg of sample protein with trypsin (overnight at 25 ℃) and injecting the sample onto an RPC column (reverse phase chromatography; elution by applying an acetonitrile gradient) followed by ESI-mass spectrometry, where LC-MS and LC-MS/MS data were used for quantification and identification, respectively.

In contrast to VHH-64, VHH-84 did not show any substantial high temperature (40 ℃) dependency problems after 4 weeks of storage, such as Asn/Gln deamination, met/Trp oxidation or Asp isomerization upon 4 weeks of storage at 40 ℃. No other problems were found.

These sequence-optimizing mutations were found to have substantially no effect on target binding parameters. The E1D mutation in all of VHH-57, VHH-64 and VHH-67 was found to eliminate pyroglutamate formation. In addition, the mutation D99Q or S100T in VHH-67 was found to reduce aspartate isomerisation. Interestingly, mutations T74A and A78V in VHH-64 were found to increase the thermal stability of the binding agent by 5 ℃. It was also found that after 4 weeks of storage at 40℃the deamidation was significantly reduced after substitution of the asparagine residue at position 27 in VHH-57 with serine. Substitution of proline at position 62 with serine was also found to increase the thermal stability of VHH-57 by 3 ℃. Substitution of valine at position 65 in VHH-67 with glycine increases thermal stability by 10 ℃. The other amino acid substitutions listed above mainly lead to increased humanisation of the binding agent.

Example 13.Synthesis and purification of optimized VHH-Fc fusions

Fc-fusions of optimized VHH sequences were generated as in example 8. Thus, VHH-84 was fused directly to the IgG1 short hinge domain (SEQ ID NO: 48) or through the 10GS linker (SEQ ID NO: 56) or through the 20GS linker (SEQ ID NO: 64). Similarly, VHH-119 was fused directly to the IgG1 short hinge domain (SEQ ID NO: 50) or through the 10GS linker (SEQ ID NO: 58) or through the 20GS linker (SEQ ID NO: 66). VHH-120 was fused directly to the IgG1 short hinge domain (SEQ ID NO: 52) or through a 10GS linker (SEQ ID NO: 60) or through a 20GS linker (SEQ ID NO: 68). VHH-121 was fused directly to the IgG1 short hinge domain (SEQ ID NO: 53) or through a 10GS linker (SEQ ID NO: 61) or through a 20GS linker (SEQ ID NO: 69). VHH-122 was fused directly to the IgG1 short hinge domain (SEQ ID NO: 54) or through a 10GS linker (SEQ ID NO: 62) or through a 20GS linker (SEQ ID NO: 70). These constructs that retain binding capacity are most suitable for the treatment of the diseases mentioned herein.

Example 14.Optimized ADCC efficacy of VHH-Fc fusions

In the ADCC assay, one of the Fc-fusions of the optimized sequence obtained in example 13 (VHH-120 fused directly to the IgG1 short hinge domain, SEQ ID NO:52, hereinafter VHH-Fc-256) and isotype control were tested at a ratio of 40:1 effector cells to target cells using human PBMC from three independent healthy donors. The ADCC efficacy of the afucosylated and nonfucosylated forms of VHH-Fc-256 was evaluated.

Briefly, HEK293 cells expressing human CCR8 were labeled with DiO and 5X 10 per well ³ Individual cells were seeded in 96-well round bottom plates. Binding agents were titrated in duplicate at 8-spots. The labeled target cells were conditioned with titrating binders and then incubated with effector cells for 3 hours. Specific lysis on target cells was monitored by PI live/dead staining. Samples were collected on a NxT flow cytometer (Attune).

Both the afucosylated and nonfucosylated forms of the VHH-Fc-256 fusion showed potent ADCC activity compared to isotype control (see figure 12). The observed ADCC activity of the afucosylated form of VHH-Fc-256 showed the strongest ADCC activity. These data show that Fc fusions of optimized VHH sequences exhibit potent ADCC activity, while the afucosylated forms of the Fc fusions perform better.

Sequence listing

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Gly Ser Ile Phe Ser Leu Leu Asp

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Ile Thr Ser Gly Gly Ser Thr

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Asn Gly Arg Gln Thr Gly Trp Arg Thr Arg Val Asp Tyr

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Asn Ala Ala Pro Tyr Tyr Trp Gly Ala Tyr Arg Arg Gln Glu Ser

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Tyr Ala Gln Asp Ser Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp

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Tyr

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Tyr Ala Gln Gln Ser Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp

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Tyr

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Tyr Ala Gln Asp Thr Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp

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Tyr

<210> 12

<211> 25

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<223> FR1-P120/P121/P122

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Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Thr Val Ser

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Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala

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Thr

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<212> PRT

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<220>

<223> FR2-P57/P119

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Met Lys Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala

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Asp

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<212> PRT

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Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

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Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

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Thr Ala Val Tyr Tyr Cys

35

<210> 16

<211> 11

<212> PRT

<213> artificial sequence

<220>

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<400> 16

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 17

<211> 119

<212> PRT

<213> artificial sequence

<220>

<223> VHH-P84

<400> 17

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Gly Ile Arg Ser Ile Ile

20 25 30

Pro Ala Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Ser Thr Ala Gly Ser Ala Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Gly Arg Gln Thr Gly Trp Arg Thr Arg Val Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 18

<211> 121

<212> PRT

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<220>

<223> VHH-P119

<400> 18

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Ser Ile Phe Ser Leu Leu

20 25 30

Asp Met Lys Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Asp Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Ala Pro Tyr Tyr Trp Gly Ala Tyr Arg Arg Gln Glu Ser Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 19

<211> 123

<212> PRT

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<220>

<223> VHH-P120

<400> 19

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ser Ile Phe Ser Leu Arg

20 25 30

Thr Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Ser Ala Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Ala Gln Asp Ser Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 20

<211> 123

<212> PRT

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<220>

<223> VHH-P121

<400> 20

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ser Ile Phe Ser Leu Arg

20 25 30

Thr Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Ser Ala Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Ala Gln Gln Ser Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 21

<211> 123

<212> PRT

<213> artificial sequence

<220>

<223> VHH-P122

<400> 21

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ser Ile Phe Ser Leu Arg

20 25 30

Thr Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Ser Ala Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Ala Gln Asp Thr Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 22

<211> 8

<212> PRT

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<223> CDR1-IMGT-P57

<400> 22

Gly Asn Ile Phe Ser Leu Leu Asp

1 5

<210> 23

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> FR1-P64

<400> 23

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser

20 25

<210> 24

<211> 25

<212> PRT

<213> artificial sequence

<220>

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<400> 24

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser

20 25

<210> 25

<211> 25

<212> PRT

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<220>

<223> FR1-P67

<400> 25

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Val Ser

20 25

<210> 26

<211> 17

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<223> FR2-P64

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Ala Gly Trp Tyr Arg Gln Val Pro Gly Lys Gln Arg Glu Leu Val Ala

1 5 10 15

Ala

<210> 27

<211> 38

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<223> FR3-P64

<400> 27

Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Thr Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 28

<211> 38

<212> PRT

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<223> FR3-P57

<400> 28

Asn Tyr Ala Asp Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 29

<211> 38

<212> PRT

<213> artificial sequence

<220>

<223> FR3-P67

<400> 29

Asp Tyr Ala Asp Ser Val Lys Val Arg Phe Thr Val Ser Arg Asp Asn

1 5 10 15

Gly Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 30

<211> 119

<212> PRT

<213> artificial sequence

<220>

<223> VHH-P64

<400> 30

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Gly Ile Arg Ser Ile Ile

20 25 30

Pro Ala Gly Trp Tyr Arg Gln Val Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Ser Thr Ala Gly Ser Ala Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Ala Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Gly Arg Gln Thr Gly Trp Arg Thr Arg Val Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 31

<211> 121

<212> PRT

<213> artificial sequence

<220>

<223> VHH-P57

<400> 31

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Asn Ile Phe Ser Leu Leu

20 25 30

Asp Met Lys Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Asp Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Pro Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Ala Pro Tyr Tyr Trp Gly Ala Tyr Arg Arg Gln Glu Ser Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 32

<211> 123

<212> PRT

<213> artificial sequence

<220>

<223> VHH-P67

<400> 32

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ser Ile Phe Ser Leu Arg

20 25 30

Thr Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Ser Ala Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Val Arg Phe Thr Val Ser Arg Asp Asn Gly Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Ala Gln Asp Ser Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 33

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> CDR1-Kabat-P64/P84

<400> 33

Gly Gly Ile Arg Ser Ile Ile Pro Ala Gly

1 5 10

<210> 34

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> CDR1-Kabat-P57

<400> 34

Gly Asn Ile Phe Ser Leu Leu Asp Met Lys

1 5 10

<210> 35

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> CDR1-Kabat-P67/P120/P121/P122

<400> 35

Gly Ser Ile Phe Ser Leu Arg Thr Val Gly

1 5 10

<210> 36

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> CDR2-Kabat-P64/P84

<400> 36

Ala Ile Ser Thr Ala Gly Ser Ala Asp Tyr Ala Asp Ser Val Lys Gly

1 5 10 15

<210> 37

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> CDR2-Kabat-P57

<400> 37

Asp Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Pro Val Lys Gly

1 5 10 15

<210> 38

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> CDR2-Kabat-P67

<400> 38

Thr Ile Ser Ala Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys Val

1 5 10 15

<210> 39

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> FR1-P84

<400> 39

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser

20 25

<210> 40

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> FR1-P119

<400> 40

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser

20 25

<210> 41

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> FR2-P84

<400> 41

Ala Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala

1 5 10 15

Ala

<210> 42

<211> 38

<212> PRT

<213> artificial sequence

<220>

<223> FR3-P119

<400> 42

Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 43

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> CDR1-Kabat-P119

<400> 43

Gly Ser Ile Phe Ser Leu Leu Asp Met Lys

1 5 10

<210> 44

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> CDR2-Kabat-P119

<400> 44

Asp Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly

1 5 10 15

<210> 45

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> CDR2-Kabat-P120/P121/P122

<400> 45

Thr Ile Ser Ala Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys Gly

1 5 10 15

<210> 46

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> GS linker

<400> 46

Gly Gly Gly Gly Ser

1 5

<210> 47

<211> 345

<212> PRT

<213> artificial sequence

<220>

<223> P64-Fc

<400> 47

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Gly Ile Arg Ser Ile Ile

20 25 30

Pro Ala Gly Trp Tyr Arg Gln Val Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Ser Thr Ala Gly Ser Ala Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Ala Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Gly Arg Gln Thr Gly Trp Arg Thr Arg Val Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

115 120 125

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

130 135 140

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

145 150 155 160

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

165 170 175

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

180 185 190

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

195 200 205

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

210 215 220

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

225 230 235 240

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

245 250 255

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

260 265 270

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

275 280 285

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

290 295 300

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

305 310 315 320

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

325 330 335

Gln Lys Ser Leu Ser Leu Ser Pro Gly

340 345

<210> 48

<211> 345

<212> PRT

<213> artificial sequence

<220>

<223> P84-Fc

<400> 48

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Gly Ile Arg Ser Ile Ile

20 25 30

Pro Ala Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Ser Thr Ala Gly Ser Ala Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Gly Arg Gln Thr Gly Trp Arg Thr Arg Val Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

115 120 125

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

130 135 140

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

145 150 155 160

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

165 170 175

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

180 185 190

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

195 200 205

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

210 215 220

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

225 230 235 240

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

245 250 255

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

260 265 270

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

275 280 285

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

290 295 300

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

305 310 315 320

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

325 330 335

Gln Lys Ser Leu Ser Leu Ser Pro Gly

340 345

<210> 49

<211> 347

<212> PRT

<213> artificial sequence

<220>

<223> P57-Fc

<400> 49

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Asn Ile Phe Ser Leu Leu

20 25 30

Asp Met Lys Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Asp Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Pro Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Ala Pro Tyr Tyr Trp Gly Ala Tyr Arg Arg Gln Glu Ser Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Asp Lys Thr His Thr Cys Pro

115 120 125

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

130 135 140

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

145 150 155 160

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

165 170 175

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

180 185 190

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

195 200 205

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

210 215 220

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

225 230 235 240

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

245 250 255

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

260 265 270

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

275 280 285

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

290 295 300

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

305 310 315 320

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

325 330 335

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

340 345

<210> 50

<211> 347

<212> PRT

<213> artificial sequence

<220>

<223> P119-Fc

<400> 50

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Ser Ile Phe Ser Leu Leu

20 25 30

Asp Met Lys Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Asp Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Ala Pro Tyr Tyr Trp Gly Ala Tyr Arg Arg Gln Glu Ser Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Asp Lys Thr His Thr Cys Pro

115 120 125

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

130 135 140

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

145 150 155 160

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

165 170 175

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

180 185 190

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

195 200 205

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

210 215 220

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

225 230 235 240

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

245 250 255

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

260 265 270

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

275 280 285

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

290 295 300

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

305 310 315 320

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

325 330 335

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

340 345

<210> 51

<211> 349

<212> PRT

<213> artificial sequence

<220>

<223> P67-Fc

<400> 51

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ser Ile Phe Ser Leu Arg

20 25 30

Thr Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Ser Ala Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Val Arg Phe Thr Val Ser Arg Asp Asn Gly Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Ala Gln Asp Ser Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Asp Lys Thr His Thr

115 120 125

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

130 135 140

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

145 150 155 160

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

165 170 175

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

180 185 190

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

195 200 205

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

210 215 220

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

225 230 235 240

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

245 250 255

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

260 265 270

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

275 280 285

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

290 295 300

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

305 310 315 320

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

325 330 335

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

340 345

<210> 52

<211> 349

<212> PRT

<213> artificial sequence

<220>

<223> P120-Fc

<400> 52

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ser Ile Phe Ser Leu Arg

20 25 30

Thr Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Ser Ala Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Ala Gln Asp Ser Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Asp Lys Thr His Thr

115 120 125

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

130 135 140

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

145 150 155 160

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

165 170 175

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

180 185 190

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

195 200 205

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

210 215 220

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

225 230 235 240

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

245 250 255

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

260 265 270

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

275 280 285

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

290 295 300

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

305 310 315 320

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

325 330 335

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

340 345

<210> 53

<211> 349

<212> PRT

<213> artificial sequence

<220>

<223> P121-Fc

<400> 53

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ser Ile Phe Ser Leu Arg

20 25 30

Thr Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Ser Ala Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Ala Gln Gln Ser Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Asp Lys Thr His Thr

115 120 125

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

130 135 140

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

145 150 155 160

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

165 170 175

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

180 185 190

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

195 200 205

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

210 215 220

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

225 230 235 240

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

245 250 255

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

260 265 270

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

275 280 285

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

290 295 300

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

305 310 315 320

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

325 330 335

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

340 345

<210> 54

<211> 349

<212> PRT

<213> artificial sequence

<220>

<223> P122-Fc

<400> 54

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ser Ile Phe Ser Leu Arg

20 25 30

Thr Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Ser Ala Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Ala Gln Asp Thr Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Asp Lys Thr His Thr

115 120 125

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

130 135 140

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

145 150 155 160

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

165 170 175

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

180 185 190

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

195 200 205

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

210 215 220

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

225 230 235 240

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

245 250 255

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

260 265 270

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

275 280 285

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

290 295 300

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

305 310 315 320

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

325 330 335

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

340 345

<210> 55

<211> 355

<212> PRT

<213> artificial sequence

<220>

<223> P64-10GS-Fc

<400> 55

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Gly Ile Arg Ser Ile Ile

20 25 30

Pro Ala Gly Trp Tyr Arg Gln Val Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Ser Thr Ala Gly Ser Ala Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Ala Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Gly Arg Gln Thr Gly Trp Arg Thr Arg Val Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

130 135 140

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

145 150 155 160

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

165 170 175

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

180 185 190

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

195 200 205

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

210 215 220

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

225 230 235 240

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

245 250 255

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

260 265 270

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

275 280 285

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

290 295 300

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

305 310 315 320

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

325 330 335

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

340 345 350

Ser Pro Gly

355

<210> 56

<211> 355

<212> PRT

<213> artificial sequence

<220>

<223> P84-10GS-Fc

<400> 56

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Gly Ile Arg Ser Ile Ile

20 25 30

Pro Ala Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Ser Thr Ala Gly Ser Ala Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Gly Arg Gln Thr Gly Trp Arg Thr Arg Val Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

130 135 140

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

145 150 155 160

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

165 170 175

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

180 185 190

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

195 200 205

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

210 215 220

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

225 230 235 240

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

245 250 255

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

260 265 270

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

275 280 285

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

290 295 300

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

305 310 315 320

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

325 330 335

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

340 345 350

Ser Pro Gly

355

<210> 57

<211> 357

<212> PRT

<213> artificial sequence

<220>

<223> P57-10GS-Fc

<400> 57

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Asn Ile Phe Ser Leu Leu

20 25 30

Asp Met Lys Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Asp Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Pro Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Ala Pro Tyr Tyr Trp Gly Ala Tyr Arg Arg Gln Glu Ser Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

130 135 140

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

145 150 155 160

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

165 170 175

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

180 185 190

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

195 200 205

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

210 215 220

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

225 230 235 240

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

245 250 255

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

260 265 270

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

275 280 285

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

290 295 300

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

305 310 315 320

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

325 330 335

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

340 345 350

Ser Leu Ser Pro Gly

355

<210> 58

<211> 357

<212> PRT

<213> artificial sequence

<220>

<223> P119-10GS-Fc

<400> 58

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Ser Ile Phe Ser Leu Leu

20 25 30

Asp Met Lys Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Asp Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Ala Pro Tyr Tyr Trp Gly Ala Tyr Arg Arg Gln Glu Ser Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

130 135 140

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

145 150 155 160

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

165 170 175

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

180 185 190

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

195 200 205

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

210 215 220

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

225 230 235 240

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

245 250 255

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

260 265 270

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

275 280 285

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

290 295 300

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

305 310 315 320

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

325 330 335

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

340 345 350

Ser Leu Ser Pro Gly

355

<210> 59

<211> 359

<212> PRT

<213> artificial sequence

<220>

<223> P67-10GS-Fc

<400> 59

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ser Ile Phe Ser Leu Arg

20 25 30

Thr Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Ser Ala Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Val Arg Phe Thr Val Ser Arg Asp Asn Gly Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Ala Gln Asp Ser Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly

355

<210> 60

<211> 359

<212> PRT

<213> artificial sequence

<220>

<223> P120-10GS-Fc

<400> 60

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ser Ile Phe Ser Leu Arg

20 25 30

Thr Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Ser Ala Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Ala Gln Asp Ser Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly

355

<210> 61

<211> 359

<212> PRT

<213> artificial sequence

<220>

<223> P121-10GS-Fc

<400> 61

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ser Ile Phe Ser Leu Arg

20 25 30

Thr Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Ser Ala Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Ala Gln Gln Ser Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly

355

<210> 62

<211> 359

<212> PRT

<213> artificial sequence

<220>

<223> P122-10GS-Fc

<400> 62

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ser Ile Phe Ser Leu Arg

20 25 30

Thr Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Ser Ala Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Ala Gln Asp Thr Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly

355

<210> 63

<211> 365

<212> PRT

<213> artificial sequence

<220>

<223> P64-20GS-Fc

<400> 63

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Gly Ile Arg Ser Ile Ile

20 25 30

Pro Ala Gly Trp Tyr Arg Gln Val Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Ser Thr Ala Gly Ser Ala Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Ala Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Gly Arg Gln Thr Gly Trp Arg Thr Arg Val Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr

130 135 140

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

145 150 155 160

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

165 170 175

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

180 185 190

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

195 200 205

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

210 215 220

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

225 230 235 240

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

245 250 255

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

260 265 270

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

275 280 285

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

290 295 300

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

305 310 315 320

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

325 330 335

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

340 345 350

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

355 360 365

<210> 64

<211> 365

<212> PRT

<213> artificial sequence

<220>

<223> P84-20GS-Fc

<400> 64

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Gly Ile Arg Ser Ile Ile

20 25 30

Pro Ala Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Ser Thr Ala Gly Ser Ala Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Gly Arg Gln Thr Gly Trp Arg Thr Arg Val Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr

130 135 140

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

145 150 155 160

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

165 170 175

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

180 185 190

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

195 200 205

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

210 215 220

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

225 230 235 240

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

245 250 255

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

260 265 270

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

275 280 285

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

290 295 300

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

305 310 315 320

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

325 330 335

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

340 345 350

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

355 360 365

<210> 65

<211> 367

<212> PRT

<213> artificial sequence

<220>

<223> P57-20GS-Fc

<400> 65

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Asn Ile Phe Ser Leu Leu

20 25 30

Asp Met Lys Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Asp Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Pro Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Ala Pro Tyr Tyr Trp Gly Ala Tyr Arg Arg Gln Glu Ser Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr

130 135 140

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

145 150 155 160

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

165 170 175

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

180 185 190

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

195 200 205

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

210 215 220

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

225 230 235 240

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

245 250 255

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

260 265 270

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

275 280 285

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

290 295 300

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

305 310 315 320

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

325 330 335

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

340 345 350

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

355 360 365

<210> 66

<211> 367

<212> PRT

<213> artificial sequence

<220>

<223> P119-20GS-Fc

<400> 66

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Ser Ile Phe Ser Leu Leu

20 25 30

Asp Met Lys Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Asp Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Ala Pro Tyr Tyr Trp Gly Ala Tyr Arg Arg Gln Glu Ser Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr

130 135 140

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

145 150 155 160

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

165 170 175

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

180 185 190

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

195 200 205

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

210 215 220

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

225 230 235 240

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

245 250 255

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

260 265 270

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

275 280 285

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

290 295 300

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

305 310 315 320

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

325 330 335

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

340 345 350

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

355 360 365

<210> 67

<211> 369

<212> PRT

<213> artificial sequence

<220>

<223> P67-20GS-Fc

<400> 67

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ser Ile Phe Ser Leu Arg

20 25 30

Thr Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Ser Ala Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Val Arg Phe Thr Val Ser Arg Asp Asn Gly Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Ala Gln Asp Ser Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

130 135 140

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

145 150 155 160

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

165 170 175

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

180 185 190

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

195 200 205

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

210 215 220

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

225 230 235 240

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

245 250 255

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

260 265 270

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

275 280 285

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

290 295 300

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

305 310 315 320

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

325 330 335

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

340 345 350

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

355 360 365

Gly

<210> 68

<211> 369

<212> PRT

<213> artificial sequence

<220>

<223> P120-20GS-Fc

<400> 68

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ser Ile Phe Ser Leu Arg

20 25 30

Thr Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Ser Ala Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Ala Gln Asp Ser Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

130 135 140

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

145 150 155 160

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

165 170 175

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

180 185 190

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

195 200 205

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

210 215 220

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

225 230 235 240

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

245 250 255

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

260 265 270

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

275 280 285

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

290 295 300

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

305 310 315 320

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

325 330 335

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

340 345 350

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

355 360 365

Gly

<210> 69

<211> 369

<212> PRT

<213> artificial sequence

<220>

<223> P121-20GS-Fc

<400> 69

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ser Ile Phe Ser Leu Arg

20 25 30

Thr Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Ser Ala Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Ala Gln Gln Ser Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

130 135 140

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

145 150 155 160

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

165 170 175

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

180 185 190

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

195 200 205

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

210 215 220

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

225 230 235 240

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

245 250 255

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

260 265 270

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

275 280 285

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

290 295 300

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

305 310 315 320

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

325 330 335

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

340 345 350

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

355 360 365

Gly

<210> 70

<211> 369

<212> PRT

<213> artificial sequence

<220>

<223> P122-20GS-Fc

<400> 70

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ser Ile Phe Ser Leu Arg

20 25 30

Thr Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Ser Ala Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Ala Gln Asp Thr Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

130 135 140

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

145 150 155 160

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

165 170 175

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

180 185 190

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

195 200 205

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

210 215 220

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

225 230 235 240

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

245 250 255

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

260 265 270

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

275 280 285

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

290 295 300

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

305 310 315 320

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

325 330 335

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

340 345 350

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

355 360 365

Gly

<210> 71

<211> 226

<212> PRT

<213> artificial sequence

<220>

<223> IgG1 short hinge

<400> 71

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 72

<211> 355

<212> PRT

<213> Chile person

<400> 72

Met Asp Tyr Thr Leu Asp Leu Ser Val Thr Thr Val Thr Asp Tyr Tyr

1 5 10 15

Tyr Pro Asp Ile Phe Ser Ser Pro Cys Asp Ala Glu Leu Ile Gln Thr

20 25 30

Asn Gly Lys Leu Leu Leu Ala Val Phe Tyr Cys Leu Leu Phe Val Phe

35 40 45

Ser Leu Leu Gly Asn Ser Leu Val Ile Leu Val Leu Val Val Cys Lys

50 55 60

Lys Leu Arg Ser Ile Thr Asp Val Tyr Leu Leu Asn Leu Ala Leu Ser

65 70 75 80

Asp Leu Leu Phe Val Phe Ser Phe Pro Phe Gln Thr Tyr Tyr Leu Leu

85 90 95

Asp Gln Trp Val Phe Gly Thr Val Met Cys Lys Val Val Ser Gly Phe

100 105 110

Tyr Tyr Ile Gly Phe Tyr Ser Ser Met Phe Phe Ile Thr Leu Met Ser

115 120 125

Val Asp Arg Tyr Leu Ala Val Val His Ala Val Tyr Ala Leu Lys Val

130 135 140

Arg Thr Ile Arg Met Gly Thr Thr Leu Cys Leu Ala Val Trp Leu Thr

145 150 155 160

Ala Ile Met Ala Thr Ile Pro Leu Leu Val Phe Tyr Gln Val Ala Ser

165 170 175

Glu Asp Gly Val Leu Gln Cys Tyr Ser Phe Tyr Asn Gln Gln Thr Leu

180 185 190

Lys Trp Lys Ile Phe Thr Asn Phe Lys Met Asn Ile Leu Gly Leu Leu

195 200 205

Ile Pro Phe Thr Ile Phe Met Phe Cys Tyr Ile Lys Ile Leu His Gln

210 215 220

Leu Lys Arg Cys Gln Asn His Asn Lys Thr Lys Ala Ile Arg Leu Val

225 230 235 240

Leu Ile Val Val Ile Ala Ser Leu Leu Phe Trp Val Pro Phe Asn Val

245 250 255

Val Leu Phe Leu Thr Ser Leu His Ser Met His Ile Leu Asp Gly Cys

260 265 270

Ser Ile Ser Gln Gln Leu Thr Tyr Ala Thr His Val Thr Glu Ile Ile

275 280 285

Ser Phe Thr His Cys Cys Val Asn Pro Val Ile Tyr Ala Phe Val Gly

290 295 300

Glu Lys Phe Lys Lys His Leu Ser Glu Ile Phe Gln Lys Ser Cys Ser

305 310 315 320

Gln Ile Phe Asn Tyr Leu Gly Arg Gln Met Pro Arg Glu Ser Cys Glu

325 330 335

Lys Ser Ser Ser Cys Gln Gln His Ser Ser Arg Ser Ser Ser Val Asp

340 345 350

Tyr Ile Leu

355

<210> 73

<211> 366

<212> PRT

<213> artificial sequence

<220>

<223> hCCR8-δ18-3XHA

<400> 73

Met Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ala Tyr Pro Tyr Asp Val

1 5 10 15

Pro Asp Tyr Ala Gly Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ile Phe

20 25 30

Ser Ser Pro Cys Asp Ala Glu Leu Ile Gln Thr Asn Gly Lys Leu Leu

35 40 45

Leu Ala Val Phe Tyr Cys Leu Leu Phe Val Phe Ser Leu Leu Gly Asn

50 55 60

Ser Leu Val Ile Leu Val Leu Val Val Cys Lys Lys Leu Arg Ser Ile

65 70 75 80

Thr Asp Val Tyr Leu Leu Asn Leu Ala Leu Ser Asp Leu Leu Phe Val

85 90 95

Phe Ser Phe Pro Phe Gln Thr Tyr Tyr Leu Leu Asp Gln Trp Val Phe

100 105 110

Gly Thr Val Met Cys Lys Val Val Ser Gly Phe Tyr Tyr Ile Gly Phe

115 120 125

Tyr Ser Ser Met Phe Phe Ile Thr Leu Met Ser Val Asp Arg Tyr Leu

130 135 140

Ala Val Val His Ala Val Tyr Ala Leu Lys Val Arg Thr Ile Arg Met

145 150 155 160

Gly Thr Thr Leu Cys Leu Ala Val Trp Leu Thr Ala Ile Met Ala Thr

165 170 175

Ile Pro Leu Leu Val Phe Tyr Gln Val Ala Ser Glu Asp Gly Val Leu

180 185 190

Gln Cys Tyr Ser Phe Tyr Asn Gln Gln Thr Leu Lys Trp Lys Ile Phe

195 200 205

Thr Asn Phe Lys Met Asn Ile Leu Gly Leu Leu Ile Pro Phe Thr Ile

210 215 220

Phe Met Phe Cys Tyr Ile Lys Ile Leu His Gln Leu Lys Arg Cys Gln

225 230 235 240

Asn His Asn Lys Thr Lys Ala Ile Arg Leu Val Leu Ile Val Val Ile

245 250 255

Ala Ser Leu Leu Phe Trp Val Pro Phe Asn Val Val Leu Phe Leu Thr

260 265 270

Ser Leu His Ser Met His Ile Leu Asp Gly Cys Ser Ile Ser Gln Gln

275 280 285

Leu Thr Tyr Ala Thr His Val Thr Glu Ile Ile Ser Phe Thr His Cys

290 295 300

Cys Val Asn Pro Val Ile Tyr Ala Phe Val Gly Glu Lys Phe Lys Lys

305 310 315 320

His Leu Ser Glu Ile Phe Gln Lys Ser Cys Ser Gln Ile Phe Asn Tyr

325 330 335

Leu Gly Arg Gln Met Pro Arg Glu Ser Cys Glu Lys Ser Ser Ser Cys

340 345 350

Gln Gln His Ser Ser Arg Ser Ser Ser Val Asp Tyr Ile Leu

355 360 365

<210> 74

<211> 353

<212> PRT

<213> mice

<400> 74

Met Asp Tyr Thr Met Glu Pro Asn Val Thr Met Thr Asp Tyr Tyr Pro

1 5 10 15

Asp Phe Phe Thr Ala Pro Cys Asp Ala Glu Phe Leu Leu Arg Gly Ser

20 25 30

Met Leu Tyr Leu Ala Ile Leu Tyr Cys Val Leu Phe Val Leu Gly Leu

35 40 45

Leu Gly Asn Ser Leu Val Ile Leu Val Leu Val Gly Cys Lys Lys Leu

50 55 60

Arg Ser Ile Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ala Ser Asp Leu

65 70 75 80

Leu Phe Val Leu Ser Ile Pro Phe Gln Thr His Asn Leu Leu Asp Gln

85 90 95

Trp Val Phe Gly Thr Ala Met Cys Lys Val Val Ser Gly Leu Tyr Tyr

100 105 110

Ile Gly Phe Phe Ser Ser Met Phe Phe Ile Thr Leu Met Ser Val Asp

115 120 125

Arg Tyr Leu Ala Ile Val His Ala Val Tyr Ala Ile Lys Val Arg Thr

130 135 140

Ala Ser Val Gly Thr Ala Leu Ser Leu Thr Val Trp Leu Ala Ala Val

145 150 155 160

Thr Ala Thr Ile Pro Leu Met Val Phe Tyr Gln Val Ala Ser Glu Asp

165 170 175

Gly Met Leu Gln Cys Phe Gln Phe Tyr Glu Glu Gln Ser Leu Arg Trp

180 185 190

Lys Leu Phe Thr His Phe Glu Ile Asn Ala Leu Gly Leu Leu Leu Pro

195 200 205

Phe Ala Ile Leu Leu Phe Cys Tyr Val Arg Ile Leu Gln Gln Leu Arg

210 215 220

Gly Cys Leu Asn His Asn Arg Thr Arg Ala Ile Lys Leu Val Leu Thr

225 230 235 240

Val Val Ile Val Ser Leu Leu Phe Trp Val Pro Phe Asn Val Ala Leu

245 250 255

Phe Leu Thr Ser Leu His Asp Leu His Ile Leu Asp Gly Cys Ala Thr

260 265 270

Arg Gln Arg Leu Ala Leu Ala Ile His Val Thr Glu Val Ile Ser Phe

275 280 285

Thr His Cys Cys Val Asn Pro Val Ile Tyr Ala Phe Ile Gly Glu Lys

290 295 300

Phe Lys Lys His Leu Met Asp Val Phe Gln Lys Ser Cys Ser His Ile

305 310 315 320

Phe Leu Tyr Leu Gly Arg Gln Met Pro Val Gly Ala Leu Glu Arg Gln

325 330 335

Leu Ser Ser Asn Gln Arg Ser Ser His Ser Ser Thr Leu Asp Asp Ile

340 345 350

Leu

<210> 75

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> CDR1-Kabat-P64/P84

<400> 75

Ile Ile Pro Ala Gly

1 5

<210> 76

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> CDR1-Kabat-P57/P119

<400> 76

Leu Leu Asp Met Lys

1 5

<210> 77

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> CDR1-Kabat-P67/P120/P121/P122

<400> 77

Leu Arg Thr Val Gly

1 5

<210> 78

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> CDR3-Kabat-P64/P84

<400> 78

Arg Gln Thr Gly Trp Arg Thr Arg Val Asp Tyr

1 5 10

<210> 79

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> CDR3-Kabat-P57/P119

<400> 79

Ala Pro Tyr Tyr Trp Gly Ala Tyr Arg Arg Gln Glu Ser

1 5 10

<210> 80

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> CDR3-Kabat-P67/P120

<400> 80

Gln Asp Ser Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp Tyr

1 5 10 15

<210> 81

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> CDR3-Kabat-P121

<400> 81

Gln Gln Ser Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp Tyr

1 5 10 15

<210> 82

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> CDR3-Kabat-P122

<400> 82

Tyr Ala Gln Asp Thr Tyr Lys Ile Tyr Lys Ser Arg Tyr Thr Gln Asp

1 5 10 15

Tyr

<210> 83

<211> 98

<212> PRT

<213> Chile person

<400> 83

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Ser Ser Asp Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210> 84

<211> 11

<212> PRT

<213> Chile person

<400> 84

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

Claims

1. A human CCR8 (hCCR 8) binding agent, wherein the binding agent is cross-reactive with murine CCR 8.

2. A binding agent according to claim 1, wherein the binding agent binds to the extracellular loop of hCCR 8.

3. A binding agent according to claim 1 or claim 2, wherein the binding agent binds to the extracellular loop 2 of hCCR 8.

4. The binding agent of any one of the preceding claims, comprising a single domain antibody moiety that binds to human CCR 8.

5. The binding agent of claim 4, wherein the single domain antibody portion comprises three Complementarity Determining Regions (CDRs), CDR1, CDR2, and CDR3, wherein CDR3 is selected from the group consisting of:

a) NGRQTGWRTRVDY (SEQ ID NO: 7);

b) NAAPYYWGAYRRQES (SEQ ID NO: 8);

c) YAQDSYKIYKSRYTQDY (SEQ ID NO: 9);

d) YAQQSYKIYKSRYTQDY (SEQ ID NO: 10);

e) YAQDTYKIYKSRYTQDY (SEQ ID NO: 11);

f) An amino acid sequence having at least 80% amino acid identity to SEQ ID No. 7, 8, 9, 10 or 11; and

g) An amino acid sequence having a 3, 2 or 1 amino acid difference from SEQ ID NO 7, 8, 9, 10 or 11.

6. The binding agent of claim 5, wherein

CDR1 is selected from:

a) The amino acid sequence of GGIRSIIP (SEQ ID NO: 1);

b) The amino acid sequence of GSIFSLLD (SEQ ID NO: 2);

c) The amino acid sequence of GSIFSLRT (SEQ ID NO: 3);

d) An amino acid sequence having at least 80% amino acid identity to SEQ ID No. 1, 2 or 3; and

e) Amino acid sequence with 3, 2, 1 amino acid differences with SEQ ID NO 1, 2 or 3;

and is also provided with

CDR2 is selected from:

f) The amino acid sequence of ISTAGSA (SEQ ID NO: 4);

g) The amino acid sequence of ITSGGST (SEQ ID NO: 5);

h) The amino acid sequence of ISAGGAT (SEQ ID NO: 6);

i) An amino acid sequence having at least 80% amino acid identity to SEQ ID No. 4, 5 or 6; and

j) Amino acid sequence having 3, 2, 1 amino acid differences from SEQ ID NO 4, 5 or 6.

7. The binding agent of claim 5 or claim 6, wherein the single domain antibody portion further comprises four Framework Regions (FR), FR1, FR2, FR3 and FR4, wherein

-FR1 has at least 85% sequence identity with SEQ ID NO. 12;

FR2 has at least 85% sequence identity with SEQ ID NO. 13 or SEQ ID NO. 14:

-FR3 has at least 85% sequence identity with SEQ ID NO. 15;

FR4 has at least 85% sequence identity with SEQ ID NO. 16.

8. The binding agent of claims 4 to 7, wherein the single domain antibody moiety comprises the amino acid sequence of SEQ ID No. 17 or SEQ ID No. 18 or SEQ ID No. 19 or SEQ ID No. 20 or SEQ ID No. 21.

9. The binding agent of any one of the preceding claims, comprising a cytotoxic moiety.

10. The binding agent of claim 9, wherein the cytotoxic moiety

Inducing Antibody Dependent Cellular Cytotoxicity (ADCC),

inducing Complement Dependent Cytotoxicity (CDC),

inducing Antibody Dependent Cellular Phagocytosis (ADCP),

binding and activating T cells, or

-comprising a cytotoxic payload.

11. A nucleic acid encoding the binding agent of any one of the preceding claims.

12. The binding agent according to any one of claims 1 to 10 or the nucleic acid according to claim 11 for use as a medicament.

13. The binding agent according to any one of claims 1 to 10 or the nucleic acid according to claim 11 for use in the treatment of a tumor.

14. The binding agent or nucleic acid for use according to claim 13, wherein the tumour is selected from breast cancer, endometrial cancer, lung cancer, gastric cancer, head and neck squamous cell carcinoma, skin cancer, colorectal cancer, renal cancer and T-cell lymphoma.

15. The binding agent or nucleic acid for use according to any one of claims 11 to 14, wherein the treatment further comprises administration of a checkpoint inhibitor, such as an inhibitor that blocks PD-1 or PD-L1.