CCR8 antibody for therapeutic use

Abstract

The present invention relates to tools and methods for generating antibodies that specifically bind to chemokine receptors, such as CC or CXC chemokine receptors. To facilitate the generation of anti-human, anti-cynomolgus, and / or anti-mouse chemokine receptor antibodies, isolated sulfated polypeptides and conjugates thereof are provided, which can be used, for example, as antigens or for off-target panning, to generate antibodies with fully human CDRs and / or other desirable properties for therapeutic use. The present invention also relates to antibodies and conjugates thereof obtainable by applying the above-described tools and methods. Antibodies that specifically bind to human, cynomolgus, and / or mouse CCR8 with desirable properties for therapeutic use, such as cross-reactive antibodies, fully human antibodies, low-internalizing (including non-internalizing) antibodies, and antibodies that efficiently induce ADCC and / or ADCP in Treg cells, are provided. Medical uses of the antibodies or conjugates of the present invention and / or treatment methods comprising administering these antibodies, alone or in combination, to patients or subjects are also provided. Biomarkers, stratification methods and diagnostic methods for predicting or assessing responsiveness to anti-CCR8 antibody monotherapy or combination therapy are finally provided. The invention further provides tools and methods for producing the aforementioned antibodies, pharmaceutical compositions, diagnostic uses of the antibodies, and kits with instructions for use.

Description

[Technical Field]

[0001] The present invention relates to tools and methods for producing antibodies that specifically bind to chemokine receptors such as CC or CXC chemokine receptors. Isolated sulfated polypeptides and their conjugates are provided that can be used, for example, as antigens or for off-target panning, to facilitate the production of anti-human, anti-cynomolgus monkey and / or anti-mouse chemokine receptor antibodies, for example, for the production of antibodies having other desirable properties for full human CDR and / or therapeutic use.

[0002] The present invention further relates to antibodies and their conjugates that can be obtained by applying the tools and methods described above. Antibodies that have desirable properties for therapeutic use and that specifically bind to human, cynomolgus monkey, and / or mouse CCR8 are provided, such as cross-reactive antibodies, fully human antibodies, low-internalized (including non-internalized) antibodies, and antibodies that efficiently induce ADCC and / or ADCP in Treg cells.

[0003] The present invention also provides medical uses of the antibodies or conjugates, and / or treatment methods comprising administering these antibodies alone or in combination to a patient or subject. Ultimately, biomarkers, stratification methods, and diagnostic methods for predicting or evaluating responsiveness to anti-CCR8 antibody monotherapy or combination therapy are provided.

[0004] The present invention further provides tools and methods for producing the aforementioned antibodies, pharmaceutical compositions, diagnostic uses of the antibodies, and kits with instructions for use. [Background technology]

[0005] technical challenges Antibody production against CC and CXC chemokine receptors CXC and CC chemokine receptors are specific seven-transmembrane G protein-coupled receptors that mediate cell migration in the chemotactic gradient. Due to their unique structural, biophysical, and biological properties, chemokine receptors are a challenging target class for antibody production.

[0006] There are several reasons why obtaining optimal antibodies against chemokine receptors is difficult, and these will be discussed in an exemplary manner, with a focus on human CCR8.

[0007] Because chemokine receptors are characterized by seven domains embedded in the cell membrane, they cannot be easily purified by their native identification. Purified native chemokine receptors are removed from the membrane environment and are therefore potentially conformationally impaired. These disrupted structures are typically unsuitable for antibody production, as antibodies are required to recognize intact antigens when they are presented on the cell surface. While some chemokine receptors, such as CXCR4 and CCR5, possess some inherent stability that allows for purification with mild surfactants, this is not true for most chemokine receptors (Hutchings, Catherine J., et al. “Opportunities for therapeutic antibodies directed at G-protein-coupled receptors.” Nature reviews Drug discovery 16.11(2017):787.). This is highlighted by the fact that, to date, there are no X-ray crystal structures available for CCR8 in the Protein Data Bank (PDB) (rcsb.org).

[0008] As a result, solubilization to obtain the necessary amount of protein in the natural conformation, as well as the correct orientation and folding for use as an immunogen, is difficult for chemokine receptors (see Klarenbeek, Alex, et al. "Targeting chemokines and chemokine receptors with antibodies." Drug Discovery Today: Technologies 9.4(2012):e237-e244).

[0009] Figure 2b shows a schematic diagram of the overall structure of human CCR8. Of the 355 amino acids, residues 1-35 (N-terminus), 94-107 (ECL1), 172-202 (ECL2), and 264-280 (ECL3) are predicted to be extracellular domains (uniprot.org). These extracellular domains are thought to adopt a tertiary structure stabilized by disulfide bonds. Therefore, on average, less than 30% of the chemokine receptor is exposed on the cell surface. As a result, as described in International Publication No. 200744756, the chemokine receptor, particularly CCR8, is not readily accessible for antibody binding.

[0010] Furthermore, antibodies produced against peptides corresponding to the extracellular domain of chemokine receptors often fail to recognize intact receptors on cells, likely due to differences in secondary structure. As a result, researchers in this field have had a low success rate in antibody development (Tschammer, Nuska, ed. Chemokines: chemokines and their receptors in drug discovery. Vol. 14. Springer, 2015, Chapter by JEPease & R. Horuk, section 6, page 12, 2nd para).

[0011] Antibody generation to obtain anti-mouse CCR8 antibodies appears to be relatively easy, and conventional approaches, such as those described by Kremer and Marquez, were reproducible by the present inventors (D'Ambrosio, Daniele, and Francesco Sinigaglia. Cell Migration in Inflammation and Immunity. Springer, 2004., Chapter by Kremer and Marquez pp. 243-260). Mouse and human CCR8 have approximately 70% sequence identity, which is even lower for the extracellular domain, as described in detail in Example 2. Nevertheless, Kremer and Marquez argue that the production of anti-mouse chemokine receptor monoclonal antibodies is a challenging task, and that antibodies against mouse chemokine receptors are relatively scarce despite the time elapsed since their amino acid sequences were first reported.

[0012] Therapeutic antibodies for CCR8 and their medical use Despite the difficulties associated with their production, antibodies targeting CC chemokine receptors have been suggested as promising therapeutic tools in various diseases, based on mechanistic insights into diseases involving immune cell involvement or various cancer indications characterized by abnormal expression of chemokine receptors.

[0013] In 2004, Curiel et al. demonstrated that human tumor Treg cells suppress tumor-specific T cell immunity and contribute to human tumor growth in vivo in 104 individuals with ovarian cancer (Curiel, Tyler J., et al. "Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival." Nature medicine 10.9(2004):942-949).

[0014] Tumor cells and microenvironment macrophages produce chemokines such as CCL22, which mediate the transport of Treg cells to tumors. High levels of Treg cells in the tumor microenvironment are not only associated with poor prognosis in many cancers, including ovarian, breast, renal, and pancreatic cancers, but also suppress the immune response to these cancers, for example by inhibiting the action of effector cells in the immune system. Therefore, this specific recruitment of Treg cells represents a mechanism by which tumors can promote immune privilege. Thus, it has been suggested that blocking the migration or function of Treg cells can inactivate human cancers.

[0015] Recognizing Curiel's findings, two independent teams, around Plitas / Rudensky and De Simone / Abrignani / Pagani, found that tumor-infiltrating Treg cells are characterized by selective expression of CCR8. Indeed, the selectivity of Treg cell depletion against tumor-infiltrating Tregs is important because systemic depletion of Treg cells can trigger severe autoimmunity (Nishikawa, Hiroyoshi, and Shimon Sakaguchi. “Regulatory T cells in tumor immunity.” International journal of cancer 127.4(2010):759-767.). The search for specific Treg markers is complicated by the fact that Treg cells exhibit molecular patterns similar to effector lymphocytes (see Example 11.2). Since peripheral Tregs are important for evading autoimmunity, while tumor-specific effector cells help keep tumors under control, neither peripheral Tregs nor tumor-specific effector cells should be fighting each other during intratumor Treg depletion.

[0016] Based on these findings, various teams have suggested using CCR8 antibodies for the selective depletion of tumor-infiltrating regulatory T cells. Some have presented data confirming tumor reduction in tumor models using anti-human CCR4 or anti-mouse CCR8 antibodies, thereby confirming the mechanistic concept of Treg depletion. However, there is a need for therapeutic anti-human CCR8 antibodies with superior properties for therapeutic applications, for example.

[0017] Conventional technology 1.1 Antigen, Method and Antibody Antigen selection in antibody production is crucial for the characteristics of the resulting antibody and can cause serious problems, as discussed elsewhere in this specification for chemokine receptors such as CCR8. In some cases, antibodies are obtained using whole cells engineered to overexpress chemokine receptors as antigens. For at least some chemokine receptors, these approaches appear to result in a limited number of conjugates. Furthermore, antibodies obtained using these antigens are often characterized by off-target binding and low specificity to chemokine receptors. When whole cells are used as antigens, immunodominant epitopes can mask other low-antigenitor epitopes, which otherwise have the potential to produce the desired selective and specific antibodies.

[0018] Regarding CCR8, a successful "whole-cell" approach is described in International Publication No. 2007044756 (ICOS). Briefly, anti-CCR8 monoclonal antibodies were developed by immunizing Balb / c mice with irradiated cells transfected with CCR8 expressing high levels of CCR8 on the cell surface. Splenocytes derived from these mice were fused using standard methods to create antibody-producing hybridomas. The positive pool was identified by FACS and cloned by limiting dilution. Two of the antibodies, 433H and 459M, showed specific reactivity to human CCR8 by immunohistochemistry. Antibody 433H is still available and can be purchased from BD.

[0019] Biolegend distributes clone L263G8, a purified mouse IgG2a anti-human CCR8 antibody generated using human CCR8 transfectant as the immunogen.

[0020] Kremer and Marquez describe the generation of monoclonal antibodies against mouse CCR8 (D’Ambrosio, Daniele, and Francesco Sinigaglia. Cell Migration in Inflammation and Immunity. Springer, 2004., Chapter by Kremer and Marquez p.243-260). Briefly, while Kremer and Marquez describe the use of a peptide derived from the extracellular domain of mouse CCR8 as an immunogen, they do not suggest tyrosine sulfation. In fact, the present inventors found that although the approach described by Kremer and Marquez can be successfully applied to antibodies recognizing mouse CCR8, the success rate is low for antibodies recognizing human CCR8.

[0021] Schaerli et al. generated anti-human CCR8 antibodies by immunizing rabbits with a human CCR8 peptide conjugate corresponding to positions 1 to 34 of the N-terminal region of CCR8 coupled to either KLH or BSA, and here again do not suggest tyrosine sulfation (Schaerli, Patrick, et al. "A skin-selective homing mechanism for human immune surveillance T cells." The Journal of experimental medicine 199.9(2004):1265-1275.).

[0022] A mouse monoclonal antibody that binds to a 26-amino acid peptide from the extracellular N-terminal portion of CCR8 has been described by Haque et al. (Haque, Nasreen S., et al. "The chemokine receptor CCR8 mediates human endothelial cell chemotaxis induced by I-309 and Kaposi sarcoma herpesvirus-encoded vMIP-I and by lipoprotein(a)-stimulated endothelial cell conditioned medium." Blood, The Journal of the American Society of Hematology 97.1(2001):39-45.).

[0023] None of these methods for antibody production use isolated sulfated polypeptides containing chemokine receptors or tyrosine-rich domains (TRDs) of transmembrane proteins as antigens. The use of chemokine receptor TRDs or isolated sulfated polypeptides containing transmembrane proteins as antigens affects both the structural and functional characteristics of the resulting antibody set, as discussed elsewhere in this specification.

[0024] As a result, the antibodies according to the present invention are assumed to deviate from the aforementioned prior art antibodies in terms of structure and function, for example, in terms of their affinity for sulfated CCR8, their affinity for unsulfated CCR8, the mode and extent to which they modulate receptor signaling, their internalization behavior, their cross-reactivity, their clearance and pharmacokinetic behavior, and finally, their efficacy for Treg depletion and / or therapeutic application.

[0025] Furthermore, the use of the antigens disclosed herein has enabled the production of fully human antibodies. In contrast, the prior art antibodies discussed for CCR8 deviate from some of the antibodies of the present invention in that they are of non-human origin and do not contain at least human CDRs.

[0026] 1.2 Medical Uses and Modes of Action Cancer immunotherapy involves the targeted use of the immune system to treat or prevent cancer. Immunotherapy typically takes advantage of the fact that cancer cells often have subtly different molecules on their surface that can be detected by the immune system, cancer antigens. Therefore, immunotherapy involves inducing the immune system to attack tumor cells via these cancer antigens. However, some cancers, such as solid tumors or hematological malignancies, can evade immune surveillance. For example, tumor infiltration by regulatory T cells (Treg cells or Tregs), more specifically a low ratio of effector T cells (Teffs) to Tregs, has been proposed as an important factor in concealing tumors from the immune system (Smyth, Mark J., Shin Foong Ngiow, and Michele WL Teng. “Targeting regulatory T cells in tumor immunotherapy.” Immunology and cell biology 92.6(2014):473-474.).

[0027] Foxp3-expressing regulatory T cells are essential for preventing autoimmunity and are known to effectively suppress tumor immunity. Treg cells infiltrate tumor tissue abundantly, which is often associated with poor prognosis in cancer patients. While removal of Treg cells enhances the anti-tumor immune response, it can also induce autoimmunity. A key challenge in structuring Treg-targeted cancer immunotherapy lies in the specific depletion of Treg cells that infiltrate tumor tissue without affecting tumor-reactive effector T cells, while suppressing autoimmunity.

[0028] As far back as 2010, various groups had already investigated whether Treg removal by depletion, for example, with anti-CD25 antibodies, could improve antitumor immunity in mice. It had been shown that Treg depletion before tumor cell inoculation resulted in efficient rejection of those cells, but Treg depletion occurring simultaneously with or after tumor inoculation did not lead to tumor regression. Furthermore, it was suggested that this was due to the fact that the administered depletion antibody also removed CD25-expressing effector T cells, but CD25-Foxp3+ Tregs persisted (Klages, Katjana, et al. "Selective depletion of Foxp3+ regulatoryy T cells improves effective therapeutic vaccination against established melanoma." Cancer research 70.20(2010):7788-7799.).

[0029] In 2015, the afucosylated humanized anti-human CCR4 monoclonal antibody mogamulizumab (KW-0761) was evaluated in a clinical trial in patients with CCR4-positive cancer (Kurose, Koji, et al. "Phase Ia study of FoxP3+CD4 Treg depletion by infusion of a humanized anti-CCR4 antibody, KW-0761, in cancer patients." Clinical Cancer Research 21.19(2015):4327-4336.). CCR4 is expressed on regulatory T cells.

[0030] In fact, mogamulizumab efficiently depletes Treg cells, and enhancement or induction of a specific immune response to cancer antigens has been observed. However, mogamulizumab targets both peripheral and intratumoral Treg cells and further effector cell proliferation, thereby causing immunological side effects such as rash or Stevens-Johnson syndrome.

[0031] Therefore, while the great potential of Treg depletion in cancer treatment was clear, the concept of selectively targeting Tregs and avoiding overt autoimmunity was lacking. This was changed by studies by two teams, Plitas / Rudensky and De Simone / Abrignani / Pagani, in 2015 and 2016.

[0032] Plitas et al. demonstrated that CCR8 is selectively expressed by human breast cancer-infiltrating Treg cells and immediately concluded that targeting CCR8 is a promising immunotherapeutic approach for treating patients with breast cancer and other tumors (Plitas, G., et al. “Abstract P4-04-11: Preferred expression of the chemokine receptor 8 (CCR8) on regulatory T cells (Treg) infiltrating human breast cancers represents a novel immunotherapeutic target.” (2016): P4-04.; Plitas, George, et al. “Regulatory T cells exhibit distinct features in human breast cancer.” Immunity 45.5 (2016): 1122-1134.; U.S. Patent No. 10087259).

[0033] Shortly after the initial study by Plitas et al. was published, De Simone et al. published an analysis of the transcriptional landscape of tumor-infiltrating regulatory cells, finding that tumor-infiltrating Treg cells are highly immunosuppressive and express CCR8 on their cell surface as a specific signature molecule (De Simone, Marco, et al. “Transcriptional landscape of human tissue lymphocytes unveils uniqueness of tumor-infiltrating T regulatory cells.” Immunity 45.5(2016):1135-1147., International Publication No. 2017198631). The authors described that CCR8 correlates with poor prognosis and concluded that CCR8 could be an interesting therapeutic target for inhibiting Treg cell transport to tumor sites without interfering with the recruitment of other effector T cells that do not express CCR8.

[0034] International Publication Nos. 2018112032 and International Publication Nos. 2018112033 describe a method for reducing the number or activity of tumor-infiltrating regulatory T cells (TITRs) in a tumor, comprising administering a drug that induces cytotoxicity in cells expressing a gene product, such as CCR8.

[0035] International Publication No. 2018112033 and European Patent No. 3431105 describe molecules that can modulate the expression and / or function of at least one marker selectively deregulated in tumor-infiltrating regulatory T cells for use in the prevention and / or treatment of this tumor.

[0036] International Publication No. 2018181425 relates to an antibody against CCR8 possessing ADCC activity for use in methods of treating cancer, wherein the antibody against CCR8 is a CCR8 neutralizing antibody. International Publication No. 2018181425 does not disclose the specific antibody sequence, but refers to the rat anti-mouse CCR8 clone SA214G2 distributed by BioLegend under catalog number 150302. This antibody was used as a tool antibody to reproduce the aforementioned antitumorogenic effect of Treg depletion in mice.

[0037] The present invention provides a method for promoting the production of anti-CCR8 antibodies, resulting in antibodies with superior properties for therapeutic use. When the antibodies according to the present invention are compared to prior art antibodies, they are found to deviate in structure, function, and therapeutic efficacy, as discussed elsewhere in this specification. [Prior art documents] [Patent Documents]

[0038] [Patent Document 1] International Publication No. 200744756 [Patent Document 2] U.S. Patent No. 10087259 [Patent Document 3] International Publication No. 2017198631 [Patent Document 4] International Publication No. 2018112032 [Patent Document 5] International Publication No. 2018112033 [Patent Document 6] European Patent No. 3431105 [Patent Document 7] International Publication No. 2018181425 [Non-patent literature]

[0039] [Non-Patent Document 1] Hutchings, Catherine J., et al. “Opportunities for therapeutic antibodies directed at G-protein-coupled receptors.” Nature reviews Drug discovery 16.11(2017):787. [Non-Patent Document 2] Klarenbeek, Alex, et al. “Targeting chemokines and chemokine receptors with antibodies.” Drug Discovery TODAY:Technologies 9.4(2012):e237-e244. [Non-Patent Document 3] Tschammer,Nuska,ed.Chemokines:chemokines and their receptors in drug discovery.Vol.14.Springer,2015,Chapter by JEPease & R.Horuk,section 6,page 12,2nd para [Non-Patent Document 4] D'Ambrosio,Daniele, and Francesco Sinigaglia.Cell Migration in Inflammation and Immunity.Springer,2004.,Chapter by Kremer and Marquez p.243-260 [Non-Patent Document 5] Curiel, Tyler J., et al. “Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival.”Nature medicine 10.9(2004):942-949. [Non-Patent Document 6] Nishikawa, Hiroyoshi, and Shimon Sakaguchi.“Regulatory T cells in tumor immunity.” International Journal of Cancer 127.4(2010):759-767.

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[0040] 1.1 Antibody production against CC and CXC chemokine receptors Prior art approaches for producing antibodies against CC chemokine receptors have low success rates, and the resulting antibodies often perform poorly (see, for example, Example 3). Therefore, we have developed a novel method for producing antibodies against chemokine receptors in general, and in particular for producing anti-CCR8 antibodies.

[0041] The use of specifically modified isolated polypeptides as antigens for selecting human anti-human CCR8 antibodies solves the problem of providing an improved method for antibody production against CC and CXC chemokine receptors, thereby enabling the first acquisition of, for example, fully human anti-CCR8 antibodies (see Example 6). More specifically, a polypeptide containing the tyrosine-rich domain of human CCR8 (which may also contain a LID domain) was modified by introducing sulfuric acid modifications at specific positions to form the antigen (see Example 4, Table 4.1). A phage display approach could also be used with this specifically modified antigen to select fully human anti-human CCR8 antibodies. Alternatively, the sulfated antigen can be used in a variety of further methods, such as conventional immunization approaches.

[0042] The obtained antibodies showed excellent binding profiles for sulfated antigens and biological targets expressed under physiological conditions (see Examples 6 and 10.1.1), but showed relatively low affinity or no affinity for unmodified antigens (see Examples 10.1.2 and 10.1.3), demonstrating that sulfated residues are indeed important for antigen-antibody binding. The antibodies of the present invention also showed excellent specific binding to human CCR8, cynomolgus monkey CCR8, or cell lines engineered to express mouse CCR8, and activated human Treg (see Example 10.1.1).

[0043] Of the six CDR loops in the antibody, the H3 loop exhibits the greatest structural diversity and is located at the center of the binding site. It also acquires the most mutations through affinity maturation and, on average, has the highest number of contacts with the antigen. Therefore, it plays a crucial role in antigen binding. Analysis of the specific structure of the antibody obtained by the method according to the present invention revealed, surprisingly, that the composition of HCDR3 was structurally different from that of a normal human HCDR3 domain (see Example 9). In particular, the HCDR3 domain of the antibody with the most therapeutically beneficial properties and specific binding to CCR8 is characterized by an average frequency of approximately 21% tyrosine residues and an average histidine content of approximately 10%, highlighting the beneficial influence of these residues on the recognition of specific sulfated antigens (see Table 9.2).

[0044] While not bound by theory, the inventors believe that these structural features translate into specific functional features that make the resulting antibodies more suitable for therapeutic use. For example, in contrast to prior art antibodies tested, some antibodies according to the present invention block G protein-dependent signaling (see Example 10.4.3) but do not affect G protein-independent pathways (see Examples 10.4, 10.4.1, and 10.4.2) and do not substantially internalize in cells with endogenous expression of the target chemokine receptor (see Example 10.5). Furthermore, particularly preferred antibodies according to the present invention are specific to target receptors and target cells (see Examples 10.2 and 11) and exhibited particularly suitable / excellent properties in methods for treating cancer (see Example 12ff). In addition, the use of these synthetic polypeptides as either antigens or off-target panning facilitated or enabled the generation of cross-reactive antibodies, such as antibodies that specifically bind to human CCR8 and / or cynomolgus monkey CCR8 (see Example 10.1.1).

[0045] 1.2 Provision of therapeutic antibodies that specifically bind to chemokine receptors 1.2.1 Acquisition of chemokine receptor antibodies possessing human CDRs Humanization of antibodies using mouse CDRs can improve their immunogenicity, but residual immunogenicity resides in the CDR region (Harding, Fiona A., et al. "The immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the CDR regions." MAbs. Vol.2. No.3. Taylor & Francis, 2010). Therefore, antibodies containing human CDRs are considered to have superior suitability as human therapeutic agents compared to antibodies containing CDRs from other species.

[0046] For example, antibodies possessing human CDRs can be produced via phage display technology or using transgenic animals capable of generating fully human antibodies. However, obtaining fully human antibodies is not always straightforward. Theoretically, the diversity of the HCDR3 region is almost infinite, but in practice, the generation of diversity in the antibody repertoire appears to be carefully regulated by multiple mechanisms that result in an in vivo repertoire where that diversity is limited and constrained within the range of antigen-binding sites. This is also true for human phage display libraries, which are often designed to reflect the in vivo repertoire. Thus, in some cases, the structural requirements of a “complex” antigen may be met by a rodent CDR, but the same structure may not be readily recognized by a human CDR. Based on the observation that tyrosine presentation is, on average, about 50% lower in human HCDR3 than in mouse HCDR3, it can be assumed that finding human anti-human antibodies is clearly more difficult when the unique binding ability of tyrosine and histidine is required to enable target binding. In fact, we have not recognized any fully human anti-human CCR8 prior art antibodies. According to the present invention, CCR8 antibodies containing human-derived CDRs and fully human antibodies are also provided (see Examples 6, 7, and 8).

[0047] 1.2.2 Acquisition of cross-reactive chemokine receptor antibodies Cross-reactive anti-chemokine receptor antibodies, such as cross-reactive anti-CCR8 antibodies, are advantageous in the development of therapeutic antibodies because they can be used in non-human animal models to characterize therapeutic agents in terms of pharmacological data and safety before the antibodies are administered to humans. However, cross-reactive antibodies that have similar binding behavior in two species, for example, are difficult to produce and cannot be easily affinity-matured because the extracellular portions of chemokine receptors such as CCR8 have low homology between species (see Example 2). According to the present invention, cross-reactive antibodies against CCR8 can be produced by using small sulfated tyrosine containing a motif that has higher conservation between species, and as a result, cross-reactive antibodies that bind to chemokine receptors such as CCR8 from two or more species with similar affinity can be obtained in an easy and convenient manner (see Examples 6, 7, and 10.1.1).

[0048] 1.2.3 Acquisition of therapeutic chemokine receptor antibodies Multiple modes of action are conceivable to induce cell death of cells expressing the target chemokine receptor or CCR8. One mode of action is the conjugation of an antibody targeting the chemokine receptor or CCR8 to a drug in the form of an antibody-drug conjugate (ADC).

[0049] Other possible modes of action are the induction of antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cell-mediated cytotoxicity (CDC). In the cases of ADCC, CDC, and ADCP, a two-step mechanism is involved: on the one hand, the antibody or fragment is required to effectively bind to target cells, e.g., Tregs via CCR8; on the other hand, the FC portion of the antibody (or an alternative binding portion that can be conjugated to the antibody or fragment as described elsewhere herein) must bind to effector cells, which then mediate the death of the target cells. In the case of ADCP, binding to macrophages as effector cells typically occurs via the interaction between the antibody FC portion and FcγRIIa (CD32a) expressed by the macrophage. In contrast, ADCC is mediated via the interaction between the antibody or fragment and FcγRIIIa. In humans, FcγRIII exists in two distinct forms: FcγRIIIa (CD16a) and FcγRIIIb (CD16b). FcγRIIIa is expressed on mast cells, macrophages, and natural killer cells as a transmembrane receptor, while FcγRIIIb is expressed only on neutrophils. These receptors bind to the Fc portion of IgG antibodies and subsequently activate antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by human effector cells.

[0050] 1.2.4 Acquisition of low-internalization (including non-internalization) chemokine receptor antibodies When the inventors analyzed known anti-human CCR8 prior art antibodies, they found that these antibodies were readily internalized into cells with endogenous target expression but remained absent from the cell surface for extended periods. The internalization behavior of antibodies affects not only their clearance and pharmacological behavior but also their suitability for specific modes of action for therapeutic use.

[0051] High internalization is desirable for the formation of specific antibody-drug conjugates but undesirable for ADCC-induced depletion of tumor cells or Treg cells. More specifically, antibody-drug conjugates are required to transport the drug into the cell in order to achieve efficient and specific depletion of target cells. In contrast, the ADCC mode of action requires exposure of the antibody and its Fc domain outside the target cell, and immune effector cells can bind to the FC domain for lysis of the target cell. Thus, a low or absent rate of internalization increases the effect duration of ADCC / ADCP-inducing antibodies.

[0052] In characterizing antibodies obtained using novel methods, the inventors surprisingly found that some of the antibodies according to the present invention showed particularly low or even absent internalization profiles in cells with endogenous CCR8 expression levels (see Example 10.5), while prior art antibodies recognizing the same target had higher internalization rates. For example, prior art antibodies 433H and L263G8 readily internalized in targeted cells and remained on the cell surface for extended periods. Therefore, due to their low internalization characteristics, some of the antibodies according to the present invention are particularly useful in ADCC, ADCP, CDC, or mixed approaches (e.g., combined ADCC / ADCP approaches).

[0053] Typically, target selection is a crucial factor strongly influencing the internalization of antibodies into cells. According to Islam SA et al., CCL1 ligand binding induces Ca2+ flux and rapid receptor internalization of CCL8 (Islam, Sabina A., et al. "Identification of human CCR8 as a CCL18 receptor. CCR8 is a CCL18 receptor." The Journal of experimental medicine 210.10(2013):1889-1898). Surprisingly, we have found that the antibody itself can influence the internalization characteristics to such a considerable extent.

[0054] However, when characterizing the ability of the antibodies of the present invention to modulate the G protein-independent signaling pathway of their target chemokine receptors, we found that all tested prior art antibodies not only blocked G protein-dependent signaling but also modulated G protein-independent signaling (see Example 10.4ff). G protein-independent signaling has previously been associated with internalization behavior (see Fox, James M., et al. “Structure / function relationships of CCR8 agonists and antagonists: Amino-terminal extension of CCL1 by a single amino acid generates a partial agonist.” Journal of Biological Chemistry 281.48(2006):36652-36661).

[0055] 1.2.5 Acquisition of chemokine receptor antibodies that modulate target receptor signaling There are several different ways in which antibodies can modulate chemokine receptors. For example, antibodies can... a) Blocking G protein-independent signaling, b) Blocking G protein-dependent signaling, c) Blocking G protein-dependent and G protein-independent signaling, d) Increase G protein-independent signaling, e) Increases G protein-dependent signaling, f) It can increase G protein-dependent and G protein-independent signaling.

[0056] While not bound by theory, the inventors hypothesize that the sulfated TRDs of each chemokine receptor may be involved in ligand-induced signaling of the chemokine receptor.

[0057] Interestingly, most of the antibodies of the present invention efficiently blocked ligand-induced G protein-dependent signaling but did not affect G protein-independent signaling (see Example 10.4ff). In contrast, all prior art antibodies similarly blocked ligand-induced G protein-dependent signaling but blocked agonized G protein-independent signaling. While not theoretically bound, these differences may contribute to differences in internalization behavior.

[0058] Since nonspecific signaling can lead to uncontrollable downstream effects such as increased cell proliferation, it may be advantageous to avoid inducing G protein-independent signaling with therapeutic antibodies (see Hutchings, Catherine J., et al. (2017); Webb, David R., et al. “Opportunities for functional selectivity in GPCR antibodies.” Biochemical pharmacology 85.2(2013):147-152.; Fox, James M., et al. (2006)).

[0059] 1.2.6 Acquisition of ADCC / ADCP that induces chemokine receptor antibodies The antibodies according to the present invention are characterized by excellent induction of ADCC and ADCP, as shown, for example, in Examples 10.3.3 and 10.3.4. In some preferred embodiments, the antibodies were manipulated by fucose removal (afcosylation) at N297 to obtain a high ADCC rate (see Example 10.3.1). Interestingly, many of the anti-CCR8 antibodies of the present invention appeared to induce target cell depletion through a combined mode of action using both mechanisms, namely ADCC and ADCP.

[0060] 1.3 Treatment Method While several research groups have suggested the use of antibodies that bind to CCR8 in treatment methods based on mechanistic insight, providing antibodies with optimal therapeutic properties has been cumbersome. By providing a method for antibody production according to the present invention, antibodies for chemokine receptors with superior therapeutic properties can now be easily obtained. According to the present invention, various sequence-defined antibodies with advantageous properties are disclosed, which can be used in treatment methods, for example, as part of a conjugate, in an ADCC-based approach, an ADCP-based approach, a CDC-based approach, or a mixed ADCC / ADCP-based approach. Example 12ff. demonstrates remarkable efficacy in Treg depletion, overall response rate, and tumor-to-control ratio for surrogate antibodies produced by the method according to the present invention.

[0061] 1.4 Concomitant Treatment While monotherapy with the anti-CCR8 antibody of the present invention yielded remarkable response rates, it was surprisingly found that the therapeutic benefits could be further increased by combining the antibody with immune checkpoint inhibitors, further targeted therapies, and even nonspecific chemotherapeutic agents or radiotherapy. These combination therapies were particularly beneficial in challenging tumor models. In particular, a specific combination therapy scheme in which the second therapeutic agent or treatment was administered only after the anti-CCR8 antibody had caused substantial Treg depletion proved effective (see Example 12.6ff). Furthermore, even single anti-CCR8 antibody treatment was observed to show substantial therapeutic effects (see Example 12.4.2).

[0062] 1.5 Stratification Scheme and Diagnostic Method When evaluating the responsiveness of different syngeneic mouse models to anti-CCR8 antibody treatment, we were able to identify several mechanisms, biomarkers, and combinations of biomarkers that predict treatment efficacy and overall response. For example, surprisingly, the degree of responsiveness to immune checkpoint inhibitors such as PD-1, PD-L1, or CTLA4 antibodies was also found to predict the response to anti-CCR8 antibody treatment. Since PD-L1 expression is sometimes used as a surrogate marker to predict responsiveness to immune checkpoint inhibitors, we evaluated whether PD-L1 could also be used to predict responsiveness to anti-CCR8 antibody treatment. This hypothesis was ultimately confirmed by correlation data. See Example 12.7.1. In a further attempt to identify the subjects most likely to benefit from anti-CCR8 treatment, immune cells and further marker genes such as Treg markers were evaluated as biomarkers (see Examples 12.1.2, 12.7.2, and 12.8). [Brief explanation of the drawing]

[0063] [Figure 1-1] Alignment of human CC chemokine receptors and CXC chemokine receptors. [Figure 1-2] Alignment of human CC chemokine receptors and CXC chemokine receptors. [Figure 1-3] Alignment of human CC chemokine receptors and CXC chemokine receptors. [Figure 2a] a. Human CCR8, cynomolgus monkey CCR8, and mouse CCR8 sequences were recovered from Uniprot and aligned with Clustal Omega. b. Schematic diagram of the CCR8 receptor structure, including seven transmembrane domains (black squares), three extracellular loops (ECLs), several disulfide crosslinks (SSs), an intracellular C-terminal region, and an extracellular N-terminal domain composed of a "LID" domain and a tyrosine-rich domain (TRD). [Figure 2b]a. Human CCR8, cynomolgus monkey CCR8, and mouse CCR8 sequences were recovered from Uniprot and aligned with Clustal Omega. b. Schematic diagram of the CCR8 receptor structure, including seven transmembrane domains (black squares), three extracellular loops (ECLs), several disulfide crosslinks (SSs), an intracellular C-terminal region, and an extracellular N-terminal domain composed of a "LID" domain and a tyrosine-rich domain (TRD). [Figure 3] Evaluation of three prior art antibodies by FACS staining in CCR8-positive target cells. None of the prior art antibodies shown showed a shift in FACS staining compared to isotype controls. hAP-0068 and MAB1429-100 were tested in 293T cells stably transfected with human CCR8, while MAB8324 was tested in BW5147.3 cells expressing mouse CCR8. [Figure 4] Panning strategies for lead discovery: Two main strategies for selection of sulfated peptides are presented. In all strategies, a depletion step for relevant or irrelevant biotinylated proteins was included before each selection. [Figure 5] Panning strategies for obtaining anti-mouse CCR8 antibodies: The main strategies for selection based on a mixture of sulfated / unsulfated peptides are presented. A depletion step against the relevant biotinylated protein was performed before each selection. [Figure 6] FACS data of the antibody of the present invention that binds to human CCR8-expressing CHO cells. [Figure 7] FACS data of the antibody of the present invention that binds to CHO cells expressing cynomolgus monkey CCR8. [Figure 8] FACS data of candidate organisms that bind to activated human Treg (donor 1). Right: Percentage of CCR8 expression determined using BioLegend clone L263G8. [Figure 9] FACS data for the antibodies TPP-21181 and TPP-23411 of the present invention that bind to human CCR8-expressing CHO cells. [Figure 10]FACS data for the antibodies TPP-21181 and TPP-23411 of the present invention that bind to CHO cells expressing cynomolgus monkey CCR8. [Figure 11] Binding of TPP-21360, L263G8, and 433H to CHO cells expressing human CCR8. [Figure 12] Binding of TPP-21360, L263G8, and 433H to CHO cells expressing cynomolgus monkey CCR8. Prior art antibodies showed very low overall binding to cynomolgus monkey CCR8 (low saturation). Nevertheless, EC50 values ​​could be determined for each antibody. [Figure 13] Activated human Treg staining and gating strategy using the antibody TPP-23411 of the present invention. CD4+ and CD25+ sorted cells from peripheral blood mononuclear cells (PBMCs) were activated with anti-CD3 and anti-CD28 beads (Treg expansion kit, Miltenyi). Center panel: Cells that are CD4+CD25+CD127 low and Foxp3+. Bottom panel: CCR8 expression on Tregs after staining with TPP-23411 or L263G8. [Figure 14] Binding of TPP-21360 to CHO cells using a pseudo-transfectant. No nonspecific binding was observed. [Figure 15] Off-target binding in HEK cells transiently transfected with human CCR1. Most of the candidate compounds of the present invention showed only low off-target binding to CCR1. [Figure 16] Off-target binding in HEK cells transiently transfected with human CCR4. Most of the candidate compounds of the present invention showed only low off-target binding to CCR4. [Figure 17-1] Staining for CCR8 expression in different immune cell populations derived from healthy donor PBMCs (CD4+ T cells, CD8+ T cells, B cells expressed by CD19+, myeloid cells expressed by CD11b+, CD4 T cells activated with anti-CD3 and anti-CD28 beads for 4 days, and CD8+ T cells activated with anti-CD3 and anti-CD28 beads for 4 days). [Figure 17-2] Staining for CCR8 expression in different immune cell populations derived from healthy donor PBMCs (CD4+ T cells, CD8+ T cells, B cells expressed by CD19+, myeloid cells expressed by CD11b+, CD4 T cells activated with anti-CD3 and anti-CD28 beads for 4 days, and CD8+ T cells activated with anti-CD3 and anti-CD28 beads for 4 days). [Figure 17-3] Staining for CCR8 expression in different immune cell populations derived from healthy donor PBMCs (CD4+ T cells, CD8+ T cells, B cells expressed by CD19+, myeloid cells expressed by CD11b+, CD4 T cells activated with anti-CD3 and anti-CD28 beads for 4 days, and CD8+ T cells activated with anti-CD3 and anti-CD28 beads for 4 days). [Figure 18] CCR8 is upregulated on human Tregs after activation. After activation, the percentage of CCR8+ cells was significantly increased in CD4+CD25+ purified cells with increased MFI, while the percentage of CCR8+ cells was only slightly increased in the PBMC population. Anti-CCR8 antibodies from BioLegend were used in these experiments. N=4 donors. [Figure 19] ADCC induced by wild-type or afucosylated antibodies TPP-19546, TPP-21360, or TPP-23411 of the present invention in HEK cells expressing human CCR8 as target cells. Isotype control: TPP-9808. [Figure 20] Left: CCR8+ expression percentage on day 3 in activated human Treg cells as target cells. Right: ADCC induced in activated human Treg cells as target cells by wild-type or afucosylated antibodies TPP-21360 and TPP-23411 of the present invention. Isotype control: TPP-9808. Results were reproducible in different donors (data not shown). [Figure 21] Protocol for ADCP assay. [Figure 22]ADCP assays using wild-type and afucosylated versions of the antibodies of the present invention demonstrate the induction of phagocytosis in HEK cells expressing human CCR8 as target cells by in vitro differentiated M2c macrophages as effector cells. Wild-type and afucosylated versions of the antibodies TPP-19546, TPP-21360, and TPP-23411 of the present invention induced ADCP. TPP-9808 is an isotype control. Mogamulizumab is a commercially available anti-CCR4 antibody. [Figure 23] ADCP assays of the wild-type and afucosylated versions of the antibodies TPP-21360 and TPP-23411 of the present invention demonstrate the induction of phagocytosis in activated human Treg cells from two different donors as target cells (upper or lower panel) and M2c macrophages as effector cells. TPP-9808 is the isotype control. [Figure 24] ADCP assays of the wild-type and afucosylated versions of the antibodies TPP-21360 and TPP-23411 of the present invention demonstrate the induction of phagocytosis in activated human Treg cells as target cells and M1 macrophages as effector cells. [Figure 25] Activation of β-arrestin signaling was measured by the DiscoverX assay for CCL1, TPP-23411, L263G8, or 433H on CHO cells co-expressing human CCR8 tagged with ProLink and EA. As expected, the CCR8 ligand CCL1 (human) induced β-arrestin signaling. Further time point analysis for TPP-23411 confirmed that β-arrestin activation did not occur at earlier time points. None of the antibodies analyzed induced β-arrestin signaling activation. [Figure 26]Evaluation of CCL1-induced β-arrestin signaling blockade by TP-23411, L263G8, or 433H, as measured by the DiscoverX assay. CCL1 was used at its EC80 (20 ng / ml). Both prior art antibodies L263G8 and 433H blocked CCL1-induced β-arrestin signaling at low antibody concentrations, but the present antibody TPP-23411 showed no effect at these concentrations. β-arrestin signaling is suggested to play a role in internalization. [Figure 27] PhosphoErk1 / 2 ELISA assay. Human CCR8-expressing CHO cells were treated with CCL1, TPP-23411, Biolegend L263G8, or BD antibody 433H, and cell lysates were collected at each time point. [Figure 28] Phospho-Erk1 / 2 ELISA assay. Activated human Treg cells expressing CCR8 were treated with CCL1, TPP-23411, Biolegend L263G8, or BD antibody 433H, and cell lysates were collected at each time point. Both prior art antibodies, Biolegend L263G8 and BD antibody 433H, induced a significant increase in phosphorylated Erk1 / 2 levels in activated human Treg cells, for example, after 15 minutes, but this was not the case with the antibody of the present invention. [Figure 29] Phospho-AKT ELISA assay. Human CCR8-expressing CHO cells were treated with CCL1, TPP-23411, Biolegend L263G8, or BD antibody 433H, and cell lysates were collected at each time point. [Figure 30] Phospho-AKT ELISA assay. Activated human Treg cells expressing CCR8 were treated with CCL1, TPP-23411, Biolegend L263G8, or BD antibody 433H, and cell lysates were collected at each time point. Both prior art antibodies, Biolegend L263G8 and BD 433H, induced a significant increase in phospho-AKT levels in activated human Tregs, for example, after 15 minutes, but this was not the case with the antibody of the present invention. [Figure 31a]Internalization study. a. FACS analysis of commercially available anti-human CCR8 antibodies Biolegend L263G8 or BD antibody 433H using the endogenous CCR8-expressing cell line HuT78. b. Internalization study of commercially available anti-human CCR8 antibodies using the endogenous CCR8-expressing cell line HuT78 based on cytoplasmic intensity spots. TPP-5657: Isotype control. [Figure 31b] Internalization study. a. FACS analysis of commercially available anti-human CCR8 antibodies Biolegend L263G8 or BD antibody 433H using the endogenous CCR8-expressing cell line HuT78. b. Internalization study of commercially available anti-human CCR8 antibodies using the endogenous CCR8-expressing cell line HuT78 based on cytoplasmic intensity spots. TPP-5657: Isotype control. [Figure 32a] Internalization study. a. FACS analysis of the commercially available anti-mouse CCR8 antibody SA214G2 using the mouse endogenous CCR8-expressing cell line BW5147.3. b. Internalization study of the commercially available anti-mouse CCR8 antibody SA214G2 using the mouse endogenous CCR8-expressing cell line BW5147.3. The prior art anti-mouse CCR8 antibody SA214G2 induces internalization. [Figure 32b] Internalization study. a. FACS analysis of the commercially available anti-mouse CCR8 antibody SA214G2 using the mouse endogenous CCR8-expressing cell line BW5147.3. b. Internalization study of the commercially available anti-mouse CCR8 antibody SA214G2 using the mouse endogenous CCR8-expressing cell line BW5147.3. The prior art anti-mouse CCR8 antibody SA214G2 induces internalization. [Figure 33a] Internalization studies of the anti-human CCR8 antibodies TPP-21360 and TPP-23411 of the present invention using endogenous CCR8-expressing cell lines TALL-1(a) and HuT78(b). Both antibodies exhibit identical internalization behavior, comparable to that of the isotype control TPP-5657. [Figure 33b] Internalization studies of the anti-human CCR8 antibodies TPP-21360 and TPP-23411 of the present invention using endogenous CCR8-expressing cell lines TALL-1(a) and HuT78(b). Both antibodies exhibit identical internalization behavior, comparable to that of the isotype control TPP-5657. [Figure 34a]FACS analysis of the anti-human CCR8 antibodies TPP-21360 and TPP-23411 of the present invention using endogenous CCR8-expressing cell lines TALL-1(a) and HuT78(b). Both antibodies exhibit the same binding efficacy to the cell lines. [Figure 34b] FACS analysis of the anti-human CCR8 antibodies TPP-21360 and TPP-23411 of the present invention using endogenous CCR8-expressing cell lines TALL-1(a) and HuT78(b). Both antibodies exhibit the same binding efficacy to the cell lines. [Figure 35-1] CCR8 mRNA expression across 11,642 samples representing a comprehensive set of human tissues and cell types, measured by the affymetrix probe 208059_at. All samples were conormalized using the refRMA method (Katz, Simon, et al. "A summarization approach for Affymetrix GeneChip data using a reference training set from a large, biologically diverse database." BMC bioinformatics 7.1(2006):1-11.). Dark gray boxes indicate that the median sample of the corresponding group has a probe signal intensity significantly above the background noise (estimated by the affymetrix' MAS5 algorithm; see Pepper, Stuart D., et al. "The utility of MAS5 expression summary and detection call algorithms." BMC bioinformatics 8.1(2007):1-12.). Median CCR8 expression significantly above background noise is observed only in activated regulatory T cells and tumor-infiltrating lymphocytes. The groups are classified based on the decrease in average expression. [Figure 35-2]CCR8 mRNA expression across 11,642 samples representing a comprehensive set of human tissues and cell types, measured by the affymetrix probe 208059_at. All samples were conormalized using the refRMA method (Katz, Simon, et al. "A summarization approach for Affymetrix GeneChip data using a reference training set from a large, biologically diverse database." BMC bioinformatics 7.1(2006):1-11.). Dark gray boxes indicate that the median sample of the corresponding group has a probe signal intensity significantly above the background noise (estimated by the affymetrix' MAS5 algorithm; see Pepper, Stuart D., et al. "The utility of MAS5 expression summary and detection call algorithms." BMC bioinformatics 8.1(2007):1-12.). Median CCR8 expression significantly above background noise is observed only in activated regulatory T cells and tumor-infiltrating lymphocytes. The groups are classified based on the decrease in average expression. [Figure 36-1]The graph shows CCR8 mRNA expression as measured by RNA-seq (light gray squares) across 50 different TCGA (https: / / www.cancer.gov / tcga) tumor indications (dark gray squares), corresponding normal tissues (white squares), and a similarly grouped CCLE tumor cell line panel by tumor indication (Barretina, Jordi, et al. “The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity.” Nature 483.7391(2012):603-607.). Groups are classified based on mean expression. The indications with the highest CCR8 expression are breast cancer, lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC), head and neck malignancies, and esophageal tumors. In all indications except pancreatic adenocarcinoma and melanoma, expression appears higher in tumors compared to corresponding normal tissues. CCR8 expression is rarely or never observed in corresponding tumor cell lines of epithelial origin, indicating that CCR8 is not expressed by tumor cells but only by tumor-infiltrating T cells. In principle, each tumor indication involving CCR8+ Treg cell infiltration is considered eligible for anti-CCR8 antibody treatment. [Figure 36-2]The graph shows CCR8 mRNA expression as measured by RNA-seq (light gray squares) across 50 different TCGA (https: / / www.cancer.gov / tcga) tumor indications (dark gray squares), corresponding normal tissues (white squares), and a similarly grouped CCLE tumor cell line panel by tumor indication (Barretina, Jordi, et al. “The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity.” Nature 483.7391(2012):603-607.). Groups are classified based on mean expression. The indications with the highest CCR8 expression are breast cancer, lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC), head and neck malignancies, and esophageal tumors. In all indications except pancreatic adenocarcinoma and melanoma, expression appears higher in tumors compared to corresponding normal tissues. CCR8 expression is rarely or never observed in corresponding tumor cell lines of epithelial origin, indicating that CCR8 is not expressed by tumor cells but only by tumor-infiltrating T cells. In principle, each tumor indication involving CCR8+ Treg cell infiltration is considered eligible for anti-CCR8 antibody treatment. [Figure 37] Immunohistochemistry using staining for the Treg markers FOXP3 and CCR8 (clone 433H) in human non-small cell lung cancer (NSCLC) or human melanoma tissue. [Figure 38] Staining of FACS-sorted T cell populations based on CCR4, CCR8, OX40, GITR, and CD25. Only CCR8 is specific to activated Tregs. OX40, GITR, and CD25 are significantly expressed on stimulated CD8+ Teff cells and CD4+ T cells (CD4+CD25+Foxp3-). Dark gray: isotype control, light gray: targeted staining. [Figure 39-1]CCR8 expression across different T cell subsets extracted from different tumor entities, as measured by single-cell RNA-seq. In these tumors, CCR8 mRNA expression is primarily restricted to regulatory T cells present in tumor tissue (light gray squares), but is largely absent in normal tissue, as well as regulatory T cells from CD4 helper T cells and CD8 cytotoxic T cells (medium and dark gray squares, respectively). Top panel: Colorectal tumor tissue (tumor) or adjacent normal tissue (normal). Middle panel: Hepatocellular carcinoma tissue (tumor) or adjacent normal tissue (normal). Bottom panel: Lung cancer tissue (tumor) or adjacent normal lung tissue and peripheral blood (normal). Sample size N indicates the number of cells in each category. Cells were named regulatory T cells, T helper cells, or cytotoxic T cells based on the expression of the marker genes FOXP3, CD4 (except FOXP3), and CD8A / B, respectively. [Figure 39-2] CCR8 expression across different T cell subsets extracted from different tumor entities, as measured by single-cell RNA-seq. In these tumors, CCR8 mRNA expression is primarily restricted to regulatory T cells present in tumor tissue (light gray squares), but is largely absent in normal tissue, as well as regulatory T cells from CD4 helper T cells and CD8 cytotoxic T cells (medium and dark gray squares, respectively). Top panel: Colorectal tumor tissue (tumor) or adjacent normal tissue (normal). Middle panel: Hepatocellular carcinoma tissue (tumor) or adjacent normal tissue (normal). Bottom panel: Lung cancer tissue (tumor) or adjacent normal lung tissue and peripheral blood (normal). Sample size N indicates the number of cells in each category. Cells were named regulatory T cells, T helper cells, or cytotoxic T cells based on the expression of the marker genes FOXP3, CD4 (except FOXP3), and CD8A / B, respectively. [Figure 40]Tregs, macrophages, T cells, or tumor cell populations from NSCLC, CRC (colorectal cancer), or RCC (renal carcinoma) were stained with anti-CCR8 antibody or isotype control and analyzed by flow cytometry. X-axis: log-shifted to 10⁵; Y-axis: intensity normalized to mode. Light gray: isotype control. Dark gray: CCR8. Specific CCR8 expression on intratumoral human Tregs is indicated by arrows. [Figure 41] CT26 tumor growth after treatment with various doses of anti-CCR8 antibody or anti-PDL1 antibody. [Figure 42-1] Spider plots of CT26 tumor-bearing mice after treatment with various doses of anti-CCR8 antibody, anti-PDL1 antibody, or isotype control. [Figure 42-2] Spider plots of CT26 tumor-bearing mice after treatment with various doses of anti-CCR8 antibody, anti-PDL1 antibody, or isotype control. [Figure 43] FACS analysis of intratumor immune cells 24 hours after the second antibody treatment. Analysis of intratumor Tregs. Anti-CCR8 antibody TPP15285 resulted in an average reduction of intratumor Tregs to 13%, 33%, or 47% of isotype control Tregs at doses of 10 mg / kg, 1 mg / kg, or 0.1 mg / kg, respectively. Anti-CCR8 antibody TPP15286 resulted in an average reduction of intratumor Tregs to 10%, 9%, or 14% of isotype control Tregs at doses of 10 mg / kg, 1 mg / kg, or 0.1 mg / kg, respectively (see Table 12.2.1). Analysis of intratumor CD8+ T cells. The average percentage increase compared to isotype control is shown in Table 12.2.1. Analysis of the ratio of CD8+ T cells to Treg cells. The anti-CCR8 antibody TPP15286 resulted in an average increase in the ratio of CD8+ cells to Treg cells at 44 (10 mg / kg), 87 (1 mg / kg), or 68 (0.1 mg / kg). The anti-CCR8 antibody TPP15285 resulted in an average increase in the ratio of CD8+ cells to Treg cells at 64 (10 mg / kg), 16 (1 mg / kg), or 10 (0.1 mg / kg). Analysis of the ratio of CD4+ convolutional T cells to Treg cells. [Figure 44]CT26 tumor growth after treatment with glycosylated (TPP-14095, TPP-14099, TPP-15285, TPP-15286) or aglycosylated (TPP-18208, TPP-18209) anti-CCR8 antibodies. Aglycosylation significantly negated the antitumor effect. [Figure 45] Spider plots of CT26 tumor-bearing mice treated with glycosylated (TPP-14095, TPP-14099, TPP-15285, TPP-15286) or aglycosylated (TPP-18208, TPP-18209) anti-CCR8 antibodies. Aglycosylation significantly negated the antitumor effect, suggesting an ADCC / ADCP-dependent mechanism of antitumor efficacy. [Figure 46-1] FACS analysis of immune cells 24 hours after the second antibody treatment. Absolute number of CD45+ cells in the tumor. Absolute number of CD45+CD8+ T cells in the tumor. Absolute number of CD8+ T cells in the tumor. Absolute number of CD4+ convolutional cells in the tumor. Analysis of intratumoral Treg depletion by flow cytometry showing absolute cell counts. The mean percentage of remaining Tregs compared to each isotype control after antibody administration was 15.6% for TPP-14095, 28.2% for TPP-14099, 8.7% for TPP-15285, and 36.5% for TPP-15286. No decrease in Tregs was observed for aglycosylated antibodies. % CD8+ T cells among CD45+ cells in the tumor. CD8+ T cell:Treg ratio. CD4+ convolution:Treg ratio. Percentage of Treg cells among CD4+ T cells. Absolute number of 4-1BB+ Treg cells in the tumor. Absolute number of 4-1BB+CD4+convolutional cells within the tumor. [Figure 46-2]FACS analysis of immune cells 24 hours after the second antibody treatment. Absolute number of CD45+ cells in the tumor. Absolute number of CD45+CD8+ T cells in the tumor. Absolute number of CD8+ T cells in the tumor. Absolute number of CD4+ convolutional cells in the tumor. Analysis of intratumoral Treg depletion by flow cytometry showing absolute cell counts. The mean percentage of remaining Tregs compared to each isotype control after antibody administration was 15.6% for TPP-14095, 28.2% for TPP-14099, 8.7% for TPP-15285, and 36.5% for TPP-15286. No decrease in Tregs was observed for aglycosylated antibodies. % CD8+ T cells among CD45+ cells in the tumor. CD8+ T cell:Treg ratio. CD4+ convolution:Treg ratio. Percentage of Treg cells among CD4+ T cells. Absolute number of 4-1BB+ Treg cells in the tumor. Absolute number of 4-1BB+CD4+convolutional cells within the tumor. [Figure 47] EMT6 tumor growth after treatment with different doses of the anti-CCR8 antibody TPP-15285. Significance was determined by a log-transformed one-way ANOVA + Sidak post-hoc test. [Figure 48] EMT6 tumor growth 19 days after treatment with different doses of anti-CCR8 antibody TPP-15285 or anti-CTLA4 antibody. [Figure 49] Spider plots of EMT6 tumor-carrying mice treated with different doses of anti-CCR8 antibody TPP-15285 or anti-CTLA4 antibody. [Figure 50-1] FACS analysis of immune cells in EMT6 tumor-bearing mice after treatment with different doses of CCR8 antibody TPP-15285 or anti-CTLA4 antibody, 24 hours after the second treatment or at the end of the study. Absolute number of intratumor Treg cells. Significant differences were observed at both time points, e.g., for 10 mg / kg of anti-CCR8 antibody. CD8-positive cell to Treg ratio. CD4+ convolutional cell to Treg ratio. Percentage of CD4+ T cells to Treg cells. [Figure 50-2]FACS analysis of immune cells in EMT6 tumor-bearing mice after treatment with different doses of CCR8 antibody TPP-15285 or anti-CTLA4 antibody, 24 hours after the second treatment or at the end of the study. Absolute number of intratumor Treg cells. Significant differences were observed at both time points, e.g., for 10 mg / kg of anti-CCR8 antibody. CD8-positive cell to Treg ratio. CD4+ convolutional cell to Treg ratio. Percentage of CD4+ T cells to Treg cells. [Figure 51-1] FACS analysis of immune cells in EMT6 tumor-bearing mice 24 hours after the second treatment or at the end of the study, following treatment with different doses of the anti-CCR8 antibody TPP-15285 or anti-CTLA4 antibody. Absolute number of intratumor CD45+ cells. Absolute number of intratumor CD4+ convolutional cells. Absolute number of intratumor CD4+ T cells. Absolute number of intratumor activated CD8+ T cells. Absolute number of intratumor NK cells. Absolute number of intratumor CD8+ T cells. For example, a difference was observed at the end of the study for 10 mg / kg of anti-CCR8 antibody. [Figure 51-2] FACS analysis of immune cells in EMT6 tumor-bearing mice 24 hours after the second treatment or at the end of the study, following treatment with different doses of the anti-CCR8 antibody TPP-15285 or anti-CTLA4 antibody. Absolute number of intratumor CD45+ cells. Absolute number of intratumor CD4+ convolutional cells. Absolute number of intratumor CD4+ T cells. Absolute number of intratumor activated CD8+ T cells. Absolute number of intratumor NK cells. Absolute number of intratumor CD8+ T cells. For example, a difference was observed at the end of the study for 10 mg / kg of anti-CCR8 antibody. [Figure 51-3] FACS analysis of immune cells in EMT6 tumor-bearing mice 24 hours after the second treatment or at the end of the study, following treatment with different doses of the anti-CCR8 antibody TPP-15285 or anti-CTLA4 antibody. Absolute number of intratumor CD45+ cells. Absolute number of intratumor CD4+ convolutional cells. Absolute number of intratumor CD4+ T cells. Absolute number of intratumor activated CD8+ T cells. Absolute number of intratumor NK cells. Absolute number of intratumor CD8+ T cells. For example, a difference was observed at the end of the study for 10 mg / kg of anti-CCR8 antibody. [Figure 52] F9 tumor growth after treatment with different doses of anti-CCR8 antibody TPP-15285 or anti-PDL1 antibody. [Figure 53]F9 tumor volume at day 16 after initiation of treatment with different doses of anti-CCR8 antibody TPP-15285 or anti-PDL1 antibody. Significant improvement was observed with antibody doses of at least 10 mg / kg. [Figure 54] Spider plots of F9 tumor-bearing mice treated with different doses of anti-CCR8 antibody or anti-PDL1 antibody. [Figure 55] The effect of anti-CCR8 antibody or anti-PDL1 antibody on immune cell populations and their ratios in F9 tumors, analyzed by FACS 24 hours after the second antibody treatment. Anti-CCR8 antibody increased the absolute number of intratumoral CD45+ T cells, intratumoral CD4+ T cells, intratumoral CD8+ T cells, intratumoral CD4+ convolutional T cells, and intratumoral NK cells compared to matched isotype controls. 10 mg / kg of anti-CCR8 antibody decreased the absolute number of intratumoral Treg cells (CD4+, CD25+, FoxP3+). [Figure 56] The effect of anti-CCR8 antibody or anti-PDL1 antibody on immune cell populations and their ratios in F9 tumors, analyzed by FACS 24 hours after the second treatment, plotted as CD8+ T cell to Treg ratio, Treg to CD4+ T cell frequency, CD8+ T cell 4-1BB+ cell frequency, Treg 4-1BB+ cell frequency, and conventional CD4+ T cell 4-1BB+ cell frequency. At 10 mg / kg, anti-CCR8 antibody increased the ratio of CD8+ cells to Treg cells to approximately 54 or higher (see Table 12.5.1). [Figure 57a] Efficacy of combination therapy in C38 tumor-bearing mice. a. C38 tumor growth after treatment with anti-CCR8 surrogate antibody TPP-14099 or TPP-15285 or anti-PD-L1 antibody, or after combination therapy with 3 mg / kg anti-PDL1 antibody and 10 mg / kg TPP-15285. Significance determined by log-transformed one-way ANOVA + Sidak post-hoc test. b. Survival plots of C38 tumor-bearing mice after treatment with anti-mouse CCR8 antibody TPP-15285 (10 mg / kg), anti-mouse PD-L1 antibody (PDL1, 3 mg / kg), or a combination of TPP-15285 (10 mg / kg) and anti-mouse PD-1 antibody (3 mg / kg). [Figure 57b] Efficacy of combination therapy in C38 tumor-bearing mice. a. C38 tumor growth after treatment with anti-CCR8 surrogate antibody TPP-14099 or TPP-15285 or anti-PD-L1 antibody, or after combination therapy with 3 mg / kg anti-PDL1 antibody and 10 mg / kg TPP-15285. Significance determined by log-transformed one-way ANOVA + Sidak post-hoc test. b. Survival plots of C38 tumor-bearing mice after treatment with anti-mouse CCR8 antibody TPP-15285 (10 mg / kg), anti-mouse PD-L1 antibody (PDL1, 3 mg / kg), or a combination of TPP-15285 (10 mg / kg) and anti-mouse PD-1 antibody (3 mg / kg). [Figure 58] Analysis of tumor Treg depletion in C38 tumors by flow cytometry (sampling 24 hours after the second antibody treatment). C38 tumor-bearing mice were treated with the anti-CCR8 surrogate antibody TPP-14099 or TPP-15285 as monotherapy, or with an anti-PD-L1 antibody, or with TPP-15285 in combination with an anti-PD-L1 antibody. Absolute Treg depletion CD8+ T cell / Treg cell ratio. [Figure 59] CD11+F4 / 80+ macrophages in C38 tumors analyzed by flow cytometry (sampling at the end of the study). [Figure 60] B16 F10-OVA tumor growth after treatment with TPP-15285, anti-CTLA4 antibody, or both. [Figure 61] EMT-6 tumor growth after treatment with anti-mouse CCR8 antibody TPP-15285 (1 mg / kg), anti-mouse PD-1 antibody (CDR: atezolizumab, 10 mg / kg), or a combination of TPP-15285 (1 mg / kg) and anti-mouse PD-1 antibody (10 mg / kg) (intravenous therapy twice weekly). Mean and standard deviation. [Figure 62-1] FACS analysis of immune cell populations in EMT-6 tumors, sampled 24 hours after the second antibody treatment and analyzed by flow cytometry. CD4+ T cells, Tregs, CD8+ T cells, NK cells, and the ratio of CD8+ T cells to Tregs. Square and whiskers, minimum to maximum, median (GraphPad). [Figure 62-2] FACS analysis of immune cell populations in EMT-6 tumors, sampled 24 hours after the second antibody treatment and analyzed by flow cytometry. CD4+ T cells, Tregs, CD8+ T cells, NK cells, and the ratio of CD8+ T cells to Tregs. Square and whiskers, minimum to maximum, median (GraphPad). [Figure 63a] a. Analysis of C38 tumor-bearing mice treated with anti-mouse CCR8 antibody TPP-15285 (5 mg / kg), anti-mouse PD-1 antibody (CDR: atezolizumab, 5 mg / kg), or a combination of TPP-15285 (5 mg / kg) and anti-mouse PD-1 antibody (5 mg / kg) (intravenous injection twice weekly for each). a. Tumor volume. Mean and standard deviation. b. Survival plot. [Figure 63b] a. Analysis of C38 tumor-bearing mice treated with anti-mouse CCR8 antibody TPP-15285 (5 mg / kg), anti-mouse PD-1 antibody (CDR: atezolizumab, 5 mg / kg), or a combination of TPP-15285 (5 mg / kg) and anti-mouse PD-1 antibody (5 mg / kg) (intravenous injection twice weekly for each). a. Tumor volume. Mean and standard deviation. b. Survival plot. [Figure 64-1] Flow cytometry analysis of tumor CD4+ T cells, Treg cells, CD8+ T cells, NK cells, or CD8+ T cell to Treg cell ratios in C38 tumors (sampling 24 hours after the second antibody treatment). [Figure 64-2] Flow cytometry analysis of tumor CD4+ T cells, Treg cells, CD8+ T cells, NK cells, or CD8+ T cell to Treg cell ratios in C38 tumors (sampling 24 hours after the second antibody treatment). [Figure 65] MB49 tumor growth after treatment with anti-CCR8 antibody TPP-15285 (10 mg / kg) alone, anti-PD-1 antibody (aPD-1, CDR: atezolizumab, 10 mg / kg) alone, or sequential combination therapy with TPP-15285 (10 mg / kg) and anti-PD-1 antibody (10 mg / kg) (both administered twice weekly as intravenous therapy). [Figure 66-1]Analysis of tumor CD45+ cells, Treg cells, CD8+ T cells, NK cells, and CD8 to Treg ratios in MB49 tumors by flow cytometry (sampling at the end of the study). [Figure 66-2] Analysis of tumor CD45+ cells, Treg cells, CD8+ T cells, NK cells, and CD8 to Treg ratios in MB49 tumors by flow cytometry (sampling at the end of the study). [Figure 67] EMT-6 tumor growth after treatment with anti-CCR8 antibody TPP-15285 (5 mg / kg, q3 / 4d ip), oxaliplatin (5 mg / kg, q4d ip), doxorubicin (6 mg / kg, ivSD), docetaxel (10 mg / kg, q2dx5, iv), or a combination of TPP-15285 and any of oxaliplatin, doxorubicin, or docetaxel. [Figure 68] MB49 tumor growth after treatment with anti-CCR8 antibody TPP-15285, gemcitabine, or a combination of TPP-15285 and gemcitabine. [Figure 69] Lewis lung tumor growth after treatment with anti-CCR8 antibody TPP-15285 (10 mg / kg) alone, anti-PD-1 antibody (aPD-1, CDR: atezolizumab, 10 mg / kg) alone, anti-PD-L1 antibody (10 mg / kg) alone, anti-CTLA4 antibody alone, TPP-15285 (10 mg / kg) in combination with anti-mouse PD-1 antibody (10 mg / kg), or TPP-15285 (10 mg / kg) in combination with anti-PD-L1 antibody (10 mg / kg). [Figure 70] EMT-6 tumor growth after treatment with anti-CCR8 antibody TPP-15285 (3 mg / kg) or radiotherapy (RT, 3 × 2 Gy) alone or in combination. Significance was determined by a log-transformed one-way ANOVA + Sidak post-hoc test. [Figure 71]mRNA expression levels of different immune cell markers in different syngeneic tumor models treated with either an isotype control (TPP-9809) or an anti-CCR8 antibody (TPP-14099). The median expression in the isotype-treated control group was set to 0. Gene expression was measured in transcript permillions (TPM) estimated by the RSEM algorithm. Treg marker Foxp3. Significantly elevated Foxp3 levels were observed in CT26, H22, Hepa1-6, and RM1 models treated with TPP-14099, indicating increased Treg infiltration after administration of at least three doses of TPP-14099. [Figure 72] The inflammatory marker lfng. Significantly high lfng levels were observed in H22, CT26, and RM1 models treated with TPP-14099, indicating strong pro-inflammatory activity induced by TPP-14099. [Figure 73] Macrophage marker Ms4a7. Significantly elevated Ms4a7 levels were observed in CT26, H22, and Hepa1-6 models treated with TPP-14099, indicating increased macrophage infiltration induced by TPP-14099. [Figure 74] Cytotoxic T cell markers Cd8a and Cd8b1. Significantly higher levels of cytotoxic T cells were observed in CT26, H22, RM1, and Hepa1-6 models treated with TPP-14099, indicating induction of cytotoxic T cell infiltration and / or proliferation by TPP-14099. [Figure 75] Natural killer (NK) cell marker Ncr1. Significantly elevated NK cell levels were observed in CT26, H22, and Hepa1-6 models treated with TPP-14099. [Figure 76] Pan T cell marker Cd3e / d / g. Significantly elevated T cell levels were observed in CT26, H22, and Hepa1-6 models treated with TPP-14099. [Figure 77]Activated Treg marker and antibody target Ccr8. Significantly elevated Ccr8 levels were observed in CT26, H22, and Hepa1-6 models treated with TPP-14099. [Figure 78a] Acod1 (a), a highly specific pro-inflammatory M1 macrophage marker, and Mrc1 (b), a highly specific anti-inflammatory M2 macrophage marker. Significantly elevated M1 macrophage levels were observed in CT26, 4T1, H22, Hepa1-6, and RM1 models treated with TPP-14099, while significantly elevated M2 macrophage levels were not observed across any of the models treated with TPP-14099. [Figure 78b] Acod1 (a), a highly specific pro-inflammatory M1 macrophage marker, and Mrc1 (b), a highly specific anti-inflammatory M2 macrophage marker. Significantly elevated M1 macrophage levels were observed in CT26, 4T1, H22, Hepa1-6, and RM1 models treated with TPP-14099, while significantly elevated M2 macrophage levels were not observed across any of the models treated with TPP-14099. [Figure 79] The ratio of the highly specific pro-inflammatory M1 macrophage marker Acod1 to the highly specific anti-inflammatory M2 macrophage marker Mrc1. In summary, multiple doses of the anti-CCR8 antibody TPP-14099 increased the M1 / M2 macrophage ratio in these tumor models. [Figure 80] B cell markers Cd19 and Cd22. Significantly high B cell levels were observed in CT26, H22, RM1, and Hepa1-6 models treated with TPP-14099. Anti-CCR8 antibodies appear to recruit B cells to tumors. While not theoretically bound, B cell recruitment may influence the antitumor response induced by TPP-14099. [Figure 81-1]Expression levels of LTta / b and Cxcr5 and its ligand Cxcl13. These markers are important for the development of lymph nodes and tertiary lymphoid structures, which are key drivers of antitumor effects in humans (see Cyster, Jason G. "Blown away: the unexpected role of lymphotoxin in lymphoid organ development." The Journal of Immunology 192.5(2014):2007-2009. and Cupedo, Tom, et al. "Induction of secondary and tertiary lymphoid structures in the skin." Immunity 21.5(2004):655-667.) (see Dieu-Nosjean, Marie-Caroline, et al. "Tertiary lymphoid structures, drivers of the anti-tumor responses in human cancers." Immunological reviews 271.1(2016):260-275.). All three publications are incorporated herein by reference in their entirety. [Figure 81-2]Expression levels of LTta / b and Cxcr5 and its ligand Cxcl13. These markers are important for the development of lymph nodes and tertiary lymphoid structures, which are key drivers of antitumor effects in humans (see Cyster, Jason G. "Blown away: the unexpected role of lymphotoxin in lymphoid organ development." The Journal of Immunology 192.5(2014):2007-2009. and Cupedo, Tom, et al. "Induction of secondary and tertiary lymphoid structures in the skin." Immunity 21.5(2004):655-667.) (see Dieu-Nosjean, Marie-Caroline, et al. "Tertiary lymphoid structures, drivers of the anti-tumor responses in human cancers." Immunological reviews 271.1(2016):260-275.). All three publications are incorporated herein by reference in their entirety. [Figure 81-3]Expression levels of LTta / b and Cxcr5 and its ligand Cxcl13. These markers are important for the development of lymph nodes and tertiary lymphoid structures, which are key drivers of antitumor effects in humans (see Cyster, Jason G. "Blown away: the unexpected role of lymphotoxin in lymphoid organ development." The Journal of Immunology 192.5(2014):2007-2009. and Cupedo, Tom, et al. "Induction of secondary and tertiary lymphoid structures in the skin." Immunity 21.5(2004):655-667.) (see Dieu-Nosjean, Marie-Caroline, et al. "Tertiary lymphoid structures, drivers of the anti-tumor responses in human cancers." Immunological reviews 271.1(2016):260-275.). All three publications are incorporated herein by reference in their entirety. [Figure 82-1] Spider plots of tumor growth over time (measured in mm³) in the TPP-14099 treatment group (shown as triangles) and the isotype control treatment group (shown as circles) for different tumor models. The level of T cell infiltration at the end of the study is indicated by gray shading (black and light gray correspond to the highest and lowest Cd8a levels, respectively), as determined by the Cd8a mRNA levels in the corresponding bulk tumor samples. In H22 and CT26, tumor size showed a strong inverse correlation with Cd8a levels. [Figure 82-2]Spider plots of tumor growth over time (measured in mm³) in the TPP-14099 treatment group (shown as triangles) and the isotype control treatment group (shown as circles) for different tumor models. The level of T cell infiltration at the end of the study is indicated by gray shading (black and light gray correspond to the highest and lowest Cd8a levels, respectively), as determined by the Cd8a mRNA levels in the corresponding bulk tumor samples. In H22 and CT26, tumor size showed a strong inverse correlation with Cd8a levels. [Figure 83-1] Correlation between tumor infiltration by different immune cell populations, as determined by mRNA levels of different immune cell markers and tumor size. TPP-14099-treated tumors are shown as triangles, and isotype-treated tumors are shown as circles. Tumor size is measured in mm³. A strong negative correlation between cytotoxic T cell infiltration (determined by Cd8a+Cd8b1 mRNA levels) and CT26 tumor size. TPP-14099-treated tumors were smaller in size and showed higher Cd8a+Cd8b1 T cell infiltration levels than controls, indicating that increased T cell infiltration during TPP-14099 treatment leads to reduced tumor growth. A strong negative correlation between T cell infiltration (measured by Cd3 mRNA levels) and CT26 tumor size in TPP-14099-treated tumors. Tumors treated with TPP-14099 were smaller in size and showed higher T cell infiltration levels, i.e., higher Cd3 mRNA levels resulted in smaller tumors. A negative correlation was observed between NK cell infiltration (determined by NK marker Ncr1 expression) and CT26 tumor size. TPP-14099-treated tumors were smaller in size and showed higher NK cell infiltration. Tumor size was strongly negatively correlated with NK infiltration, with higher NK levels associated with smaller tumors. Correlation was observed between Lta / Ltb / Cxcr5 and Cxcl13 mRNA levels and the assumed induction of tertiary lymphoid structure, as determined by CT26 tumor size, in TPP-14099-treated tumors. For each of the four markers, levels increased strongly after TPP-14099 treatment, and tumor size was strongly negatively correlated with expression levels, indicating that increased formation of tertiary lymphoid structure during TPP-14099 treatment leads to decreased tumor growth. [Figure 83-2]Correlation between tumor infiltration by different immune cell populations, as determined by mRNA levels of different immune cell markers and tumor size. TPP-14099-treated tumors are shown as triangles, and isotype-treated tumors are shown as circles. Tumor size is measured in mm³. A strong negative correlation between cytotoxic T cell infiltration (determined by Cd8a+Cd8b1 mRNA levels) and CT26 tumor size. TPP-14099-treated tumors were smaller in size and showed higher Cd8a+Cd8b1 T cell infiltration levels than controls, indicating that increased T cell infiltration during TPP-14099 treatment leads to reduced tumor growth. A strong negative correlation between T cell infiltration (measured by Cd3 mRNA levels) and CT26 tumor size in TPP-14099-treated tumors. Tumors treated with TPP-14099 were smaller in size and showed higher T cell infiltration levels, i.e., higher Cd3 mRNA levels resulted in smaller tumors. A negative correlation was observed between NK cell infiltration (determined by NK marker Ncr1 expression) and CT26 tumor size. TPP-14099-treated tumors were smaller in size and showed higher NK cell infiltration. Tumor size was strongly negatively correlated with NK infiltration, with higher NK levels associated with smaller tumors. Correlation was observed between Lta / Ltb / Cxcr5 and Cxcl13 mRNA levels and the assumed induction of tertiary lymphoid structure, as determined by CT26 tumor size, in TPP-14099-treated tumors. For each of the four markers, levels increased strongly after TPP-14099 treatment, and tumor size was strongly negatively correlated with expression levels, indicating that increased formation of tertiary lymphoid structure during TPP-14099 treatment leads to decreased tumor growth. [Figure 84-1] Correlation between Cd8a invasion level, as determined by Cd8a mRNA expression, and tumor size in TPP-14099-treated tumors (indicated by triangles) and isotype-treated control groups (indicated by circles) for different tumor models. TPP-14099-treated tumors are smaller in size and show higher levels of Cd8a invasion. Tumor size is strongly negatively correlated with Cd8a invasion, with higher levels of Cd8a associated with smaller tumors. [Figure 84-2]Correlation between Cd8a invasion level, as determined by Cd8a mRNA expression, and tumor size in TPP-14099-treated tumors (indicated by triangles) and isotype-treated control groups (indicated by circles) for different tumor models. TPP-14099-treated tumors are smaller in size and show higher levels of Cd8a invasion. Tumor size is strongly negatively correlated with Cd8a invasion, with higher levels of Cd8a associated with smaller tumors. [Figure 85] Efficacy of the CCR8 antibody TPP-15285 in EMT6 tumor-bearing mice treated with four different treatment schemes: single treatment, two BIW treatments, three BIW treatments, or four BIW treatments. Each group was treated with a vehicle control or with 0.1 mg / kg, 1 mg / kg, or 5 mg / kg of TPP-15285. [Figure 86] The therapeutic efficacy of the anti-CCR8 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, or paclitaxel of the present invention, tested individually or in combination, in MBT2 syngeneic tumor-carrying mice as shown in Table 12.6.9.1. [Figure 87] Therapeutic efficacy of the anti-CCR8 antibody of the present invention after quantitative depletion of CD8a expression in diphtheria toxin-treated DTR mice. Depleted Cd8+ T cells render the antitumor growth effect of TPP15825 ineffective (see group 02b vs. group 01b). [Figure 88] Therapeutic efficacy of the anti-CCR8 antibody of the present invention after quantitative depletion of CD19+ expression in diphtheria toxin-treated DTR mice. Depleted Cd19+ B cells enhance the antitumor growth effect of TPP15825 (see group 02b vs group 01b). [Figure 89] Internalization curves of various anti-CCR8 antibodies of the present invention in HUVEC cells. Anti-CD71 antibodies with known internalization profiles were used as positive controls. Corresponding data are shown in Table 10.5.2. None of the antibodies obtained by the method according to the present invention showed substantial internalization, making them particularly useful for ADCC / ADCP-based approaches. [Modes for carrying out the invention]

[0064] A brief explanation of array keys The sequence listing provided with this application via electronic filing is included in its entirety herein.

[0065] Sequence IDs 1-108 and 157-168 relate to isolated polypeptides that can be used as antigens or for off-target panning.

[0066] Sequence IDs 109 to 156 relate to chemokine receptor proteins from different species. Sequence IDs 201 to 965 relate to the antibodies of the present invention. The sulfation columns are provided for convenience only and are not intended to restrict the use of any particular sequence.

[0067] [Table 1] TIFF0007927604000002.tif252160TIFF0007927604000003.tif254161TIFF0007927604000004.tif188161

[0068] [Table 2] TIFF0007927604000006.tif254164TIFF0007927604000007.tif254163TIFF0007927604000008.tif254162TIFF000 7927604000009.tif252163TIFF0007927604000010.tif254165TIFF0007927604000011.tif253163TIFF00079276040 00012.tif253162TIFF0007927604000013.tif253163TIFF0007927604000014.tif252163TIFF0007927604000015.t if253162TIFF0007927604000016.tif252164TIFF0007927604000017.tif253163TIFF0007927604000018.tif219160

[0069] definition Unless otherwise defined, all scientific and technical terms used herein, in the figures and claims have their ordinary meanings as generally understood by those skilled in the art. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. In case of conflict, this specification, including its definitions, shall prevail. If two or more documents incorporated by reference are in conflict and / or contain conflicting disclosures, the document with the later effective date shall prevail. When referring to databases, unless otherwise indicated, the effective data shall be the version number applicable to 26.05.2021. Materials, methods, and examples are illustrative and not intended to limit. Unless otherwise specified, the following terms used herein (including in the descriptions and claims) have the definitions set forth below.

[0070] As used herein, the expressions “approximately” or “~” refer to a value that is within an acceptable margin of error for a particular value as determined by those skilled in the art, which in part depends on how the value is measured or determined, i.e., the limitations of the measuring system. For example, “approximately” may mean within or above one standard deviation, according to convention in the art. The term “approximately” is also used to indicate that the quantity or value in question may be a specified value or any other value that is approximately the same. This phrase is intended to convey that similar values ​​will promote equivalent results or effects described herein. In this context, “approximately” may refer to a range of up to 10% above and / or below. Whenever the term “approximately” is specified for a particular assay or embodiment, its definition takes precedence over the specific context.

[0071] Terms such as “comprising,” “including,” “containing,” and “having” shall be read broadly and openly without limitation. As used herein, “comprising” includes “consisting of.”

[0072] Singular forms such as "a," "an," or "the" include multiple references unless the context clearly indicates otherwise. Therefore, for example, a reference to "monoclonal antibody" includes a single monoclonal antibody as well as multiple identical or different monoclonal antibodies. Similarly, a reference to "cell" includes a single cell and multiple cells.

[0073] Unless otherwise specified, the term "at least" preceding a set of elements should be understood to refer to all elements of that set. The terms "at least one" and "at least one" include, for example, one, two, three, four, five, or more elements.

[0074] Furthermore, it is understood that by using slight variations above and below the stated range, substantially the same results can be achieved as with values ​​within that range. Also, unless otherwise specified, the disclosure of a range is intended as a continuous range encompassing all values ​​between the minimum and maximum values.

[0075] As used herein, the terms “amino acid” or “amino acid residue” typically refer to naturally occurring amino acids. Single-letter codes are used herein to refer to each amino acid. As used herein, “charged amino acid” refers to an amino acid that is negatively or positively charged. “Negatively charged amino acids” are aspartic acid (D) and glutamic acid (E). “Positively charged amino acids” are arginine (R), lysine (K), and histidine (H). “Polar amino acids” are all amino acids that form hydrogen bonds as donors or acceptors. These are all charged amino acids and are asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), and cysteine ​​(C). “Polar uncharged amino acids” are asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), and cysteine ​​(C). "Amphiphilic amino acids" are tryptophan (W), tyrosine (Y), and methionine (M). "Aromatic amino acids" are phenylalanine (F), tyrosine (Y), and tryptophan (W). "Hydrophobic amino acids" are glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), and cysteine. "Minor amino acids" are glycine (G), alanine (A), serine (S), proline (P), threonine (T), aspartic acid (D), and asparagine (N).

[0076] As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, this term refers to both short chains, also commonly called peptides, oligopeptides, and oligomers in the art, and long chains, also commonly called proteins in the art, of which there are many types. A “polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0077] When referring in general to genes or proteins derived from a specific species such as mice, human analogues are also included unless otherwise documented or clearly incompatible. This is especially true in the context of biomarkers.

[0078] The term "isolation," when applied to nucleic acids, polypeptides, proteins, or antibodies, indicates that the nucleic acids, polypeptides, proteins, or antibodies are essentially free from other cellular components that associate with them in their natural state. This is preferably in a homogeneous state. This can be either a dry solution or an aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The protein, polypeptide, or antibody that is the dominant species present in the preparation is substantially purified. In particular, isolated genes are separated from open reading frames adjacent to the gene and encoding proteins other than the gene of interest. However, isolated polypeptides can be immobilized, for example, on beads or particles via a suitable linker.

[0079] The term "purification" means that nucleic acids or proteins produce essentially one band in an electrophoresis gel. Specifically, this means that the nucleic acids or proteins are at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

[0080] As used herein, the term “synthetic” refers to nucleic acid molecules or polypeptide molecules produced by recombinant and / or chemical synthesis methods, for example, with respect to synthetic nucleic acid molecules, synthetic genes, or synthetic peptides. As used herein, production by recombinant means, using recombinant DNA methods, means the use of well-known molecular biology methods for expressing proteins encoded by cloned DNA.

[0081] Post-translational modifications (PTMs) refer to covalent modifications of peptides or proteins introduced after protein biosynthesis under natural conditions. This term includes, but is not limited to, glycosylation, phosphorylation, acylation, adenylation, farnesylation, ubiquitination, and sulfated proteins. Post-translational modifications can affect the activity of peptides or proteins. In 2004, Gutierrez et al. described the sulfated and glycosylated states of the mouse CCR8 chemokine receptor and how these post-translational modifications affect CCR8 activity. They suggest that tyrosine at positions 14 and 15 of mouse CCR8 is a sulfated amino acid residue, while asparagine 8 and threonine 10 and 12 are glycosylated. Furthermore, these studies demonstrate that sulfation is important for CCR8 activity (Gutierrez, Julio, et al. “Analysis of post-translational CCR8 modifications and their influence on receptor activity.” Journal of Biological Chemistry 279.15(2004):14726-14733.).

[0082] Sulfation is a post-translational modification in which a sulfate group is added to an amino acid, such as a tyrosine residue, in a polypeptide or protein. Tyrosine sulfate occurs in all multicellular organisms. Under physiological conditions, this is catalyzed by tyrosylprotein sulfotransferases (TPSTs) 1 and 2, which are Golgi-resident enzymes that transfer sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to context-dependent tyrosine in the protein substrate. Synthetic sulfated tyrosine can be carried out using techniques known in the art, for example, as described in Bunschoten, Anton, et al. "A general sequence independent solid phase method for the site specific synthesis of multiple sulfated-tyrosine containing peptides." Chemical Communications 21(2009):2999-3001. A sulfated polypeptide is a polypeptide containing at least one sulfated molecule. A non-sulfated polypeptide is a polypeptide that does not contain sulfated molecules.

[0083] The "tyrosine-rich domain" (TRD) is a conserved domain that characterizes seven-transmembrane proteins such as CXC and CC chemokine receptors. The TRD is typically located at the N-terminus of the chemokine receptor and is typically linked to the LID domain via cysteine ​​(see Figure 2b). Therefore, as used herein, the term TRD refers to the amino acid or protein sequence of the CXC or CC chemokine receptor located at the N-terminus of the first cysteine, counting from the N-terminus. The TRD may or may not contain a signal peptide. The TRD may or may not be modified. In addition to tyrosine, the TRD often contains negatively charged amino acid residues such as aspartic acid. The TRD has been proposed to be a crucial structure for the interaction between the chemokine receptor and its endogenous ligand. Under physiological conditions, tyrosine residues in the TRD can be sulfated, unsulfated, or partially sulfated. Specific sequences of the TRD for each chemokine receptor are provided in Table 4.1. However, it is clear that mutations can be introduced into the TRD sequence without altering the overall charge and interaction pattern. Preferably, the TRD has at least 90%, 95%, or 99% sequence identity or similarity to at least one TRD sequence as shown in Table 4.1.

[0084] As used herein, the terms “N-terminus” or “N-term” of a chemokine receptor refer to the N-terminal amino acid of the chemokine receptor, including at least the TRD. If the polypeptide or protein contains a signal peptide, the N-terminus may also refer to the N-terminal sequence following the native cleavage site of the polypeptide or protein.

[0085] According to some preferred embodiments, the N-terminus includes the LID domain and TRD domain of the chemokine receptor, but does not contain native cysteine ​​between these two domains.

[0086] Alternatively, cysteine ​​can be removed or replaced with a different amino acid.

[0087] As used herein, the "LID" domain of a chemokine receptor refers to the amino acid sequence located at the C-terminus of the TRD of the chemokine receptor. The TRD and LID domains are typically separated by a single cysteine.

[0088] Sequence identity, or percentage identity, is a number that describes how similar a query sequence is to a target sequence, or more precisely, how many characters in each sequence are identical after alignment. The most common tool for calculating sequence identity is BLAST (basic local alignment search tool, https: / / blast.ncbi.nlm.nih.gov / ), which compares pairs of sequences and searches for regions of local similarity. Appropriate alignment methods are known in the art, for example, the Needleman-Wunsch algorithm for global-global alignment using the BLOSUM62 matrix with an 11-second gap-opening penalty and a 1-second gap-expanding penalty. The pairs of identical residues in the aligned sequence can then be counted and divided by the total length of the alignment (including gaps, internal, and external) to arrive at the percentage identity value.

[0089] For "percent similarity" or "sequence similarity" values, the same approach can be used as for percentage identity values, except that instead of pairs of identical residues, what is counted is aligned residue pairs with non-negative (i.e., ≥0) BLOSUM62 values.

[0090] The "seven-transmembrane receptor" (7-TM receptor) is an endogenous membrane protein containing seven transmembrane helices. As used herein, the 7-TM receptor is a G protein-coupled receptor.

[0091] Chemokine receptors are seven-transmembrane receptors. The chemokine receptor family in humans comprises 24 members and can be subdivided into four subfamilies based on the class of chemokines they bind to: CX3CR, CXCR, CCR, and XCR. All of these activate G proteins, while ACKR, which contains six atypical receptors, cannot activate G proteins upon ligand binding.

[0092] CXC chemokine receptors (CXCRs) are endogenous membrane proteins that specifically bind to and respond to cytokines of the CXC chemokine family. They constitute a subfamily of chemokine receptors and are a large family of G protein-linked receptors known as 7-transmembrane (7-TM) proteins because they cross the cell membrane seven times. Currently, there are seven known CXC chemokine receptors in mammals, called CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, and CXCR6. CXCR6 is structurally more closely related to CC chemokine receptors than the other CXC chemokine receptors.

[0093] CC chemokine receptors (CCRs, beta-chemokine receptors) are endogenous membrane proteins that specifically bind to and respond to cytokines of the CC chemokine family. They constitute one subfamily of chemokine receptors and are a large family of G protein-linked receptors known as 7-transmembrane (7-TM) proteins because they cross the cell membrane seven times. The CC chemokine receptor subfamily includes CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10.

[0094] The term "CCR8" refers to CC chemokine receptor type 8. The CCR8 protein is encoded by the gene CCR8 (NCBI gene ID 1237). Synonyms for CCR8 include, among others, CC-CKR-8, CCR-8, CDw198, CKRL1, CMKBR8, CMKBRL2, GPRCY6, CY6, and TER1. The CCR8 protein includes human, mouse, rat, rhesus monkey, and further mammalian and non-mammalian homologs. One or more sequences of human CCR8 are accessible via the UniProt identifier P51685 (CCR8_HUMAN), e.g., human isoforms P51685-1 or P51685-2 (UniProt, November 29, 2019). One or more sequences of mouse CCR8 are accessible via the UniProt identifier P56484 (CCR8_MOUSE). The sequences of rhesus macaque CCR8 (one or more) are accessible via the UniProt identifier O97665 (CCR8_MACMU). Different isoforms and mutants may exist for different species and are all included in the term CCR8. This also includes pre- and post-maturity CCR8 molecules, i.e., CCR8 molecules unrelated to the cleavage of one or more prodomains. Furthermore, synthetic mutants of the CCR8 protein can be generated and are included in the term CCR8. The protein CCR8 can also undergo various modifications, such as synthetic or naturally occurring modifications, and post-translational modifications.

[0095] Recombinant human CCR8 is commercially available or can be manufactured as known in the art. CCR8 is the receptor for the chemokine CCL1 / SCYA1 / I-309. Barington et al. reported the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) for ligand binding and activation in the chemokine receptor CCR8 (Barington, Line, et al. "Role of conserved disulfide bridges and aromatic residues in extracellular loop 2 of chemokine receptor CCR8 for chemokine and small molecule binding." Journal of Biological Chemistry 291.31(2016):16208-16220). Furthermore, Barington et al. found that two different aromatic residues in ECL-2, Tyr184 (Cys+1) and Tyr187 (Cys+4), are important for the binding of CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but are not important for small molecule binding.

[0096] "Programmed death-1 (PD-1)" refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is primarily expressed on previously activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes, but is not limited to, human PD-1 (hPD-1), variants, isoforms and species homologs of hPD-1, and analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence can be found under GenBank accession number U64863 (November 29, 2019).

[0097] Programmed death ligand-1 (PD-L1) is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that, upon binding to PD-1, downregulate T cell activation and cytokine secretion. As used herein, the term "PD-L1" includes, but is not limited to, human PD-L1 (hPDL1), variants, isoforms and species homologs of hPD-L1, and analogs having at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found under GenBank accession number Q9NZQ7 (November 29, 2019).

[0098] The Tumor Proportion Score (TPS) is the percentage of surviving tumor cells that exhibit partial or complete membrane staining at any given intensity. For example, a specimen should be considered to have PD-L1 expression if TPS ≥ 1%, and high PD-L1 expression if TPS ≥ 50%. For instance, PD-L1 protein expression in NSCLC is typically measured using the Tumor Proportion Score (TPS).

[0099] The "composite positivity score" (CPS) is calculated by dividing the number of stained cells (tumor cells, lymphocytes, macrophages) by the total number of surviving tumor cells and multiplying by 100. For example, a sample should be considered to have PD-L1 expression if CPS ≥ 1, and high PD-L1 expression if CPS ≥ 10. The FDA has approved the use of the PD-L1 IHC 22C3 pharmDx assay to determine patient eligibility for the therapeutic antibody pembrolizumab.

[0100] FOXP3, also known as forkhead box protein P3, Scurfin, JM2, or IPEX, is a 50-55 kD transcription factor. FOXP3 has been proposed to be a master regulatory gene and a more specific marker for regulatory T cells than most cell surface markers. Transduction of FOXP3 in CD4+ / CD25- cells has been shown to induce GITR, CD103, and CTLA4, conferring a regulatory T cell phenotype. Biological gene antibody clones 206D and 259D recognize the human FOXP3 epitope in the amino acid region 105-235. Poly6238 recognizes both human and mouse FOXP3 and was induced against the N-terminal portion of FOXP3.

[0101] The term "regulation" refers to any change in an existing process or behavior, such as blockade (antagonism) and induction (activation). For example, regulation of G protein-independent signaling refers to any significant change in G protein-independent signaling.

[0102] The term “internalization” of an antibody, fragment, or conjugate refers to the uptake of the antibody, fragment, or conjugate into a cell. Preferably, internalization is determined for cell lines having endogenous target expression, for example, as described elsewhere herein for human or mouse CCR8. Preferably, internalization is determined by measuring the total internalization fluorescence intensity per cell and quantified in comparison to an isotype control, for example, as described in Example 10.5. Briefly, the antibody, fragment, or conjugate and a matching isotype control are labeled with a dye, the internalization fluorescence is determined, and the antibody, fragment, or conjugate is quantified in comparison to the isotype control. A “non-internalized antibody” is defined as an antibody that exhibits substantially the same internalization as the corresponding isotype control. "Low internalization antibodies" are defined as antibodies exhibiting internalization at 10 times or less the internalization of the isotype control, preferably 9, 8, 7, 6, 5, 4, 3, 2, 1.5, 1.4, 1.3, 1.2, or less than 1.1 times the internalization of the isotype control. "Moderate internalization antibodies" are defined as antibodies exhibiting internalization at 21 times or less the internalization of the isotype control, and higher than 10 times the internalization of the isotype control. "High internalization antibodies" are defined as antibodies exhibiting internalization at higher than 21 times the internalization of the isotype control.

[0103] Alternatively, internalization can be further quantified based on t(1 / 2), i.e., as the time until half the amount of antibody, fragment, or conjugate is internalized. Preferably, the antibody according to the present invention is characterized in that the time until half the amount of antibody, fragment, or conjugate is internalized is more than 2 hours, preferably more than 4 hours, more than 5 hours, more than 6 hours, more than 7 hours, more than 8 hours, more than 9 hours, more than 10 hours, more than 11 hours, more than 12 hours, more than 13 hours, more than 14 hours, more than 15 hours, more than 16 hours, more than 17 hours, more than 18 hours, more than 19 hours, more than 20 hours, more than 21 hours, more than 22 hours, more than 23 hours, more than 24 hours, more than 26 hours, more than 28 hours, more than 30 hours, or more than 48 hours. Most preferably, the antibody according to the present invention is not internalized at all, i.e., the time until half the amount of antibody, fragment, or conjugate is internalized cannot be specified.

[0104] An "isotype control" is an antibody or fragment that does not bind to the target but has the same class and type as a reference antibody or fragment that recognizes the target.

[0105] When an antibody or fragment binds to antigens from two or more different species with a KD value of, for example, 10⁻⁷M or less, more preferably less than 10⁻⁸M, and even more preferably in the range of 10⁻⁹M to 10⁻¹¹M, the antibody or fragment is called "cross-reactive" or "cross-reactive."

[0106] Where used herein with respect to antibodies, the term “specifically binding” means an antibody that recognizes a specific antigen but substantially does not recognize or bind to other molecules in the sample. Antibodies characterized by substantial nonspecific binding lack therapeutic applicability, and consequently, these embodiments are excluded. However, as is known in the art, specific binding of an antibody or binder does not necessarily preclude the binding of the antibody or binder to further antigen / target molecules. An antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more further species. Such cross-species reactivity does not in itself alter the specific classification of the antibody.

[0107] In some cases, the terms "specific binding" or "specifically binding" can be used in reference to the interaction between an antibody, protein, or peptide and a second chemical species, meaning that the interaction depends on the presence of a specific structure on the chemical species (e.g., an antigenic determinant or epitope). For example, an antibody generally recognizes and binds to a specific protein structure rather than the protein itself. If an antibody is specific to epitope "A", then in a reaction involving labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) reduces the amount of labeled A bound to the antibody.

[0108] If suspected, specific binding of the antibody or binder preferably represents the binding of the antibody, antibody fragment, or binder to its antigen / target with an affinity of at least 10⁻⁷M (as a KD value; i.e., preferably having a KD value of less than 10⁻⁷M), and the antibody or binder has at least a 50% affinity to a nonspecific antigen that is not the given antigen / target molecule or a closely related antigen / target molecule.

[0109] "Polyspecificity," "polyreactivity," or "nonspecific binding" refers to the ability of a binder or antibody to bind to a defined set of unrelated antigens. Nonspecific binding is substantial if the (therapeutic) applicability of the antibody is impaired. The polyspecificity of non-protein structures, including but not limited to target-negative cell lines or tissues, baculovirus particles (BVP), insulin, or DNA, is known in the art and can be evaluated as described herein. For example, nonspecific binding to target-negative human cell lines can be determined, for example, by FACS analysis using pseudo-transfected CHO or HEK cells. In a second example, nonspecific binding to different tissues can be analyzed by FACS analysis of cell lines or panels of cell lines derived from each tissue. In a third example, nonspecific binding to immune cell populations can be analyzed by FACS after selecting the immune cell population as known in the art.In the fourth example, nonspecific binding to BVP, insulin, or DNA can be analyzed using ELISAs as described in, for example, Hotzel, Isidro, et al. "A strategy for risk mitigation of antibodies with fast clearance." MAbs. Vol. 4. No. 6. Taylor & Francis, 2012.; Avery, Lindsay B., et al. "Establishing in vitro in vivo correlations to screen monoclonal antibodies for physicochemical properties related to favorable human pharmacokinetics." MAbs. Vol. 10. No. 2. Taylor & Francis, 2018.; and Jain, Tushar, et al. "Biophysical properties of the clinical-stage antibody landscape." Proceedings of the National Academy of Sciences 114.5(2017):944-949 (these in whole are incorporated herein, in particular, with respect to the technical details necessary to analyze and quantify nonspecific binding). Antibodies that do not exhibit substantial nonspecific binding are preferably characterized by nonspecific binding lower than that of the reference antibody gantenerumab (Roche), and most preferably lower than that of the reference antibody remicade (Janssen Biotech).

[0110] The term "off-target binding" refers to the ability of an antibody to bind to individual proteins other than its intended target, such as proteins in the target protein family. Off-target binding can be evaluated using commercially available assays known in the art, such as the Retrogenix off-target profiling assay. Briefly, the antibody is tested in a microarray containing HEK293 cells that individually express thousands of human membrane proteins and secretory proteins. Binding of the antibody to potential off-targets should be confirmed by FACS using cells that overexpress the potential off-target.

[0111] The term "affinity" is a term used in the art and represents the strength of binding between a binder, antibody, or antibody fragment and a target. The "affinity" of an antibody and its fragment for a target can be determined using techniques known in the art or described herein, for example, by ELISA, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), flow cytometry, or fluorescence polarization assay. Preferably, affinity is provided as a dissociation constant KD.

[0112] The dissociation constant (KD) is expressed in molar units (M) and corresponds to the concentration of the binder / antibody at which half of the target protein is occupied in equilibrium. A smaller dissociation constant indicates a higher affinity between the binder or antibody and its target.

[0113] According to the present invention, the antibody preferably has a target affinity in the range of at least 10⁻⁷ M (as a KD value), more preferably at least 10⁻⁸ M, and even more preferably in the range of 10⁻⁹ M to 10⁻¹¹ M. The KD value can preferably be determined by surface plasmon resonance spectroscopy, for example, as described elsewhere in this specification. If it is found that the assay conditions affect the determined KD, the assay setting having the smallest standard deviation should be used.

[0114] The "maximum half-dose effective concentration" (EC50) refers to the concentration of a drug, antibody, fragment, conjugate, or molecule that induces an intermediate response between baseline and maximum after a specific incubation period. Therefore, in relation to antibody binding, EC50 reflects the antibody concentration required for half of the maximum binding. EC50 can be determined if the inflection point can be determined by mathematical modeling (e.g., nonlinear regression) of the dose-response curve that describes the relationship between the applied drug, antibody, fragment, conjugate, or molecule concentration and the signal. For example, if the dose-response curve follows a sigmoid curve, EC50 can be determined. If the response is inhibitory, EC50 is called the maximum half-dose inhibitory concentration (IC50). EC80 can be determined with necessary modifications.

[0115] The term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds to a particular antigen or is immunologically reactive with a particular antigen (e.g., but not limited to, human IgG1, IgG2, IgG3, IgG4, IgM, IgD, IgE, IgA1, IgA2, mouse IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgA, IgD, IgE, or IgM, rat IgG1, IgG2a, IgG2b, IgG2c, IgA, IgD, IgE, or IgM, rabbit IgA1, IgA2, IgA3, IgE, IgG, IgM, goat IgA, IgE, IgG1, IgG2, IgE, IgM, or chicken IgY). Antibodies or antibody fragments contain complementarity-determining regions (CDRs), also known as hypervariable regions, in both the light chain variable domain and the heavy chain variable domain. The more highly conserved portion of the variable domain is called the framework (FR). As is known in the art, the amino acid positions / boundaries describing the hypervariable region of an antibody can vary depending on the context and various definitions known in the art. Where used herein, immunoglobulin amino acid residue numbering is performed according to the immunoglobulin amino acid residue numbering system of Kabat et al. The variable domains of the native heavy and light chains each contain four FR regions. Three CDRs in each chain are held together with CDRs from the other chain, adjacent to each other by the FR regions, contributing to the formation of the antibody's antigen-binding site (see Kabat, EA, et al. “Sequences of Proteins of Immunological Interest (Natl. Inst. Health, Bethesda, MD), GPO Publ.” No. 165-462 (1987)). Where used herein, the term antibody also refers to an antibody fragment unless otherwise specified. Depending on the context, the term antibody may also refer to any protein-binding molecule having immunoglobulin-like function.

[0116] The term "CDR" refers to the complementarity-determining region of an antibody. As is well known in the art, the complementarity-determining region (CDR) is part of the variable chain of antibodies and T cell receptors. A set of CDRs constitutes a paratope. CDRs are crucial for the diversity of antigen specificity. There are three CDRs (CDR1, CDR2, and CDR3) discontinuously positioned on the amino acid sequence of the variable domain of the antigen receptor. Since an antigen receptor typically consists of two variable domains (on two different polypeptide chains, a heavy chain and a light chain), there are usually six CDRs for each antigen receptor that can collectively contact the antigen. The CDRs of the light chain are LCDR1, LCDR2, and LCDR3. The CDRs of the heavy chain are called HCDR1, HCDR2, and HCDR3. HCDR3 is the most variable complementarity-determining region (see, for example, Chothia, Cyrus, and Arthur M. Lesk. "Canonical structures for the hypervariable regions of immunoglobulins." Journal of molecular biology 196.4(1987):901-917.; Kabat, EA, et al. "Sequences of proteins of immunological interest. Bethesda, MD: US Department of Health and Human Services." Public Health Service, National Institutes of Health (1991):103-511).

[0117] The "constant region" refers to the portion of the antibody molecule that imparts effector function. The heavy chain constant region can be selected from one of five isotypes: alpha (α), delta (δ), epsilon (ε), gamma (g), or mu (μ).

[0118] As used herein, the terms “Fc domain,” “Fc region,” or “Fc portion” refer to the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. This term includes both the native sequence Fc region and the variant Fc region. For example, the human IgG heavy chain Fc region may extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain.

[0119] The antibody or binding fragment according to the present invention may be modified to alter at least one constant region-mediated biological effector function. For example, in some embodiments, the antibody may be modified to reduce or enhance at least one constant region-mediated biological effector function compared to the unmodified antibody, for example, by reducing or improving binding to the Fc receptor (FcγR). FcγR binding can be reduced, for example, by mutating the immunoglobulin constant region segment of an antibody in a specific region required for FcγR interaction (see, e.g., Canfield, Stephen M., and Sherie L. Morrison. "The binding affinity of human IgG for its high affinity Fc receptor is determined by multiple amino acids in the CH2 domain and is modulated by the hinge region." The Journal of experimental medicine 173.6(1991):1483-1491; and Lund, John, et al. "Human Fc gamma RI and Fc gamma RII interact with distinct but overlapping sites on human IgG." The Journal of Immunology 147.8(1991):2657-2662). FcγR binding can also be enhanced, for example, by afucosylation. A decrease in FcγR binding can also reduce other effector functions that depend on FcγR interaction, such as opsonization, phagocytosis, and antigen-dependent cell-mediated cytotoxicity ("ADCC").

[0120] Furthermore, by addressing the interaction between Fc and FcRn, it becomes possible to modulate the half-life of the antibody in vivo. For example, disrupting the interaction by introducing the H435A mutation results in an extremely short half-life because the antibody is no longer protected from lysosomal degradation by FcRn recycling. In several preferred embodiments of the present invention, the antibody according to the present invention contains the H435A mutation or is manipulated to shorten its half-life.

[0121] In contrast, antibodies containing equivalent mutations such as the "YTE" mutation (M252Y / S254T / T256E) and / or the "LS" mutation (M428L / N434S) have been shown to significantly extend half-life through more efficient recycling from endosomes in both preclinical species and humans (Dall'Acqua, William F., et al. "Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences." The Journal of Immunology 169.9(2002):5171-5180.; Zalevsky, Jonathan, et al. "Enhanced antibody half-life improves in vivo activity." Nature biotechnology 28.2(2010):157-159.). In several preferred embodiments of all aspects, the antibodies according to the present invention contain YTE mutations (M252Y / S254T / T256E) and / or equivalent mutations, such as LS (M428L / N434S), or are engineered for an improved half-life. Appropriate Fc engineering approaches for extending half-life can be found in Haraya, Kenta, Tatsuhiko Tachibana, and Tomoyuki Igawa. "Improvement of pharmacokinetic properties of therapeutic antibodies by antibody engineering." Drug metabolism and pharmacokinetics 34.1(2019):25-41. and / or Lee, Chang-Han, et al. "An engineered human Fc domain that behaves like a pH-toggle switch for ultra-long circulation persistence." Nature communications 10.1(2019):1-11 (both incorporated herein by reference).

[0122] Afucosylated antibodies are antibodies that have been manipulated so that the oligosaccharide in the Fc region of the antibody does not contain a fucose sugar unit. Glycosylation of an antibody can alter its function. For example, complete elimination of glycosylation at N297 in the CH2 domain of IgG results in loss of binding to FcγR. However, modification of the specific carbohydrate composition at N297 has the opposite effect and can enhance the ADCC activity of the antibody. Briefly, the affinity of an antibody for activated FcγR depends on the composition of the N297 N-linked oligosaccharide. There are 32 different possible combinations of oligosaccharides that can occur at this site. Human IgG, both naturally occurring and produced by hybridomas or other common expression systems, typically consists of N-acetylglucosamine (GlcNAc) and three mannose residues that form a core carbohydrate. This core is branched by attachment to two further GlcNAc groups. Galactose addition at each branch, as well as sialic acid terminal addition to these galactose molecules, can occur. Fucose is often part of the core GlcNAc. This fucose interferes with the interaction between the antibody and FcγRIIIA via steric hindrance. Therefore, removing this fucose molecule while maintaining other forms of glycosylation at this site increases antibody binding to activated FcγR, enhancing its ability to induce ADCC and / or ADCP (Almagro, Juan C., et al. “Progress and challenges in the design and clinical development of antibodies for cancer therapy.” Frontiers in immunology 8(2018):1751.). Methods for preparing fucose-free antibodies include growth in rat myeloma YB2 / 0 cells (ATCC CRL 1662). YB2 / 0 cells express low levels of FUT8 mRNA encoding α-1,6-fucosyltransferase, an enzyme necessary for polypeptide fucosylation. Afucosylated antibodies are preferred for this invention.

[0123] Antibody-dependent cell-mediated cytotoxicity (ADCC), also known as antibody-dependent cell-mediated cytotoxicity, is a mechanism of cell-mediated immune defense in which immune cells actively lyse target cells whose membrane surface antigens are bound by specific antibodies. ADCC is mediated through the interaction of antibodies or fragments with FcγRIIIa. In humans, FcγRIII exists in two different forms: FcγRIIIa (CD16a) and FcγRIIIb (CD16b). FcγRIIIa is expressed on monocytes, neutrophils, mast cells, macrophages, and natural killer cells as a transmembrane receptor, while FcγRIIIb is expressed only on neutrophils. These receptors bind to the Fc portion of IgG antibodies and then activate antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by human effector cells.

[0124] Various assay systems for determining ADCC induction in human subjects have been described in the literature and are suitable for characterizing the subject disclosed herein. For example, Yao-Te Hsieh et al. studied assays based on different ADCC assay systems, namely (i) human donor-derived natural killer cells (FcγRIIIA+ primary NK), (ii) FcγRIIIA-engineered NK-92 cells, and (iii) FcγRIIIA / NFAT-RE / luc2-engineered Jurkat T cells (Hsieh, Yao-Te, et al. “Characterization of FcγRIIIA effector cells used in in vitro ADCC bioassay: comparison of primary NK cells with engineered NK-92 and Jurkat T cells.” Journal of Immunological Methods 441(2017):56-66, the whole is incorporated herein; see in particular the description of the methods of these assays). In short, all three effector cell lines differentially express FcγRIIIA and provide dose-dependent ADCC pathway activity, but only primary NK cells and engineered NK-92 cells can induce ADCC-mediated cytolysis. Therefore, for functional evaluation of ADCC activity, primary NK or NK-92(V-158) cells better reflect the physiologically relevant ADCC mechanism of action. As an engineered cell line, NK-92 cells can behave more reproducibly than primary NK cells and are therefore a preferred assay system for determining the ADCC response in human subjects, for example, when suspected.

[0125] The antibody or antigen-binding fragment that induces ADCC is an antibody capable of inducing the lysis of a substantial amount of target cells in the presence of NK effector cells. Preferably, ADCC induction results in the lysis of at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of target cells.

[0126] Antibody-dependent phagocytosis (ADCP) is a mechanism in which antibody-opsonized target cells activate FcγR on the surface of macrophages, inducing phagocytosis and leading to the internalization and degradation of target cells. In ADCP, binding to macrophages as effector cells typically occurs through the interaction between the antibody FC portion and FcγRIIa(CD32a) expressed by macrophages.

[0127] The ADCP-inducing antibody or antigen-binding fragment is an antibody capable of inducing a significant amount of phagocytosis in target cells in the presence of macrophages. Preferably, ADCP induction results in phagocytosis of at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of target cells.

[0128] Complement-dependent cell-mediated cytotoxicity ("CDC") is an effector function of IgG and IgM antibodies. When they bind to surface antigens on target cells (e.g., bacterial or virus-infected cells), the classical complement pathway is triggered by the binding of protein C1q to these antibodies, leading to the formation of membrane attack complexes (MACs) and target cell lysis. The complement system is efficiently activated by human IgG1, IgG3, and IgM antibodies, weakly activated by IgG2 antibodies, and not activated by IgG4 antibodies. The complement system is one of the mechanisms by which therapeutic antibodies (which are also specific embodiments of antibodies according to the present invention) can achieve antitumor effects. Several experimental methods exist and are known in the art for determining the efficacy of CDC.

[0129] An antibody or antigen-binding fragment that induces CDC is an antibody capable of inducing the formation of a significant amount of membrane attack complexes and the lysis of target cells. Preferably, CDC induction results in the lysis of at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of target cells.

[0130] Antibodies containing an Fc region may or may not contain modifications that promote the association of the first and second subunits of the Fc domain.

[0131] "Modifications that promote the association of the first and second subunits of the Fc domain" are operations on the peptide backbone or post-translational modifications of the Fc domain subunit that reduce or prevent association between the polypeptide containing the Fc domain subunit and the polypeptide identical to the Fc domain subunit, thereby forming a homodimer. Antibodies containing the Fc region may or may not contain modifications that promote the association of the first and second subunits of the Fc domain. The association-promoting modifications used herein include, in particular, separate modifications performed on each of the two Fc domain subunits that are desired to associate (i.e., the first and second subunits of the Fc domain), and the modifications are complementary to each other in order to promote the association of the two Fc domain subunits. For example, the association-promoting modifications may change the structure or charge of one or both of the Fc domain subunits to make their association sterically or electrostatically favorable. Thus, (hetero)dimerization occurs between the polypeptide containing the first Fc domain subunit and the polypeptide containing the second Fc domain subunit, which may not be identical in the sense that, for example, the further components fused to each subunit (e.g., antigen-binding moieties) are not the same. In some embodiments, the modification that promotes association includes amino acid mutations, specifically amino acid substitutions, in the Fc domain. In certain embodiments, the modification that promotes association includes separate amino acid mutations, specifically amino acid substitutions, in each of the two subunits of the Fc domain.

[0132] The antibody “fragments” used herein are necessary to substantially retain the desired affinity of the full-length antibody. Therefore, a suitable fragment of an anti-human CCR8 antibody retains the ability to bind to a target chemokine receptor, such as the human CCR8 receptor. An antibody fragment includes a portion of a full-length antibody, generally its antigen-binding region or variable region. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, single-chain antibody molecules, diabodies, and domain antibodies (see Holt, Lucy J., et al. “Domain antibodies: proteins for therapy.” Trends in biotechnology 21.11(2003):484-490).

[0133] The "Fab fragment" includes a constant domain of the light chain and a first constant domain (CH2) of the heavy chain.

[0134] The "Fab' fragment" differs from the Fab fragment by adding several residues to the carboxyl terminus of a heavy chain CH2 domain containing one or more cysteines derived from the antibody hinge region.

[0135] The "F(ab') fragment" is produced by the cleavage of the disulfide bond at the hinge cysteine ​​of the F(ab')2 pepsin digestion product. Further chemical coupling of antibody fragments is known to those skilled in the art. Fab and F(ab')2 fragments lack the Fc fragment of the intact antibody, disappear more rapidly from the animal circulation, and may exhibit less nonspecific tissue binding than the intact antibody (e.g., Wahl, Richard L., Charles W. Parker, and Gordon W. Philpott. "Improved radioimaging and tumor localization with monoclonal F(ab')2." Journal of nuclear medicine: official publication, Society of Nuclear Medicine 24.4(1983):316-325).

[0136] The "Fv fragment" is the smallest fragment of an antibody that contains the complete target recognition and binding site. This region consists of a dimer (VH-VL dimer) of one heavy-chain variable domain and one light-chain variable domain in a tight, non-covalent association. In this configuration, three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Often, six CDRs confer antigen-binding specificity to the antibody. However, in some cases, even a single variable domain (or half of the Fv containing only three target-specific CDRs) may have the ability to recognize and bind to an antigen, albeit with lower affinity than the entire binding site.

[0137] A "single-chain Fv" or "scFv" antibody fragment contains the VH and VL domains of the antibody in a single polypeptide chain. Generally, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains, which allows the scFv to form a desired structure for antigen binding.

[0138] A "single-domain antibody" consists of a single VH or VL domain that exhibits sufficient affinity for the target. In certain embodiments, the single-domain antibody is a camelized antibody; see, for example, Riechmann, Lutz, and Serge Muyldermans. "Single domain antibodies: comparison of camel VH and camelised human VH domains." Journal of immunological methods 231.1-2(1999):25-38.

[0139] A "bispecific antibody" is a monoclonal antibody that has binding specificity to at least two different epitopes on the same or different antigen. In this disclosure, one of the binding specificities may be directed to a target chemokine receptor such as CCR8, and the other may be directed to any other antigen, for example, but not limited to, cell surface proteins, receptors, receptor subunits, tissue-specific antigens, virus-derived proteins, virus-encoded envelope proteins, bacterial-derived proteins, or bacterial surface proteins. The bispecific antibody constructs according to the present invention also encompass multispecific antibody constructs, such as a triplicate antibody construct, which includes multiple binding domains / binding sites, and the construct includes three binding domains.

[0140] "Derivatized antibodies" are typically modified by glycosylation, acetylation, pegylation, phosphorylation, sulfation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or binding to cell ligands or other proteins. Any of the many chemical modifications can be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, or the metabolic synthesis of tunicamycin. Furthermore, derivatives may contain one or more unnatural amino acids, for example, using ambrx technology (see, e.g., Wolfson, Wendy. "Amber codon flashing ambrx augments proteins with unnatural amino acids." Chemistry & Biology 13.10(2006):1011-1012). The antibodies according to the present invention may be derivatized, for example, glycosylated or sulfated.

[0141] A "monoclonal antibody" is a substantially homogeneous population of antibodies that bind to a specific antigen.

[0142] Monoclonal immunoglobulins can be obtained by methods well known to those skilled in the art (see, for example, Kohler, Georges, and Cesar Milstein. "Continuous cultures of fused cells secreting antibody of predefined specificity." Nature 256.5517(1975):495-497., and US Patent No. 4, 376, 110). Immunoglobulins or immunoglobulin fragments having specific binding affinity can be isolated, concentrated, or purified from prokaryotes or eukaryotes. Routine methods known to those skilled in the art enable the production of both immunoglobulins or immunoglobulin fragments and protein-binding molecules having immunoglobulin-like function in both prokaryotes and eukaryotes. The antibodies according to the present invention are preferably monoclonal.

[0143] A "humanized antibody" includes a CDR region derived from a non-human species, such as mouse, transplanted into a V region derived from a human sequence, along with, for example, a back mutation in any necessary framework. Therefore, in most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's hypervariable region are replaced by residues from the hypervariable region of a non-human species (donor antibody) having the desired specificity, affinity, and capability, such as mouse, rat, rabbit, or non-human primate. See, for example, U.S. Patents 5,225,539, 5,585,089, 5,693,761, 5,693,762, and 5,859,205, incorporated herein by reference. In some examples, framework residues of human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody may contain residues not found in the recipient antibody or donor antibody. These modifications are made to further improve the performance of the antibody (e.g., to obtain the desired affinity). Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains, with all or substantially all of the hypervariable region corresponding to that of a non-human immunoglobulin, and all or substantially all of the framework region being a human immunoglobulin sequence. A humanized antibody may also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin. For further details, see Peter T., et al. “Replacing the complementarity-determining regions in a human antibody with those from a mouse.” Nature 321.6069(1986):522-525.; Riechmann, Lutz, et al. “Reshaping human antibodies for therapy.” Nature 332.6162(1988):323-327.; and Presta, Leonard G. “Antibody engineering.” Current Opinion in Structural Biology 2.4(1992):593-596, which are incorporated herein by reference.

[0144] Fully human antibodies (human antibodies) include human-derived CDRs, i.e., CDRs of human origin. Preferably, the fully human antibodies according to the present invention are antibodies that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with the nearest human VH germline gene (e.g., a sequence extracted from a recommended list and analyzed by IMGT / domain gap alignment).

[0145] As recognized by conventional nomenclature systems such as the INN species subsystem, which was valid until 2017, fully human antibodies may contain fewer germline deviations compared to the nearest human germline reference determined based on the IMGT database (http: / / www.imgt.org, November 29, 2019). For example, fully human antibodies according to the present invention may contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, or 15 germline deviations in the CDR compared to the nearest human germline reference. Fully human antibodies can be developed from human-derived B cells by cloning techniques combined with cell enrichment or immortalization steps. However, the majority of fully human antibodies used in clinical practice are isolated from either immunized mice transfected with the human IgG locus, or from sophisticated combinatorial libraries created by phage display (Bruggemann, Marianne, et al. "Human antibody production in transgenic animals." Archivum immunologiae et therapiae experimentalis 63.2(2015):101-108.; Carter, Paul J. "Potent antibody therapeutics by design." Nature reviews immunology 6.5(2006):343-357.; Frenzel, Andre, Thomas Schirrmann, and Michael Hust. "Phage display-derived human antibodies in clinical development and therapy." MAbs. Vol.8. No.7. Taylor & Francis, 2016.; Nelson, Aaron L., Eugen Dhimolea, and Janice M. Reichert. "Development trends for human monoclonal antibody therapeutics." Nature reviews drug discovery 9.10(2010):767-774.).

[0146] Several techniques are available for producing fully human antibodies or antibodies containing human-derived CDRs (International Publication No. 2008112640). Cambridge Antibody Technologies (CAT) and Dyax have devised a phage display library for obtaining antibody cDNA sequences from peripheral B cells isolated from immunized humans and identifying human variable region sequences of specific specificity. Briefly, the antibody variable region sequence is fused with either the gene III or gene VIII structure of the M13 bacteriophage. These antibody variable region sequences are expressed as either a Fab or single-stranded Fv (scFv) structure at the tip of the phage possessing the respective sequence. Through a series of panning processes using varying levels of antigen-binding conditions (stringency), phages expressing Fab or scFv structures specific to the target antigen can be selected and isolated. The antibody variable region cDNA sequences of the selected phages can then be elucidated using standard sequencing procedures. These sequences can then be used to reconstruct fully antibodies with the desired isotype using established antibody engineering techniques. Antibodies constructed according to this method are considered fully human antibodies (including CDRs). To improve the immunoreactivity (antigen-binding affinity and specificity) of selected antibodies, in vitro maturation processes can be introduced, including combinatorial association of different heavy and light chains, deletion / addition / mutation of heavy and light chains in CDR3 (to mimic recombination of VJ and VDJ), and random mutations (to mimic somatic high-frequency mutations). An example of a "fully human" antibody produced by this method is the antitumor necrosis factor α antibody, Humira (adalimumab).

[0147] The term "polynucleotide" refers to polymeric deoxyribonucleotides or their analogues, or modified polynucleotides, produced by recombinant or synthetic means. This term includes double-stranded and single-stranded DNA or RNA. Polynucleotides can be incorporated into, for example, minicircles, plasmids, cosmids, minichromosomes, or artificial chromosomes. Polynucleotides can be isolated or incorporated into other nucleic acid molecules, such as expression vectors or chromosomes in eukaryotic host cells.

[0148] As used herein, the term “vector” refers to a nucleic acid molecule capable of amplifying the nucleic acid molecule to which it is ligated. This term further includes plasmids (non-viral) and viral vectors. Certain vectors can direct the expression of nucleic acids or polynucleotides to which they are operably ligated. Such vectors are referred herein to as “expression vectors.” Expression vectors for eukaryotic use can be constructed by inserting a polynucleotide sequence encoding at least one protein of interest (POI) into a suitable vector backbone. The vector backbone may include elements necessary to ensure the maintenance of the vector and, if desired, provide amplification in a host. Viral vectors, such as lentiviral vectors or retroviral vectors, may require further virus-specific elements, such as structural elements or other elements, which are well known in the art. These elements may be provided, for example, in cis (on the same plasmid) or trans (on separate plasmids). Viral vectors may require helper viruses or packaging lines for large-scale transfection. Vectors may contain further elements such as plasmid replication enhancer elements (e.g., viruses, eukaryotes), introns, and viral replication origins for replication in mammalian cells. According to the present invention, the expression vector typically has a promoter sequence that drives the expression of POI. The expression of POI and / or a select marker protein may be constitutive or regulated (e.g., induced by the addition or removal of small molecule inducers). Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of POI expression in mammalian cells, such as regulatory elements, promoters and / or enhancers derived from cytomegalovirus (CMV), Simian virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter Ad LP), or polyoma. For further descriptions of viral regulatory elements and their sequences, see, for example, U.S. Patents 5,168,062, 4,510,245 and 4,968,615.

[0149] As used herein, the terms “linker” or “spacer” refer to any molecule that enables a direct topological connection between two parts. The parts may, among other things, be polypeptides, proteins, antibodies, antibody fragments, cytotoxic parts, binding parts, detection parts such as fluorophores, immobilization or recovery parts such as beads or magnetic beads, reactive parts, or any other molecule. The two parts may be of the same type or different. A linker may be part of a conjugate and contribute to its function. For example, in a conjugate containing a polypeptide and biotin, the presence of a spacer of approximately 4 Å (about 5 atoms) between the carboxyl group of biotin and the first bulky amino acid of the peptide allows biotin to reach the (strept)avidin binding pocket. Various linkers are known in the art and can be selected based on the parts to be linked. Linker lengths typically range from 4 atoms to over 200 atoms. Linkers with lengths exceeding 60 atoms generally comprise a group of compounds with an average length.

[0150] A "linker to polypeptide" may be attached via an amide bond or any other functional residue. The linker to polypeptide may be attached to the N-terminus or C-terminus of the polypeptide, or via a reactive functional group or amino acid side chain. Polypeptides may be coupled to proteins such as biotin or human serum albumin (HSA), carrier proteins such as keyhole limpet hemocyanin (KLH), ovalbumin (OVA), or bovine serum albumin (BSA), fluorescent dyes, short amino acid sequences such as Flag tags, HA tags, Myc tags, or His tags, reactive tags such as maleimide, iodoacetamide, alkyl halides, 3-mercaptopropyl, or 4-azidobutylic acid, or various other suitable parts. Suitable linkers, such as non-limiting examples of linkers suitable for polypeptide conjugation, include β-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy)acetic acid (AEA), 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx), PEG2 spacer (8-amino-3,6-dioxaoctanoic acid), PEG3 spacer (12-amino-4,7,10-trioxadodecanoic acid), PEG4 spacer (15-amino-4,7,10,13-tetraoxapentadecanoic acid), and Ttds (trioxatridecane-succinic acid). In some cases, the linker may be derived from a reactive moiety, such as maleimide, iodoacetamide, alkyl halides, 3-mercaptopropyl, or 4-azidobutyric acid. In some cases, the linker may include polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol or polypropylene glycol.

[0151] A "linker for antibodies" is a linker that establishes covalent bonds between different antibody moieties, and includes, but is not limited to, peptide linkers and non-protein polymers (polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol).

[0152] To "treat" a disease in a subject, or to "treat" a subject with a disease, means to subject the subject to medical treatment, such as the administration of drugs, so that at least one symptom of the disease is reduced or its worsening is prevented.

[0153] Terms such as "prevent," "preventing," and "prevention" refer to reducing the probability of developing a disease, disability, or condition in individuals who do not currently have the disease, disability, or condition but are at risk of developing it or are prone to developing it.

[0154] The terms “effective dose” or “therapeutic effective dose” are used interchangeably herein and refer to an amount sufficient to achieve a specific biological outcome or to modulate or improve symptoms in a subject, or typically at least about 10%, usually at least about 20%, preferably at least about 30%, or more preferably at least about 50% of the time to symptom onset. The effectiveness of antibody use in cancer treatment can be evaluated based on changes in tumor load. Both tumor reduction (objective response) and time to disease progression are important endpoints in cancer clinical trials. Standardized response criteria (known as Response Evaluation Criteria in Solid Tumors (RECIST)) were published in 2000. An updated version (RECIST 1.1) was published in 2009. RECIST criteria are typically used in clinical trials where objective response is the primary trial endpoint, as well as in trials where stable disease, tumor progression, or time to progression analysis is evaluated, because these outcome measures are based on an evaluation of anatomical tumor load and its changes over the course of the trial. The effective dose for a particular subject may vary depending on factors such as the condition being treated, the subject's overall health, the method, route and dosage of administration, and the severity of side effects. When combined, the effective dose is proportional to the combination of components, and the effect is not limited to the individual components alone.

[0155] Unless otherwise defined, "complete response" (CR) is defined as the disappearance of all target lesions. All pathological lymph nodes (target or non-target) must be reduced to less than 10 mm in their short axis. For "partial response" (PR), the total diameter of the target lesions must be reduced by at least 30% relative to the total diameter at baseline.

[0156] For "progressive disease" (PD), the minimum total diameter during the study is used as a reference, and the total diameter of the target lesion is increased by at least 20% (including the baseline total, if it is the minimum during the study). In addition to a 20% relative increase, the total must also show an absolute increase of at least 5 mm. For "stable disease" (SD), no contraction sufficient to qualify for a partial reduction (PR) or an increase sufficient to qualify for PD is observed, based on the minimum total diameter during the study.

[0157] Secondary outcome measures that may be used to demonstrate the therapeutic benefits of the antibodies of the present invention as described herein include: “Objective Response Rate” (ORR) is defined as the proportion of subjects achieving a complete response (CR) or partial response (PR). “Progression-Free Survival” (PFS) is defined as the time from the first administration of the antibody to the first occurrence of disease progression or death. “Overall Survival” (OS) is defined as the length of time from the date of diagnosis of the disease or the date of initiation of treatment that a patient diagnosed with the disease is still alive. “Duration of Overall Response” (DOR) is defined as the time from the first CR or PR in a participant to the point of disease progression. “Depth of Response” (DpR) is defined as the percentage of tumor reduction observed at the point of maximum response compared to the baseline tumor loading. Both the ORR and PFS clinical endpoints can be determined based on the RECIST 1.1 criteria described above.

[0158] When non-human subjects are analyzed, the aforementioned parameters for determining therapeutic efficacy and benefits must be adapted as discussed elsewhere in this specification (see Example 12ff).

[0159] Typical "subjects" according to the present invention include human subjects and non-human subjects. Subjects may be mammals such as mice, rats, cats, dogs, primates, and / or humans.

[0160] A “pharmaceutical composition” (or “therapeutic preparation”) of an antibody, fragment, or conjugate can be prepared by mixing an antibody of the desired purity with any physiologically acceptable carrier, excipient, or stabilizer, for example, in the form of a lyophilized preparation or aqueous solution, according to Remington's Pharmaceutical Sciences (18th ed.; Mack Pub. Co.: Eaton, Pa., 1990). Acceptable carriers, excipients, or stabilizers are nontoxic to the recipient at the dosage and concentration used and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; and serum. The material contains proteins such as albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, and lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as Tween®, Pluronic®, or polyethylene glycol (PEG).

[0161] A "host cell" is a cell used to receive, maintain, regenerate, and amplify a vector. A polypeptide, such as an antibody or fragment thereof encoded by the vector, can also be expressed using a host cell. The nucleic acids contained in the vector are replicated and amplified when the host cell divides. Preferred host cells are mammalian cells such as CHO cells or HEK cells. A more preferred host cell is rat myeloma YB2 / 0 cells.

[0162] "Cells with endogenous target expression" are cells that express the target protein at levels comparable to those in physiological or pathological conditions. Typically, cells manipulated for overexpression express the target protein at much higher levels.

[0163] In the context of cells, structures, proteins, antibodies, or markers, the terms “intratumor,” “intratumor,” “tumor infiltration,” or “tumor” refer to their localization within tumor tissue.

[0164] Cells that are “positive” or “+” for a particular marker or protein are cells characterized by substantial expression of that marker or protein. The expression of a marker or protein can be determined and quantified, for example, to define different cell populations, as is known in the art. For the characterization of (immune) cell populations, marker expression can be determined by FACS or using any other techniques described herein.

[0165] "White blood cells" are immune cells that express CD45. As used herein, "CD45+ cells" refers to all white blood cells. CD45 can be used as a marker to distinguish between immune cells and non-immune cells.

[0166] The term "lymphocyte" refers to all immature, mature, undifferentiated, and differentiated white lymphocyte populations, including tissue-specific and specialized varieties. It includes, in non-limiting examples, B cells, T cells, NKT cells, and NK cells. In some embodiments, lymphocytes include all B cell lineages, including pre-B cells, progenitor B cells, early pro-B cells, late pro-B cells, large pre-B cells, small pre-B cells, immature B cells, mature B cells, plasma B cells, memory B cells, B-1 cells, B-2 cells, and anergy AN1 / T3 cell populations.

[0167] "T cells" are immune cells that express TCRαβ, CD3, and CD8 or CD4. As used herein, this term includes naive T cells, CD4+ T cells, CD8+ T cells, regulatory T cells, memory T cells, activated T cells, anerious T cells, resistant T cells, chimeric B cells, and antigen-specific T cells, as well as further T cell populations known in the art. In some embodiments, the presence of a T cell receptor (TCR) on the cell surface distinguishes T cells from other lymphocytes.

[0168] CD8+ T cells (also known as cytotoxic T cells, TCs, cytotoxic T lymphocytes, CTLs, T-killer cells, cytolytic T cells, CD8+ T cells, or killer T cells) are T cells that express CD3, CD45, and CD8. CD8+ T cells can kill cancer cells, cells infected (especially by viruses), or cells damaged by other means.

[0169] CD4+ T cells (also known as T helper cells or Th cells) are immune cells that express CD3, CD4, and CD45. Several subsets of T helper cells exist, including, but are not limited to, Th1, Th2, and Th17. CD4+ T cells play a role in suppressing or regulating the immune response. CD4+ T cells are essential for maximizing B cell antibody class switching, activation and proliferation of cytotoxic T cells, and the bactericidal activity of phagocytic cells such as macrophages.

[0170] As used herein, the terms “Treg cells” (also “Treg,” “regulatory T cells,” “T regulatory cells,” and “suppressor T cells”) refer to immune cells that express CD3, CD4, CD45, and FoxP3, and also express high levels of CD25 and low levels of CD127. Identification of Treg cells may be carried out as described elsewhere herein. Treg cells also typically express high levels of CTLA-4, GITR, and LAG-3. In the literature, Tregs are further classified based on the memory marker CD45RO.

[0171] Under physiological conditions, Treg cells maintain immune tolerance. During an immune response, Treg cells suspend T cell-mediated immunity and suppress autoreactive T cells that have escaped negative selection in the thymus. Treg cells can also suppress other types of immune cells, such as NK cells and B cells. Adaptive Treg cells (called Th3 or Tr1 cells) are thought to be generated during an immune response.

[0172] Treg cells also play a crucial role in immune evasion by suppressing anti-tumor immunity, thereby providing an environment for immune tolerance. While T cells that recognize cancer cells are often abundant within tumors, their cytotoxic function is suppressed by nearby immunosuppressive cells. Tregs are abundant in many different cancers, highly concentrated in the tumor microenvironment, and their role in tumor progression is well known.

[0173] "Activated Treg cells" express CD4, CD45, FoxP3, CD69, and CCR8, and also have high expression of CD25 and low expression of CD127. CD69 is a T cell activation marker.

[0174] "CCR8-positive regulatory T cells" or "CCR8+ regulatory T cells" are Tregs that express CCR8.

[0175] "CD4conv cells" are conventional CD4+, CD25-T cells.

[0176] Gamma delta T cells are T cells that express a characteristic T cell receptor, TCRγδ, on their surface. Gamma delta T cells also express CD3.

[0177] "B cells" are immune cells that express CD19, and mature B cells express CD20 and CD22. When activated via CD40, B cells undergo differentiation, resulting in high-frequency somatic mutations and enhanced immunoglobulin class switching, leading to mature B cells or plasma cells (which can secrete Ab). B cells are involved in humoral immunity in the adaptive immune system and are antigen-presenting cells.

[0178] Macrophages are immune cells that express low CD14, high CD16, CD11b, CD68, CD163, and CD206. Macrophages engulf and digest cellular debris, foreign substances, microorganisms, or cancer cells through phagocytosis. In addition to phagocytosis, macrophages play an important role in innate immunity and help initiate adaptive immunity by recruiting other immune cells. For example, macrophages are important as antigen-presenting cells to T cells. Macrophages that promote inflammation are called M1 macrophages, while macrophages that reduce inflammation and promote tissue repair are called M2 macrophages.

[0179] As used herein, "M1 macrophages" are a subset of macrophages that express ACOD1. M1 macrophages have pro-inflammatory, bactericidal, and phagocytic functions.

[0180] As used herein, "M2 macrophages" refer to a subset of macrophages that express MRC1(CD206). M2 macrophages secrete anti-inflammatory interleukins and play a role in wound healing, which is necessary for revascularization and re-epithelialization. Tumor-associated macrophages are primarily M2 phenotype and appear to actively promote tumor growth.

[0181] "Dendritic cells" (DC) are bone marrow-derived leukocytes and are the most potent type of antigen-presenting cells. DCs are specialized to capture and process antigens, and convert proteins into peptides that are presented on major histocompatibility complex (MHC) molecules recognized by T cells. As defined herein, DCs are characterized by the expression of CD1c, CD14, CD16, CD141, CD11c and CD123. Different subsets of dendritic cells exist.

[0182] In humans, DC1 are immunogenic, while DC2 cells are tolerogenic. Mature DCs express CD83, and plasmacytoid DCs express CD123.

[0183] "NK cells" (also natural killer cells) are immune cells that express CD45, CD16, CD56, and NKG2D, but are CD3 negative. NK cells do not require activation to kill "self" cells that lack MHC class I markers. NCR1 (also called CD335 or NKp46) is expressed on NK cells and on a subset of NKT cells.

[0184] "Natural killer T (NKT) cells" are a heterogeneous group of T cells that share properties of both T cells and natural killer cells.

[0185] "iNKT cells" (also invariant natural killer T cells) express an invariant αβ TCR (Vα24-Jα18, CD24lo), CD44hi, NK1.1 (mouse), and NKG2D. The invariant TCR recognizes glycolipid antigens presented by the non-polymorphic MHC class I-like molecule CD1d. These cells can influence immune responses by rapidly producing large amounts of cytokines, namely IFNg.

[0186] As is known in the art, “effector cells” are immune cells that actively support the immune response after stimulation. As used herein, effector cells refer to immune cells that express the Fcγ receptor and are therefore capable of mediating ADCC or ADCP. Non-limiting examples of effector cells include monocytes, neutrophils, mast cells, and preferably macrophages and natural killer cells.

[0187] "Tertiary lymphoid structure" is an intratumoral structure characterized by increased expression of LTta, LTtb, Cxcr5, and Cxcl13.

[0188] As used herein, the terms “chimeric antigen receptor” or “CAR” refer to an artificial T cell surface receptor expressed on immune effector cells and engineered to specifically bind to an antigen. CARs may be used in therapies involving adoptive cell transfer. Monocytes are removed from a patient (blood, tumor, or ascites) and modified to express receptors specific to a particular form of antigen. In some embodiments, CARs are specifically expressed to tumor-associated antigens.

[0189] CARs may also include an intracellular activation domain, a transmembrane domain, and an extracellular domain containing a tumor-associated antigen-binding region. In some embodiments, a CAR comprises a fusion of a monoclonal antibody derived from a single-strand variable fragment (scFv) fused to the CD3-zeta transmembrane domain and the intracellular domain. The specificity of the CAR design may be derived from the receptor ligand (e.g., a peptide). In some embodiments, CARs can target cancer by redirecting monocytes / macrophages that express CARs specific to tumor-associated antigens.

[0190] The administration scheme is abbreviated as, for example, daily (QD), every two days (Q2D), or every three days (Q3D), as is well known in the art.

[0191] Embodiment Antigen and antibody-binding chemokine receptors Appearance 1 - Antigen According to a first aspect, isolated sulfated polypeptides containing a tyrosine-rich domain (TRD) of a seven-transmembrane receptor are provided. The tyrosine-rich domain is a conserved N-terminal domain that characterizes seven-transmembrane receptors such as CXC and CC chemokine receptors (see Example 1). As used herein, the term TRD refers to the amino acid or protein sequence of a CXC or CC chemokine receptor located at the N-terminus of the first cysteine, counting from the N-terminus. In addition to tyrosine, TRDs typically contain negatively charged amino acid residues such as aspartic acid. TRDs for all CC and CXC chemokine receptors are listed in Example 4 and Table 4.1 for mouse, monkey, and human.

[0192] Tyrosine sulfation is a ubiquitous post-translational protein modification that occurs in all multicellular organisms. It is catalyzed by tyrosylprotein sulfotransferases (TPST) 1 and 2, Golgi-resident enzymes that transfer sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to context-dependent tyrosine in the protein substrate. Currently, only a limited number of sulfated proteins are known, and the understanding of the biological sulfation mechanism and specific modification sites is still ongoing (see Example 4). Post-translational modifications such as glycosylation, phosphorylation, acylation, adenylation, farnesylation, ubiquitination, and sulfation are frequent in proteins of biological systems, but antibody production is typically based on unmodified target sequences.

[0193] The inventors have developed a unique approach for antibody production and, surprisingly, have found that using synthetically sulfated polypeptides according to the first embodiment, it is possible to increase both the success rate of antibody production of chemokine receptors themselves and the success rate of chemokine receptor antibodies with excellent functional properties for therapeutic use, as discussed elsewhere herein. The increase in the success rate of highly specific chemokine receptor antibodies was particularly surprising because research antibodies designed for sequence-independent detection of sulfated tyrosine as a post-translational modification are rare.

[0194] The seven-transmembrane receptor may be derived from any species that express a chemokine receptor characterized by TRD, such as humans, monkeys, cynomolgus macaques, rhesus macaques, rodents, mice, rats, horses, cattle, pigs, dogs, cats, and camels.

[0195] According to some first embodiments of the first aspect, an isolated polypeptide is provided comprising a tyrosine-rich domain (TRD) of a seven-transmembrane receptor, further characterized in that at least 25%, at least 50%, or at least 75% of the tyrosine residues of the TRD are sulfated.

[0196] For example, in a highly successful antibody campaign, the TRD of human or cynomolgus monkey CCR8 was sulfated at positions Y3, Y15, and Y17, with Y16 omitted, meaning 75% of the tyrosine in the TRD was sulfated (Table 6.1). In another example, the TRD of human CCR4 was sulfated at positions 19 and 22 and used for off-target binding, meaning 50% of the tyrosine was sulfated (Table 8.1). In yet another approach, the TRD of mouse CCR4 was sulfated at position 22 and used for off-target binding, meaning 25% of the tyrosine in the TRD was sulfated (Table 6.1).

[0197] Table 4.1 lists sulfated peptides containing preferred positions for tyrosine sulfate. While not bound by theory, the inventors believe that the introduction of further charge in the form of tyrosine sulfate modulates the antibody to recognize specific patterns of negative charge. This recognition appears to require an increase in the percentage of tyrosine and positively charged amino acids, at least in the HCDR3 of the antibody; i.e., the use of isolated sulfated polypeptides according to the present invention also affected the structural composition of the antibody, particularly the amino acid composition of HCDR3 (see Example 9).

[0198] According to some second embodiments of the first embodiment, which may or may not be the same as the first embodiment, an isolated polypeptide is provided in which the seven transmembrane receptor is human, cynomolgus monkey, or mouse.

[0199] In some of these second embodiments, the seven-transmembrane receptor is mouse. In some preferred embodiments of these second embodiments, the seven-transmembrane receptor is human and / or cynomolgus monkey. In some preferred embodiments of these second embodiments, the seven-transmembrane receptor is human. In some of these second embodiments, the seven-transmembrane receptor is cynomolgus monkey.

[0200] According to some third embodiments of the first embodiment, which may be the same as or different from the first and / or second embodiments of the first embodiment, an isolated polypeptide is provided, and the seven-transmembrane receptor is a chemokine receptor, preferably, a) CC chemokine receptors such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, or CCR10, b) CXC chemokine receptors such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, or CXCR6, or c) CX3CR1 or CXCR1 That is the case.

[0201] In some of these third embodiments, the seven-transmembrane receptor is a CC chemokine receptor or a CXC chemokine receptor. In some of these third embodiments, the seven-transmembrane receptor is a CC chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, or CCR10. In some preferred embodiments of these third embodiments, the seven-transmembrane receptor is CCR8 or CCR4. In some very preferred embodiments of these third embodiments, the seven-transmembrane receptor is CCR8. In some of these third embodiments, the seven-transmembrane receptor is a CXC chemokine receptor such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, or CXCR6. In some of these third embodiments, the seven-transmembrane receptor is CX3CR1 or CXCR1.

[0202] In some preferred embodiments of the first aspect, an isolated polypeptide is provided comprising a TRD of a human or cynomolgus monkey seven-transmembrane receptor, wherein at least 25%, at least 50%, or at least 75% of the tyrosine residues of the TRD are sulfated, and the seven-transmembrane receptor is a CC chemokine receptor or a CXC chemokine receptor, preferably CCR8 or CCR4.

[0203] According to some embodiments A of the third embodiment of the first aspect, the isolated sulfated polypeptide comprises the sequence described below, or a sequence having at least 90%, 95%, or 98% sequence identity: a) Preferably, at least Y10 and / or Y18 are sulfated, SEQ ID NO: 1 (CCR1_HUMAN_TRD), or b) Preferably, at least Y26 is sulfated, such as in SEQ ID NO: 7 (CCR2_HUMAN_TRD), or c) Preferably Y16 and / or Y17 are sulfated, such as SEQ ID NO: 13 (CCR3_HUMAN_TRD), or d) preferably at least Y22 is sulfated, more preferably Y16, Y19 and / or Y20 are sulfated, SEQ ID NO: 19 (CCR4_HUMAN_TRD), or e) preferably two, three or all of Y3, Y10, Y14 and Y15 are sulfated, SEQ ID NO: 25 (CCR5_HUMAN_TRD), or f) preferably at least two or three of Y18, Y26 and Y27 are sulfated, SEQ ID NO: 31 (CCR6_HUMAN_TRD), or g) preferably one or both of Y8 and Y17 are sulfated, SEQ ID NO: 37 (CCR7_HUMAN_TRD), or h) preferably at least two or all of Y3, Y15 and Y17 are sulfated, SEQ ID NO: 43 (CCR8_HUMAN_TRD), or i) at least Y28, preferably Y17 and / or Y37 are also sulfated, SEQ ID NO: 61 (CCR9_HUMAN_TRD), or j) preferably at least one or both of Y14 and Y22 are sulfated, SEQ ID NO: 67 (CCR10_HUMAN_TRD), or k) preferably Y27 is sulfated, SEQ ID NO: 73 (CXCR1_HUMAN_TRD), or l) preferably Y23 and / or Y25 are sulfated, SEQ ID NO: 79 (CXCR2_HUMAN_TRD), or m) preferably at least one or both of Y27 and Y29 are sulfated, SEQ ID NO: 85 (CXCR3_HUMAN_TRD), or n) preferably at least Y12 and / or Y21 are sulfated, SEQ ID NO: 91 (CXCR4_HUMAN_TRD), or o) preferably at least one of Y3 and Y27 is sulfated, SEQ ID NO: 97 (CXCR5_HUMAN_TRD), or p) preferably at least one or both of Y6 and Y10 are sulfated, SEQ ID NO: 103 (CXCR6_HUMAN_TRD), or q) Preferably, at least Y14 is sulfated, such as sequence number 157 (CX3CR1_HUMAN_TRD), or r) Preferably, at least Y27 is sulfated, such as SEQ ID NO: 163 (CXCR1_HUMAN_TRD).

[0204] According to some embodiments B of the third embodiment of the first aspect, the isolated sulfated polypeptide comprises the sequence described below, or comprises a sequence having at least 90%, 95%, or 98% sequence identity: a) Preferably, at least Y10 and / or Y18 are sulfated, SEQ ID NO: 3 (CCR1_MOUSE_TRD), or b) Preferably, at least Y37 and / or Y39 are sulfated, such as in SEQ ID NO: 9 (CCR2_MOUSE_TRD), or c) Preferably Y20 and / or Y22 are sulfated, such as in Sequence ID No. 15 (CCR3_MOUSE_TRD), or d) Preferably at least Y22 is sulfated, and more preferably Y16, Y19 and / or Y20 is sulfated, such as SEQ ID NO: 21 (CCR4_MOUSE_TRD), or e) Preferably two or three of Y10, Y12 and Y16 are sulfated, such as SEQ ID NO: 27 (CCR5_MOUSE_TRD), or f) Preferably, at least two or three of Y13, Y18 and Y19 are sulfated, SEQ ID NO: 33 (CCR6_MOUSE_TRD), g) Preferably, one or both of Y8 and Y17 are sulfated, and Y20 may also be sulfated, such as SEQ ID NO: 39 (CCR7_MOUSE_TRD), or h) Preferably, at least two or all of Y3, Y14 and Y15 are sulfated, such as SEQ ID NO: 45 (CCR8_MOUSE_TRD), or i) Preferably, at least Y28 is sulfated, and preferably Y19 is also sulfated, such as SEQ ID NO: 63 (CCR9_MOUSE_TRD), or j) Preferably, at least one, two or all of Y14, Y17 and Y22 are sulfated, such as sequence number 69 (CCR10_MOUSE_TRD), or k) Preferably, at least Y6 is sulfated, such as SEQ ID NO: 75 (CXCR1_MOUSE_TRD), or l) Preferably, Y24 is sulfated, such as in SEQ ID NO: 81 (CXCR2_MOUSE_TRD), or m) Preferably, at least one or both of Y27 and Y29 are sulfated, such as in SEQ ID NO: 87 (CXCR3_MOUSE_TRD), or n) Preferably, at least Y23, Y13 and / or Y14 are sulfated, such as SEQ ID NO: 93 (CXCR4_MOUSE_TRD), or o) Preferably, at least Y3 and / or Y14 and / or Y20 and / or Y26 are sulfated, such as SEQ ID NO: 99 (CXCR5_MOUSE_TRD), or p) Preferably, at least one or both of Y11 and Y15 are sulfated, such as SEQ ID NO: 105 (CXCR6_MOUSE_TRD), or q) Preferably, at least Y15 is sulfated, such as SEQ ID NO: 159 (CX3CR1_MOUSE_TRD), or r) Preferably, at least Y6 is sulfated, such as SEQ ID NO: 165 (CXCR1_MOUSE_TRD).

[0205] According to some embodiments C of the third embodiment of the first aspect, the isolated sulfated polypeptide comprises the sequence described below, or comprises a sequence having at least 90%, 95%, or 98% sequence identity: a) Preferably, at least Y10 and / or Y18 are sulfated, such as in SEQ ID NO: 2 (CCR1_MACFA_TRD), or b) Preferably, at least Y26 is sulfated, such as in SEQ ID NO: 8 (CCR2_MACMU_TRD), or c) Preferably, Y16 is sulfated, such as SEQ ID NO: 14 (CCR3_MACFA_TRD), or d) Preferably, at least Y22 is sulfated, and more preferably Y16, Y19 and / or Y20 is sulfated, such as SEQ ID NO: 20 (CCR4_MACFA_TRD), or e) Preferably two, three or all of Y3, Y10, Y14 and Y15 are sulfated, such as SEQ ID NO: 26 (CCR5_MACMU_TRD), or f) Preferably, at least two or three of Y23, Y31 and Y32 are sulfated, such as SEQ ID NO: 32 (CCR6_MACFA_TRD), or g) Preferably, one or both of Y8 and Y17 are sulfated, such as SEQ ID NO: 38 (CCR7_MACFA_TRD), or h) Preferably, at least two or all of Y3, Y15 and Y17 are sulfated, such as SEQ ID NO: 44 (CCR8_MACFA_TRD), or i) Preferably at least Y28, preferably Y17 and / or Y37 are also sulfated, SEQ ID NO: 62 (CCR9_MACFA_TRD), or j) Preferably, at least one or both of Y14 and Y22 are sulfated, such as SEQ ID NO: 68 (CCR10_MACFA_TRD), or k) Preferably, at least one of Y14 and Y28 is sulfated, such as SEQ ID NO: 74 (CXCR1_MACFA_TRD), or l) Preferably Y20 and / or Y22 are sulfated, such as in SEQ ID NO: 80 (CXCR2_MACFA_TRD), or m) Preferably, at least one or both of Y27 and Y29 are sulfated, such as in SEQ ID NO: 86 (CXCR3_MACFA_TRD), or n) Preferably, at least Y12 and / or Y21 are sulfated, such as in SEQ ID NO: 92 (CXCR4_MACFA_TRD), or o) Preferably, at least one of Y3 and Y27 is sulfated, such as SEQ ID NO: 98 (CXCR5_MACFA_TRD), or p) Preferably, at least two or all of Y4, Y7 and Y39 are sulfated, such as in SEQ ID NO: 104 (CXCR6_MACFA_TRD), or q) Preferably, at least Y20 is sulfated, such as SEQ ID NO: 158 (CX3CR1_MACFA_TRD), or r) Preferably, SEQ ID NO: 164 (CXCR1_MACMU_TRD) in which at least Y14 is sulfated.

[0206] In some fourth embodiments of the first embodiment, which may or may not be the same as the first, second and / or third embodiments of the first embodiment, the isolated polypeptide comprises the N-terminus of a seven-transmembrane receptor including a TRD domain and a LID domain, preferably with at least cysteine ​​removed or replaced with a different amino acid between the TRD domain and the LID domain.

[0207] The extracellular domain of the chemokine receptor can be composed of four regions: (i) An N-terminal domain, (a) Membrane distal tyrosine-rich domain (TRD), (b) cysteine, and (c) N-terminal domain which can be subdivided into LID domains, (iii) Extracellular domain 1 (ECL1), (iii) Extracellular domain 2 (ECL2), and (iv) Extracellular domain 3 (ECL3).

[0208] Some polypeptides according to the present invention are difficult to handle, for example, due to their high tendency to aggregate. While not bound by theory, the inventors believe that the high "stickiness" is due to an increase in the number of charged amino acids and charged sulfate residues. In the case of polypeptides containing the N-terminus of a chemokine receptor, the aggregation properties can be improved by removing cysteine ​​or exchanging amino acids between the TRD domain and the LID domain. Optimal results were obtained by changing cysteine ​​to serine (Example 5, Table 4.1).

[0209] In some of the fourth embodiments of the first embodiment, cysteine ​​may be omitted, and TRD and LID are directly linked. In some different fourth embodiments of the first embodiment, cysteine ​​may be replaced with a different polar uncharged amino acid. In some preferred embodiments of the fourth embodiment of the first embodiment, cysteine ​​is replaced with serine (see Table 4.1). In some of the fourth embodiments of the first embodiment, cysteine ​​may be replaced with at least one different amino acid.

[0210] According to some Embodiment A of the fourth embodiment of the first aspect, the isolated sulfated polypeptide comprises the sequence described below, or comprises a sequence having at least 90%, 95%, or 98% sequence identity: a) At least Y10 and / or Y18 are sulfated, SEQ ID NO: 4 (CCR1_HUMAN_N term), b) At least Y26 is sulfated, Sequence ID No. 10 (CCR2_HUMAN_N term), c) Y16 and / or Y17 are sulfated, SEQ ID NO: 16 (CCR3_HUMAN_N term), d) At least Y22 is sulfated, preferably Y16, Y19 and / or Y20 are further sulfated, SEQ ID NO: 22 (CCR4_HUMAN_N term), e) Two, three, or all of Y3, Y10, Y14, and Y15 are sulfated, Sequence ID No. 28 (CCR5_HUMAN_N term), f) At least two or three of Y18, Y26, and Y27 are sulfated, SEQ ID NO: 34 (CCR6_HUMAN_N term), g) SEQ ID NO: 40 (CCR7_HUMAN_N term), in which one or both of Y8 and Y17 are sulfated. h) At least two or all of Y3, Y15, and Y17 are sulfated, SEQ ID NO: 46 (CCR8_HUMAN_N term), i) At least Y28, preferably Y17 and / or Y37 are also sulfated, SEQ ID NO: 64 (CCR9_HUMAN_N term), j) Y14 and Y22, or at least one or both, are sulfated, SEQ ID NO: 70 (CCR10_HUMAN_N term), k) Y27 is sulfated, Sequence ID 76 (CXCR1_HUMAN_N term), l) Y23 and / or Y25 are sulfated, Sequence ID No. 82 (CXCR2_HUMAN_N term), m) At least one or both of Y27 and Y29 are sulfated, Sequence ID No. 88 (CXCR3_HUMAN_N term), n) At least Y12 and / or Y21 are sulfated, Sequence ID No. 94 (CXCR4_HUMAN_N term), o) SEQ ID NO: 100 (CXCR5_HUMAN_N term), in which at least one of Y3 and Y27 is sulfated, or p) At least one or both of Y6 and Y10 are sulfated, SEQ ID NO: 106 (CXCR6_HUMAN_N term), or q) Preferably, at least Y14 is sulfated, such as SEQ ID NO: 160 (CX3CR1_HUMAN_N term), or r) Preferably, at least Y27 is sulfated, such as SEQ ID NO: 166 (CXCR1_HUMAN_N term).

[0211] According to some embodiments B of the fourth embodiment of the first aspect, the isolated sulfated polypeptide comprises the sequence described below, or comprises a sequence having at least 90%, 95%, or 98% sequence identity: a) Preferably, at least Y10 and / or Y18 are sulfated, SEQ ID NO: 6 (CCR1_MOUSE_N term), or b) Preferably, at least Y37 and / or Y39 are sulfated, such as in SEQ ID NO: 12 (CCR2_MOUSE_N term), or c) Preferably Y20 and / or Y22 are sulfated, such as in Sequence ID No. 18 (CCR3_MOUSE_N term), or d) Preferably at least Y22 is sulfated, and more preferably Y16, Y19 and / or Y20 is sulfated, as in SEQ ID NO: 24 (CCR4_MOUSE_N term), or e) Preferably two or three of Y10, Y12, and Y16 are sulfated, such as SEQ ID NO: 30 (CCR5_MOUSE_N term), or f) Preferably, at least two or three of Y13, Y18 and Y19 are sulfated, SEQ ID NO: 36 (CCR6_MOUSE_N term), g) Preferably, one or both of Y8 and Y17 are sulfated, and Y20 may also be sulfated, such as SEQ ID NO: 42 (CCR7_MOUSE_N term), or h) Preferably, at least two or all of Y3, Y14 and Y15 are sulfated, SEQ ID NO: 48 (C=X or S, CCR8_MOUSE_N term), or i) Preferably, at least Y28 is sulfated, and preferably Y19 is also sulfated, such as SEQ ID NO: 66 (CCR9_MOUSE_N term), or j) Preferably, at least one, two or all of Y14, Y17 and Y22 are sulfated, such as SEQ ID NO: 72 (CCR10_MOUSE_N term), or k) Preferably, at least Y6 is sulfated, such as in SEQ ID NO: 78 (CXCR1_MOUSE_N term), or l) Preferably, Y24 is sulfated, such as in SEQ ID NO: 84 (CXCR2_MOUSE_N term), or m) Preferably, at least one or both of Y27 and Y29 are sulfated, such as in SEQ ID NO: 90 (CXCR3_MOUSE_N term), or n) Preferably, at least Y23 and / or Y14 are sulfated, such as in SEQ ID NO: 96 (CXCR4_MOUSE_N term), or o) Preferably, at least Y3 and / or Y14 and / or Y20 and / or Y26 are sulfated, such as SEQ ID NO: 102 (CXCR5_MOUSE_N term), or p) Preferably, at least one or both of Y11 and Y15 are sulfated, such as SEQ ID NO: 108 (CXCR6_MOUSE_N term), or q) Preferably, at least Y15 is sulfated, such as SEQ ID NO: 162 (CX3CR1_MOUSE_N term), or r) Preferably, at least Y6 is sulfated, such as SEQ ID NO: 168 (CXCR1_MOUSE_N term).

[0212] According to some embodiments C of the fourth embodiment of the first aspect, the isolated sulfated polypeptide comprises the sequence described below, or comprises a sequence having at least 90%, 95%, or 98% sequence identity: a) Preferably, at least Y10 and / or Y18 are sulfated, SEQ ID NO: 5 (CCR1_MACFA_N term), or b) Preferably, at least Y26 is sulfated, such as in SEQ ID NO: 11 (CCR2_MACMU_N term), or c) Preferably, Y16 is sulfated, such as SEQ ID NO: 17 (CCR3_MACFA_N term), or d) Preferably at least Y22 is sulfated, and more preferably Y16, Y19 and / or Y20 is sulfated, such as SEQ ID NO: 23 (CCR4_MACFA_N term), or e) Preferably two, three or all of Y3, Y10, Y14 and Y15 are sulfated, such as SEQ ID NO: 29 (CCR5_MACMU_N term), or f) Preferably, at least two or three of Y23, Y31 and Y32 are sulfated, such as SEQ ID NO: 35 (CCR6_MACFA_N term), or g) Preferably, one or both of Y8 and Y17 are sulfated, such as SEQ ID NO: 41 (CCR7_MACFA_N term), or h) Preferably, at least two or all of Y3, Y15 and Y17 are sulfated, such as SEQ ID NO: 47 (CCR8_MACFA_N term), or i) Preferably at least Y28, preferably Y17 and / or Y37 are also sulfated, SEQ ID NO: 65 (CCR9_MACFA_N term), or j) Preferably, at least one or both of Y14 and Y22 are sulfated, such as SEQ ID NO: 71 (CCR10_MACFA_N term), or k) Preferably, at least one of Y14 and Y28 is sulfated, such as in SEQ ID NO: 77 (CXCR1_MACFA_N term), or l) Preferably Y20 and / or Y22 are sulfated, such as in Sequence ID No. 83 (CXCR2_MACFA_N term), or m) Preferably, at least one or both of Y27 and Y29 are sulfated, such as in SEQ ID NO: 89 (CXCR3_MACFA_N term), or n) Preferably, at least Y12 and / or Y21 are sulfated, such as in SEQ ID NO: 95 (CXCR4_MACFA_N term), or o) Preferably, at least one of Y3 and Y27 is sulfated, such as SEQ ID NO: 101 (CXCR5_MACFA_N term), or p) Preferably, at least two or all of Y4, Y7 and Y39 are sulfated, such as SEQ ID NO: 107 (CXCR6_MACFA_N term), or q) Preferably, at least Y20 or Y22 is sulfated, such as SEQ ID NO: 161 (CX3CR1_MACFA_N term), or r) Preferably, at least Y14 or Y28 is sulfated, such as SEQ ID NO: 167 (CXCR1_MACMU_N term).

[0213] In some of the fourth embodiments of the first aspect, the isolated sulfated polypeptide comprises the sequence described below: a) Preferably, at least Y10 and / or Y18 are sulfated, such as SEQ ID NO: 4 (CCR1_HUMAN_N term), SEQ ID NO: 5 (CCR1_MACFA_N term), or SEQ ID NO: 6 (CCR1_MOUSE_N term), b) Preferably, at least Y26 is sulfated, such as SEQ ID NO: 10 (CCR2_HUMAN_N term) or SEQ ID NO: 11 (CCR2_MACMU_N term), or c) Preferably, at least Y37 and / or Y39 are sulfated, such as in SEQ ID NO: 12 (CCR2_MOUSE_N term), or d) Preferably Y16 and / or Y17 are sulfated, such as SEQ ID NO: 16 (CCR3_HUMAN_N-terminus), or e) Preferably Y16 is sulfated, SEQ ID NO: 17 (CCR3_MACFA_N term), or f) Preferably Y20 and / or Y22 are sulfated, such as in SEQ ID NO: 18 (CCR3_MOUSE_N term), or g) Preferably, at least Y22 is sulfated, and preferably Y16, Y19 and / or Y20 is further sulfated, such as SEQ ID NO: 22 (CCR4_HUMAN_N term), SEQ ID NO: 23 (CCR4_MACFA_N term), or SEQ ID NO: 24 (CCR4_MOUSE_N term), or h) Preferably, two, three, or all of Y3, Y10, Y14, and Y15 are sulfated, such as Sequence ID No. 28 (CCR5_HUMAN_N term) or Sequence ID No. 29 (CCR5_MACMU_N term), i) Preferably, two or three of Y10, Y12, and Y16 are sulfated, such as SEQ ID NO: 30 (CCR5_MOUSE_N term), or j) Preferably, at least two or three of Y18, Y26, and Y27 are sulfated, such as SEQ ID NO: 34 (CCR6_HUMAN_N term), or k) Preferably, at least two or three of Y23, Y31, and Y32 are sulfated, such as SEQ ID NO: 35 (CCR6_MACFA_N term), or l) Preferably, at least two or three of Y13, Y18 and Y19 are sulfated, such as SEQ ID NO: 36 (CCR6_MOUSE_N term), or m) Preferably, one or both of Y8 and Y17 are sulfated, such as SEQ ID NO: 40 (CCR7_HUMAN_N term) or SEQ ID NO: 41 (CCR7_MACFA_N term), or n) Preferably, one or both of Y8 and Y17 are sulfated, and Y20 may also be sulfated, as in Sequence ID No. 42 (CCR7_MOUSE_N term), or o) Preferably, at least two or all of Y3, Y15, and Y17 are sulfated, such as SEQ ID NO: 46 (C=X or S, CCR8_HUMAN_N term) or SEQ ID NO: 47 (C=X or S, CCR8_MACFA_N term), or p) Preferably, at least two or all of Y3, Y14, and Y15 are sulfated, such as SEQ ID NO: 48 (C=X or S, CCR8_MOUSE_N term), or q) Preferably at least Y28, preferably Y17 and / or Y37 are also sulfated, such as SEQ ID NO: 64 (CCR9_HUMAN_N term) or SEQ ID NO: 65 (CCR9_MACFA_N term), or r) Preferably at least Y28 is sulfated, and preferably Y19 is also sulfated, such as SEQ ID NO: 66 (CCR9_MOUSE_N term), or s) Preferably, at least one or both of Y14 and Y22 are sulfated, such as SEQ ID NO: 70 (CCR10_HUMAN_N term) or SEQ ID NO: 71 (CCR10_MACFA_N term), t) Preferably, at least one, two or all of Y14, Y17 and Y22 are sulfated, such as SEQ ID NO: 72 (CCR10_MOUSE_N term), or u) Preferably, Y27 is sulfated, such as in SEQ ID NO: 76 (CXCR1_HUMAN_N term), or v) Preferably, at least one of Y14 and Y28 is sulfated, such as SEQ ID NO: 77 (CXCR1_MACFA_N term), or w) Preferably, at least Y6 is sulfated, SEQ ID NO: 78 (CXCR1_MOUSE_N term), or x) Preferably Y23 and / or Y25 are sulfated, such as in SEQ ID NO: 82 (CXCR2_HUMAN_N term), or y) Preferably Y20 and / or Y22 are sulfated, such as in SEQ ID NO: 83 (CXCR2_MACFA_N term), or z) Preferably Y24 is sulfated, such as sequence number 84 (CXCR2_MOUSE_N term), or aa) Preferably, at least one or both of Y27 and Y29 are sulfated, such as sequence number 88 (CXCR3_HUMAN_N term), sequence number 89 (CXCR3_MACFA_N term), or sequence number 90 (CXCR3_MOUSE_N term), bb) Preferably, at least Y12 and / or Y21 are sulfated, such as SEQ ID NO: 94 (CXCR4_HUMAN_N term) or SEQ ID NO: 95 (CXCR4_MACFA_N term), or cc) Preferably, at least Y23 and / or Y14 are sulfated, such as in SEQ ID NO: 96 (CXCR4_MOUSE_N term), or dd) Preferably, at least one of Y3 and Y27 is sulfated, such as SEQ ID NO: 100 (CXCR5_HUMAN_N term) or SEQ ID NO: 101 (CXCR5_MACFA_N term), or ee) Preferably, at least Y3 and / or Y14 and / or Y20 and / or Y26 are sulfated, SEQ ID NO: 102 (CXCR5_MOUSE_N term), or ff) Preferably, at least one or both of Y6 and Y10 are sulfated, SEQ ID NO: 106 (CXCR6_HUMAN_N term), or gg) Preferably, at least two or all of Y4, Y7 and Y39 are sulfated, such as SEQ ID NO: 107 (CXCR6_MACFA_N term), or hh) Preferably, at least one or both of Y11 and Y15 are sulfated, such as SEQ ID NO: 108 (CXCR6_MOUSE_N term), or ii) Preferably, at least Y14 is sulfated, such as SEQ ID NO: 160 (CX3CR1_HUMAN_N term), or jj) Preferably, at least Y20 or Y22 is sulfated, SEQ ID NO: 161 (CX3CR1_MACFA_N term), or kk) Preferably, at least Y15 is sulfated, SEQ ID NO: 162 (CX3CR1_MOUSE_N term), or ll) Preferably, at least Y27 is sulfated, such as SEQ ID NO: 166 (CXCR1_HUMAN_N term) or (mm) Preferably, at least Y14 or Y28 is sulfated, SEQ ID NO: 167 (CXCR1_MACMU_N term), or nn) Preferably, at least Y6 is sulfated, such as SEQ ID NO: 168 (CXCR1_MOUSE_N term).

[0214] In some of the first, second, third, or fourth embodiments of the first aspect, the isolated sulfated polypeptide comprises the sequence described below or a sequence having at least 90% sequence identity with the following: a) Preferably, at least Y10 and / or Y18 is sulfated, such as SEQ ID NO: 1 (CCR1_HUMAN_TRD), SEQ ID NO: 4 (CCR1_HUMAN_N term), SEQ ID NO: 2 (CCR1_MACFA_TRD), SEQ ID NO: 5 (CCR1_MACFA_N term), SEQ ID NO: 3 (CCR1_MOUSE_TRD) or SEQ ID NO: 6 (CCR1_MOUSE_N term), or b) Preferably, at least Y26 is sulfated, such as SEQ ID NO: 7 (CCR2_HUMAN_TRD), SEQ ID NO: 10 (CCR2_HUMAN_N term), SEQ ID NO: 8 (CCR2_MACMU_TRD), or SEQ ID NO: 11 (CCR2_MACMU_N term), or c) Preferably, at least Y37 and / or Y39 are sulfated, such as Sequence ID No. 9 (CCR2_MOUSE_TRD) or Sequence ID No. 12 (CCR2_MOUSE_N term), or d) Preferably Y16 and / or Y17 are sulfated, such as SEQ ID NO: 13 (CCR3_HUMAN_TRD) or SEQ ID NO: 16 (CCR3_HUMAN_N term), or e) Preferably, Y16 is sulfated, such as sequence number 14 (CCR3_MACFA_TRD) or sequence number 17 (CCR3_MACFA_N term), or f) Preferably Y20 and / or Y22 are sulfated, such as Sequence ID No. 15 (CCR3_MOUSE_TRD) or Sequence ID No. 18 (CCR3_MOUSE_N term), or g) Preferably, at least Y22 is sulfated, and preferably Y16, Y19 and / or Y20 is further sulfated, such as SEQ ID NO: 19 (CCR4_HUMAN_TRD), SEQ ID NO: 22 (CCR4_HUMAN_N term), SEQ ID NO: 20 (CCR4_MACFA_TRD), SEQ ID NO: 23 (CCR4_MACFA_N term), SEQ ID NO: 21 (CCR4_MOUSE_TRD) or SEQ ID NO: 24 (CCR4_MOUSE_N term), or h) Preferably, two, three or all of Y3, Y10, Y14 and Y15 are sulfated, such as SEQ ID NO: 25 (CCR5_HUMAN_TRD), SEQ ID NO: 28 (CCR5_HUMAN_N term), SEQ ID NO: 26 (CCR5_MACMU_TRD), or SEQ ID NO: 29 (CCR5_MACMU_N term), or i) Preferably, two or three of Y10, Y12, and Y16 are sulfated, such as Sequence ID No. 27 (CCR5_MOUSE_TRD) or Sequence ID No. 30 (CCR5_MOUSE_N term), or j) Preferably, at least two or three of Y18, Y26, and Y27 are sulfated, such as SEQ ID NO: 31 (CCR6_HUMAN_TRD) or SEQ ID NO: 34 (CCR6_HUMAN_N term), k) Preferably, at least two or three of Y23, Y31, and Y32 are sulfated, such as Sequence ID No. 32 (CCR6_MACFA_TRD) or Sequence ID No. 35 (CCR6_MACFA_N term), or l) Preferably, at least two or three of Y13, Y18 and Y19 are sulfated, such as Sequence ID No. 33 (CCR6_MOUSE_TRD) or Sequence ID No. 36 (CCR6_MOUSE_N term), m) Preferably, one or both of Y8 and Y17 are sulfated, such as SEQ ID NO: 37 (CCR7_HUMAN_TRD), SEQ ID NO: 40 (CCR7_HUMAN_N term), SEQ ID NO: 38 (CCR7_MACFA_TRD), or SEQ ID NO: 41 (CCR7_MACFA_N term), or n) Preferably, one or both of Y8 and Y17 are sulfated, and Y20 may also be sulfated, such as Sequence ID No. 39 (CCR7_MOUSE_TRD) or Sequence ID No. 42 (CCR7_MOUSE_N term), o) Preferably, at least two or all of Y3, Y15 and Y17 are sulfated, such as SEQ ID NO: 43 (CCR8_HUMAN_TRD), SEQ ID NO: 44 (CCR8_MACFA_TRD), SEQ ID NO: 46 (C=X or S, CCR8_HUMAN_N term), or SEQ ID NO: 47 (C=X or S, CCR8_MACFA_N term), p) Preferably, at least two or all of Y3, Y14 and Y15 are sulfated, such as Sequence ID No. 45 (CCR8_MOUSE_TRD) or Sequence ID No. 48 (C=X or S, CCR8_MOUSE_N term), or q) Preferably at least Y28, preferably Y17 and / or Y37 are also sulfated, such as SEQ ID NO: 61 (CCR9_HUMAN_TRD), SEQ ID NO: 64 (CCR9_HUMAN_N term), SEQ ID NO: 62 (CCR9_MACFA_TRD) or SEQ ID NO: 65 (CCR9_MACFA_N term), or r) Preferably, at least Y28 is sulfated, and preferably Y19 is also sulfated, such as Sequence ID No. 63 (CCR9_MOUSE_TRD) or Sequence ID No. 66 (CCR9_MOUSE_N term), or s) Preferably, at least one or both of Y14 and Y22 are sulfated, such as SEQ ID NO: 67 (CCR10_HUMAN_TRD), SEQ ID NO: 70 (CCR10_HUMAN_N term), SEQ ID NO: 68 (CCR10_MACFA_TRD), or SEQ ID NO: 71 (CCR10_MACFA_N term), or t) Preferably, at least one, two or all of Y14, Y17 and Y22 are sulfated, such as Sequence ID No. 69 (CCR10_MOUSE_TRD) or Sequence ID No. 72 (CCR10_MOUSE_N term), u) Preferably Y27 is sulfated, such as sequence number 73 (CXCR1_HUMAN_TRD) or sequence number 76 (CXCR1_HUMAN_N term), or v) Preferably, at least one of Y14 and Y28 is sulfated, such as sequence number 74 (CXCR1_MACFA_TRD) or sequence number 77 (CXCR1_MACFA_N term), or w) Preferably, at least Y6 is sulfated, such as sequence number 75 (CXCR1_MOUSE_TRD) or sequence number 78 (CXCR1_MOUSE_N term), or x) Preferably Y23 and / or Y25 are sulfated, such as sequence number 79 (CXCR2_HUMAN_TRD) or sequence number 82 (CXCR2_HUMAN_N term), or y) Preferably Y20 and / or Y22 are sulfated, such as sequence number 80 (CXCR2_MACFA_TRD) or sequence number 83 (CXCR2_MACFA_N term), or z) Preferably, Y24 is sulfated, such as sequence number 81 (CXCR2_MOUSE_TRD) or sequence number 84 (CXCR2_MOUSE_N term), or aa) Preferably, at least one or both of Y27 and Y29 are sulfated, such as SEQ ID NO: 85 (CXCR3_HUMAN_TRD), SEQ ID NO: 88 (CXCR3_HUMAN_N term), SEQ ID NO: 86 (CXCR3_MACFA_TRD), SEQ ID NO: 89 (CXCR3_MACFA_N term), SEQ ID NO: 87 (CXCR3_MOUSE_TRD), or SEQ ID NO: 90 (CXCR3_MOUSE_N term), or bb) Preferably, at least Y12 and / or Y21 are sulfated, such as SEQ ID NO: 91 (CXCR4_HUMAN_TRD), SEQ ID NO: 94 (CXCR4_HUMAN_N term), SEQ ID NO: 92 (CXCR4_MACFA_TRD), or SEQ ID NO: 95 (CXCR4_MACFA_N term), or cc) Preferably, at least Y23 and / or Y14 are sulfated, such as SEQ ID NO: 93 (CXCR4_MOUSE_TRD) or SEQ ID NO: 96 (CXCR4_MOUSE_N term), or dd) Preferably, at least one of Y3 and Y27 is sulfated, such as SEQ ID NO: 97 (CXCR5_HUMAN_TRD), SEQ ID NO: 100 (CXCR5_HUMAN_N term), SEQ ID NO: 98 (CXCR5_MACFA_TRD), or SEQ ID NO: 101 (CXCR5_MACFA_N term), or ee) Preferably, at least Y3 and / or Y14 and / or Y20 and / or Y26 are sulfated, such as Sequence ID No. 99 (CXCR5_MOUSE_TRD) or Sequence ID No. 102 (CXCR5_MOUSE_N term), or ff) Preferably, at least one or both of Y6 and Y10 are sulfated, such as SEQ ID NO: 103 (CXCR6_HUMAN_TRD) or SEQ ID NO: 106 (CXCR6_HUMAN_N term), or gg) Preferably, at least two or all of Y4, Y7 and Y39 are sulfated, such as SEQ ID NO: 104 (CXCR6_MACFA_TRD) or SEQ ID NO: 107 (CXCR6_MACFA_N term), or hh) Preferably, at least one or both of Y11 and Y15 are sulfated, such as SEQ ID NO: 105 (CXCR6_MOUSE_TRD) or SEQ ID NO: 108 (CXCR6_MOUSE_N term), or ii) Preferably, at least Y14 is sulfated, such as sequence number 157 (CX3CR1_HUMAN_TRD) or sequence number 160 (CX3CR1_HUMAN_N term), or jj) Preferably, at least Y20 is sulfated, SEQ ID NO: 158 (CX3CR1_MACFA_TRD), or kk) Preferably, at least Y20 or Y22 is sulfated, SEQ ID NO: 161 (CX3CR1_MACFA_N term), or ll) Preferably, at least Y15 is sulfated, such as SEQ ID NO: 159 (CX3CR1_MOUSE_TRD) or SEQ ID NO: 162 (CX3CR1_MOUSE_N term), or (mm) Preferably, at least Y27 is sulfated, such as sequence number 163 (CXCR1_HUMAN_TRD) or sequence number 166 (CXCR1_HUMAN_N term), or nn) Preferably, SEQ ID NO: 164 (CXCR1_MACMU_TRD) in which at least Y14 is sulfated, or oo) Preferably, at least Y14 or Y28 is sulfated, SEQ ID NO: 167 (CXCR1_MACMU_N term), or pp) Preferably, at least Y6 is sulfated, such as SEQ ID NO: 165 (CXCR1_MOUSE_TRD) or SEQ ID NO: 168 (CXCR1_MOUSE_N term).

[0215] Preferably, the isolated polypeptide according to this embodiment is immobilized, for example, via a linker. Immobilization may occur with appropriate beads, particles, proteins, or solid supports, but is not limited to these.

[0216] Embodiment 2 - Conjugate containing isolated polypeptide According to a second aspect of the present invention, a conjugate comprising an isolated sulfated polypeptide according to the first aspect is provided.

[0217] For example, the conjugate may include a polypeptide according to the first embodiment of the first embodiment. For example, the conjugate may include a polypeptide according to the second embodiment of the first embodiment. For example, the conjugate may include a polypeptide according to the third embodiment of the first embodiment. For example, the conjugate may include a polypeptide according to several embodiments A, B, and C of the third embodiment of the first embodiment. For example, the conjugate may include a polypeptide according to the fourth embodiment of the first embodiment. For example, the conjugate may include a polypeptide according to several embodiments A, B, and C of the fourth embodiment of the first embodiment.

[0218] For example, isolated sulfated polypeptides can be bound to a tag or linker for immobilization or recovery. Suitable tags are selected from tags known in the art, such as small organic molecules like biotin (which binds strongly and non-covalently to streptavidin), their derivatives, or short peptide sequences such as Flag tags, HA tags, Myc tags, or His tags (see Table 4.1 or Example 10.1.2). In some applications, the tag may be a protein such as human serum albumin (see Table 4.1 or Example 10.1.2), or a carrier protein such as KLH, OVA, or BSA, or a larger structure such as beads or magnetic particles. Preferably, the tag can be bound via the C-terminus or N-terminus of the TRD or the N-terminus of the chemokine receptor, but it can also be bound via reactive residues such as lysine or amino acid side chains within the TRD. In some embodiments, the tag is bound via a linker which may be any linker known in the art. Suitable linkers include trioxatridecane succinate (Ttds) linker (see Table 4.1), β-alanine, GABA, AEA, Ava, Ahx, PEG2 spacer, PEG3 spacer, PEG4 spacer, O1Pen, O2Oc, or O1Pen-O1.

[0219] Apparatus 3 - Antigen Production Method Sulfated peptides can be difficult to synthesize, for example, because sulfates are unstable under acidic conditions (Houben-Weyl, Methods of Organic Chemistry Vol.E 22b, Synthesis of Peptides and Peptidomimetics, 4th Edition, section 6.6.1.2 Synthesis of Sulfated Tyrosine Peptides with Tyrosine O-Sulfate Synthons, p.440 ff.in: Felix, Arthur et al.: 2004).

[0220] A third aspect provides a method for producing an isolated sulfated polypeptide according to the first aspect or a conjugate according to the second aspect, the method comprising the synthesis of the isolated polypeptide and the sulfated tyrosine residues thereof.

[0221] The synthesis of the isolated polypeptide and the sulfation of each tyrosine residue according to the first embodiment may be carried out as described in Example 5, or according to any other method known in the art. In Chapter 6.6.1, "Methods of Organic Chemistry Vol. E 22b, Synthesis of Peptides and Peptidomimetics," Houben-Weyl describes various chemical approaches for the synthesis of sulfated tyrosine peptides. This chapter, and in particular these methods, are incorporated herein by reference in their entirety.

[0222] For example, the synthesis may be carried out using a sequence-independent solid-phase method previously described by Bunschoten et al. (Bunschoten, Anton, et al. “A general sequence independent solid phase method for the site specific synthesis of multiple sulfated-tyrosine containing peptides.” Chemical communications 21(2009):2999-3001.), which is incorporated entirely herein. Briefly, the peptide is synthesized according to the Fmoc-tBu strategy, followed by selective deprotection of the tyrosine residue to be sulfated and introduction of the protected sulfate group. Once the synthesis of the sulfated peptide is complete, it is cleaved from the resin by acid hydrolysis, removing the protecting group except for the sulfate protecting group, thereby preventing undesirable acid-induced removal of (one or more) sulfate groups during this step. Finally, the sulfate protecting group can be removed in a slightly acidic reduction step, leaving the sulfate group untouched.

[0223] Chen et al. reported a short, efficient one-step pathway to sY-containing peptides in which Fmoc-protected fluorosulfated tyrosine (Y(OSO2F)) is incorporated into the peptide of interest by an Fmoc-based solid-phase synthesis strategy (the entire pathway is incorporated herein; see Chen, Wentao, et al. “Synthesis of Sulfotyrosine-Containing Peptides by Incorporating Fluorosulfated Tyrosine Using an Fmoc-Based Solid-Phase Strategy.” Angewandte Chemie 128.5(2016):1867-1870.). Standard simultaneous peptide-resin cleavage and removal of acid-unstable side-chain protecting groups yield a crude peptide containing fluorosulfated tyrosine. Basic ethylene glycol, acting as both a solvent and reactant, converts the fluorosulfated tyrosine peptide to a sulfotyrosine peptide in high yield.

[0224] The entire polypeptide can also be sulfated, for example, using sulfur trioxide-pyridine. According to the present invention, this route can be used when all tyrosines must be sulfated, or when a "crude" mixture of partially sulfated polypeptides is used in a further step. Furthermore, sulfated tyrosines can be carried out enzymatically, for example, in an in vivo conversion reaction using a natural sulfase or an engineered version thereof. Preferably, the sulfated tyrosines can be carried out chemically or enzymatically. Preferably, the synthesis of isolated polypeptides is carried out using the Fmoc-tBu strategy.

[0225] According to some embodiments of the third aspect, the method includes a step of purifying the obtained polypeptide. Purification may be carried out, for example, by HPLC, using, for example, a C18 column. According to some embodiments of the third aspect, the method includes analytical characterization of the polypeptide. Analytical characterization may be carried out using spectroscopy or mass spectrometry, but is not limited to these methods.

[0226] Apparatus 4 - Use of Antigen / Method Compound Use of Antigen According to a fourth aspect, the use of the isolated sulfated polypeptide according to the first aspect or the conjugate according to the second aspect for antibody production, as an antigen, for off-target panning, and / or for antibody characterization is provided. For example, the isolated sulfated polypeptide according to the first aspect or the conjugate according to the second aspect can be used to produce a fully human antibody or a fragment thereof. For example, the isolated sulfated polypeptide according to the first aspect or the conjugate according to the second aspect can be used to produce a cross-reactive antibody.

[0227] According to several first embodiments of the fourth aspect, the use of isolated sulfated polypeptides according to the first aspect or conjugates according to the second aspect for antibody production is provided. In particular, isolated sulfated polypeptides according to the first aspect may be used to facilitate the production of antibodies that specifically recognize chemokine receptors, as described elsewhere in this specification.

[0228] According to some second embodiments of the fourth embodiment, which may be the same as or different from the first embodiment of the fourth embodiment, the isolated sulfated polypeptide according to the first embodiment or the conjugate according to the second embodiment is used, for example, as an antigen for selecting an antibody, antibody fragment, or molecule that specifically binds to a chemokine receptor (see Examples 6 and 8).

[0229] According to some third embodiments of the fourth embodiment, which may be the same as or different from the first and / or second embodiments of the fourth embodiment, the isolated sulfated polypeptide according to the first embodiment or the conjugate according to the second embodiment is used to select an antibody that does not bind to a specific seven-transmembrane receptor, such as a chemokine receptor (off-target receptor), for off-target panning (see Examples 6 and 8).

[0230] According to some fourth embodiments, the method according to the fourth embodiment includes the use of an isolated sulfated polypeptide according to the first embodiment or a conjugate according to the second embodiment for antibody characterization. Preferably, the antibody is an antibody according to the present invention. For example, antibody characterization may include the use of ELISA, surface plasmon resonance, mass spectrometry, competitive assay, staining, IHC, FACS, or various further assays known in the art.

[0231] Apparatus 5 - Antibody Production Method According to a fifth aspect, a method for obtaining an antibody or a binder is provided, the method comprising the use of an isolated sulfated polypeptide according to the first aspect or a conjugate according to the second aspect.

[0232] According to some of the first embodiments, the method according to the fifth embodiment includes the use of an isolated sulfated polypeptide according to the first embodiment or a conjugate according to the second embodiment as an antigen.

[0233] According to some preferred embodiments of the first embodiment of the fifth aspect, the method comprises the use of at least one further isolated polypeptide or its conjugate, the at least one further isolated polypeptide is a) A seven-transmembrane receptor different from the first seven-transmembrane receptor, or b) comprising a first seven-transmembrane receptor TRD derived from a different species, Preferably, at least one further isolated polypeptide is an isolated polypeptide according to the first embodiment.

[0234] According to some embodiments A of the first embodiment of the fifth aspect, the method comprises using at least one further isolated polypeptide or conjugate, preferably according to the first or second aspect, preferably as an antigen or for off-target selection. In these embodiments, the first isolated sulfated polypeptide comprises a TRD of a first seven-transmembrane receptor (e.g., a chemokine receptor), and the further isolated polypeptide or conjugate comprises a TRD of a seven-transmembrane receptor different from the first seven-transmembrane receptor. When the further isolated polypeptide or conjugate is used as an antigen, the method is a method for producing antibodies or conjugates that recognize at least two different seven-transmembrane receptors, e.g., two different chemokine receptor family members.

[0235] If further isolated polypeptides or conjugates are used for off-target panning, the method is, for example, a method for producing antibodies or conjugates that recognize only specific seven-transmembrane receptors in order to avoid off-target binding to further chemokine receptor family members.

[0236] According to some embodiments B of the first embodiment of the fifth aspect, the method includes using at least one further isolated polypeptide or conjugate, preferably according to the first or second aspect, as an antigen or for off-target selection. In these embodiments, the first isolated polypeptide comprises a TRD of the first seven-transmembrane receptor of a first species (e.g., human chemokine receptor), and the further isolated polypeptide comprises a TRD of the same seven-transmembrane receptor from a different species (e.g., cynomolgus monkey chemokine receptor). For example, the first polypeptide may comprise a TRD of the human chemokine receptor, and the second polypeptide may comprise a TRD of the cynomolgus monkey chemokine receptor.

[0237] These embodiments B are particularly advantageous because the production of chemokine receptor antibodies or conjugates that are cross-reactive to both humans and suitable model species is difficult for chemokine receptors, especially CCR8. While the overall consensus among transmembrane domains is relatively high, the extracellular domains of chemokine receptors have low consensus among different chemokine receptor family members and even among species of a given chemokine receptor (Figures 1 and 2a). However, according to the present invention, it is found here that small sulfated tyrosine motifs within the TRD are well conserved for a given chemokine receptor to be used to obtain a large number of cross-reactive antibodies. Example 6 describes the production of cross-reactive antibodies against humans and cynomolgus monkeys, and Example 10.1.1 shows excellent affinity in both species for various antibodies according to the present invention. Cynomolgus monkeys are a preferred model system because mouse models could not predict immunological side effects compared to rodents and mice.

[0238] The method according to the fifth embodiment may be any method for antibody production known in the art. For example, the method may be a conventional immunization method, for example, conjugating the polypeptide according to the first embodiment to KLH and administering it to an animal suitable for immunization.

[0239] For example, after immunization, splenocytes from immunized animals can be used to generate hybridoma cells that produce antibodies from the animal, and in a second step, they can be screened for antibodies that specifically bind to the antigen, and, if applicable, for off-target antibodies, by methods known in the art, such as ELISA. Alternatively, splenocytes can be directly screened for the production of antibodies that bind to the antigen using a droplet-based microfluidic system or a cell culture assay. Subsequently, only selected splenocytes are directly subjected to sequencing to obtain sequences for antibody or hybridoma generation. Another method may include, for example, using an antigen for panning with an antibody library, which may be a phage display library, as described elsewhere in this specification (but not limited to), or alternatively, a mammalian library. After panning the library over the antigen, the enriched antibodies can be screened for specific binding to the antigen by methods known in the art, such as ELISA or SPR, as described in more detail herein.

[0240] According to some second embodiments of the fifth embodiment, which may be the same as or different from the first embodiment of the fifth embodiment, the method for obtaining an antibody according to the fifth embodiment is a method that includes the use of any other techniques available in the art for producing antibodies, including a phage display library, a transgenic animal, or a human CDR.

[0241] In some embodiments A of the second embodiment of the fifth aspect, the method includes the use of a human phage display library (see Examples 6 and 8). For example, the phage display library may be a fully human antibody phage display library such as the BioInvent n-CoDeR Fab lambda library. In some preferred embodiments, the phage display library is enriched with tyrosine and / or histidine content. In the first step, the phage display library, which includes a bacteriophage that displays an antibody or antibody fragment on its outside, can be combined with an immobilized isolated polypeptide according to the first aspect or a conjugate according to the second aspect to enable binding to the isolated polypeptide or conjugate.

[0242] In any second depletion step that may occur before or after the first step, the off-target conjugate can be depleted by combining a phage display library containing a bacteriophage displaying an antibody or antibody fragment on its outside with an immobilized isolated polypeptide or conjugate according to the first embodiment, which is different from the isolated polypeptide in the first step.

[0243] In an optional third step, which may be performed before or after the first step, or before or after the second step, a phage display library comprising a bacteriophage displaying an antibody or antibody fragment on its outside can be combined with an immobilized isolated polypeptide according to the first embodiment or a conjugate according to the second embodiment, which is different from the isolated polypeptide of the first step and the isolated polypeptide of any second step, to obtain a cross-reactive binder.

[0244] Each step may be repeated multiple times, for example, once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more. The bound antibody or fragment can be recovered for expansion by infection of a suitable bacterial host, and the DNA can be sequenced as known in the art to obtain the sequence of the seven-transmembrane receptor antibody or fragment.

[0245] In some embodiments B of the second embodiment of the fifth aspect, the method for obtaining antibodies according to the fifth aspect is a method that includes the use of transgenic animals, as described by Lonberg (Lonberg, Nils. "Human antibodies from transgenic animals." Nature biotechnology 23.9(2005):1117-1125.), which is incorporated herein in whole. For example, the transgenic animal may be XenoMouse (Abgenix Inc., Fremont, California, e.g., U.S. Patent No. 5,939,598), HuMAb Mouse (GenPharm-Medarex, San Jose, California), RenMab Mouse (Biocytogen), or any other animal known in the art for producing fully human antibodies.

[0246] In some embodiments C of the second embodiment of the fifth aspect, the method for obtaining antibodies according to the fifth aspect is a method that includes the use of in vitro activated B cells (see U.S. Patents No. 5,567,610 and No. 5,229,275, each of which is incorporated herein by reference in whole).

[0247] According to some third embodiments of the fifth aspect, a method is provided for obtaining an antibody or antibody fragment or binder that specifically binds to the CC or CXC chemokine receptor of human and / or cynomolgus monkey and / or mouse, the method being: a) Sulfating (synthetically) a polypeptide containing a tyrosine-rich domain (TRD), b) Selecting an antibody, antibody fragment, or binder that recognizes the sulfated polypeptide, c) The process may also include manufacturing the antibody, antibody fragment, or binder.

[0248] In some embodiments, the use according to the fourth aspect or the method according to the fifth aspect is a use / method for obtaining an antibody having desirable properties as described elsewhere in this specification, preferably the antibody is a) Contains human-derived CDRs and / or b) Human, rat, or mouse IgG antibody, preferably human IgG1 antibody or mouse IgG2a antibody, and / or c) Cross-reactive to two different 7-transmembrane receptors, and / or d) Cross-reactive to human and cynomolgus monkey seven-transmembrane receptors, and / or e) An HCDR3 comprising 10-34% tyrosine and / or 2-20% histidine, preferably 7-20% histidine, and / or f) Does not regulate G protein-independent signaling of chemokine receptors, and / or g) Characterized by internalization into cells that are either non-internalized antibodies or cells with endogenous target expression lower than 1.5, 2, 3, 4, 5, 6, 7, or 10 times that of the isotype control.

[0249] The method according to this embodiment can be used to obtain a large number of antibodies that bind to chemokine receptors. Interestingly, a relatively large number of these antibodies are characterized by properties that distinguish them from known antibodies obtained by conventional methods described elsewhere in this specification.

[0250] Embodiment 6 - Antibody defined by antigen According to the present invention, isolated antibodies, antigen-binding fragments thereof, or binders obtained by methods or uses according to the preceding embodiments are provided.

[0251] As those skilled in the art will understand, antibodies and / or conjugate fragments are essentially “modules.” Throughout this disclosure, various specific aspects and embodiments of the various “modules” constituting antibodies and / or conjugate fragments are described. Various specific embodiments or functional features of VH CDRs, VH chains, VL CDRs, and VL chains are described as specific, non-limiting examples. All specific embodiments are intended to be combined with one another as if each specific combination were explicitly described individually. Various specific functional embodiments are described as specific, non-limiting examples. All specific embodiments are intended to be combined with one another as if each specific combination were explicitly described individually.

[0252] According to a sixth aspect, an isolated antibody or antigen-binding fragment thereof is provided that (specifically) binds to a first isolated sulfated polypeptide containing a tyrosine-rich domain (TRD) of a seven-transmembrane receptor, wherein at least 25%, at least 50%, or at least 75% of the tyrosine residues of the TRD are sulfated. Preferably, the first isolated sulfated polypeptide further comprises the LID domain of the seven-transmembrane receptor. Preferably, the cysteine ​​between the TRD domain and the LID domain is removed or replaced with a different amino acid.

[0253] Preferably, the first isolated sulfated polypeptide comprises the N-terminus of a seven-transmembrane receptor, which may include its tyrosine-rich domain (TRD) and optionally its LID domain, and more preferably, at least 25%, at least 50%, or at least 75% of the tyrosine residues of the TRD are sulfated.

[0254] In a preferred embodiment, the isolated antibody or its antigen-binding fragment may bind to one or more targets with KD values ​​of <5E-8M, <4E-8M, <3E-8M, <2E-8M, <1E-8M, <9E-9M, <8E-9M, <7E-9M, <6E-9M, <5E-9M, <4E-9M, <3E-9M, <2.5E-9M, <2E-9M, <1.5E-9M, <1E-9M, <9E-10M, <8E-10M, <7E-10M, <6E-10M, <5E-10M, <4E-10M, <3E-10M, <2.5E-10M, <2E-10M, <1.5E-10M, <1E-10M, or <9E-11M. For example, the antibody of the present invention may bind to one or more targets with a KD value of <8E-9M to >4E-10M. The isolated antibody or its antigen-binding fragment binds to two or more targets, most preferably with similar affinity.

[0255] According to some preferred embodiments, the isolated antibody or its antigen-binding fragment is a) Contains human-derived CDRs and / or b) Cross-reactive to humans and cynomolgus monkeys, and / or c) Characterized by an HCDR3 region containing 10-34% tyrosine and / or 7-20% histidine, and / or d) Does not regulate G protein-independent signaling of 7-transmembrane receptors, and / or e) Characterized by non-internalized antibodies or internalization into cells having an endogenous target expression 1.5, 2, 3, 4, 5, 6, 7, or 10 times lower than the internalization of the isotype control, and / or f) Induce ADCC and / or ADCP, and / or g) Human, rat, or mouse IgG antibody, preferably human IgG1 antibody or mouse IgG2a antibody, and / or h)scFv, Fab, Fab', or F(ab')2 fragment.

[0256] According to some first embodiments of the sixth embodiment, the seven-transmembrane receptor is a chemokine receptor. In some of these first embodiments, the seven-transmembrane receptor is a CC chemokine receptor or a CXC chemokine receptor. In some of these first embodiments, the seven-transmembrane receptor is a CC chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, or CCR10. In some preferred embodiments of these first embodiments, the seven-transmembrane receptor is CCR8 or CCR4. In some of the most preferred embodiments of these first embodiments, the seven-transmembrane receptor is CCR8. In some of these first embodiments, the seven-transmembrane receptor is a CXC chemokine receptor such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, or CXCR6. In some of these first embodiments, the seven-transmembrane receptor is CX3CR1 or CXCR1.

[0257] According to some second embodiments of the sixth embodiment, which may or may not be the same as the first embodiment of the sixth embodiment, the seven-transmembrane receptor may be derived from any species expressing a chemokine receptor characterized by TRD, such as humans, monkeys, cynomolgus monkeys (macaca fascicularis), rhesus macaques (macaca mulatta), rodents, mice, rats, horses, cattle, pigs, dogs, cats, and camels.

[0258] In some of these second embodiments of the sixth embodiment, the seven-transmembrane receptor is mouse. In some of the most preferred embodiments of these second embodiments of the sixth embodiment, the seven-transmembrane receptor is human. In some of these second embodiments of the sixth embodiment, the seven-transmembrane receptor is cynomolgus monkey. In some preferred embodiments of these embodiments, the seven-transmembrane receptor is human, cynomolgus monkey, or mouse. In some of these preferred second embodiments of the sixth embodiment, the seven-transmembrane receptor is human or cynomolgus monkey.

[0259] In some preferred embodiments, the seven-transmembrane receptor is a human, cynomolgus monkey, or mouse seven-transmembrane receptor, and the seven-transmembrane receptor is a) CC chemokine receptors, preferably CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, or CCR10 b) CXC chemokine receptors, preferably CXCR1, CXCR2, CXCR3, CXCR4, CXCR5 or CXCR6, c) CX3CR1 or CXCR1.

[0260] According to some third embodiments of the sixth aspect, which may be the same as or different from the first and / or second embodiments according to the sixth aspect, the (first) isolated sulfated polypeptide comprises or consists of the sequence described as follows: a) Preferably, at least Y10 and / or Y18 is sulfated, such as SEQ ID NO: 1 (CCR1_HUMAN_TRD), SEQ ID NO: 4 (CCR1_HUMAN_N term), SEQ ID NO: 2 (CCR1_MACFA_TRD), SEQ ID NO: 5 (CCR1_MACFA_N term), SEQ ID NO: 3 (CCR1_MOUSE_TRD) or SEQ ID NO: 6 (CCR1_MOUSE_N term), or b) Preferably, at least Y26 is sulfated, such as SEQ ID NO: 7 (CCR2_HUMAN_TRD), SEQ ID NO: 10 (CCR2_HUMAN_N term), SEQ ID NO: 8 (CCR2_MACMU_TRD), or SEQ ID NO: 11 (CCR2_MACMU_N term), or c) Preferably, at least Y37 and / or Y39 are sulfated, such as Sequence ID No. 9 (CCR2_MOUSE_TRD) or Sequence ID No. 12 (CCR2_MOUSE_N term), or d) Preferably Y16 and / or Y17 are sulfated, such as SEQ ID NO: 13 (CCR3_HUMAN_TRD) or SEQ ID NO: 16 (CCR3_HUMAN_N term), or e) Preferably, Y16 is sulfated, such as sequence number 14 (CCR3_MACFA_TRD) or sequence number 17 (CCR3_MACFA_N term), or f) Preferably Y20 and / or Y22 are sulfated, such as Sequence ID No. 15 (CCR3_MOUSE_TRD) or Sequence ID No. 18 (CCR3_MOUSE_N term), or g) Preferably, at least Y22 is sulfated, and preferably Y16, Y19 and / or Y20 is further sulfated, such as SEQ ID NO: 19 (CCR4_HUMAN_TRD), SEQ ID NO: 22 (CCR4_HUMAN_N term), SEQ ID NO: 20 (CCR4_MACFA_TRD), SEQ ID NO: 23 (CCR4_MACFA_N term), SEQ ID NO: 21 (CCR4_MOUSE_TRD) or SEQ ID NO: 24 (CCR4_MOUSE_N term), or h) Preferably, two, three or all of Y3, Y10, Y14 and Y15 are sulfated, such as SEQ ID NO: 25 (CCR5_HUMAN_TRD), SEQ ID NO: 28 (CCR5_HUMAN_N term), SEQ ID NO: 26 (CCR5_MACMU_TRD), or SEQ ID NO: 29 (CCR5_MACMU_N term), or i) Preferably, two or three of Y10, Y12, and Y16 are sulfated, such as Sequence ID No. 27 (CCR5_MOUSE_TRD) or Sequence ID No. 30 (CCR5_MOUSE_N term), or j) Preferably, at least two or three of Y18, Y26, and Y27 are sulfated, such as SEQ ID NO: 31 (CCR6_HUMAN_TRD) or SEQ ID NO: 34 (CCR6_HUMAN_N term), k) Preferably, at least two or three of Y23, Y31, and Y32 are sulfated, such as Sequence ID No. 32 (CCR6_MACFA_TRD) or Sequence ID No. 35 (CCR6_MACFA_N term), or l) Preferably, at least two or three of Y13, Y18 and Y19 are sulfated, such as Sequence ID No. 33 (CCR6_MOUSE_TRD) or Sequence ID No. 36 (CCR6_MOUSE_N term), m) Preferably, one or both of Y8 and Y17 are sulfated, such as SEQ ID NO: 37 (CCR7_HUMAN_TRD), SEQ ID NO: 40 (CCR7_HUMAN_N term), SEQ ID NO: 38 (CCR7_MACFA_TRD), or SEQ ID NO: 41 (CCR7_MACFA_N term), or n) Preferably, one or both of Y8 and Y17 are sulfated, and Y20 may also be sulfated, such as Sequence ID No. 39 (CCR7_MOUSE_TRD) or Sequence ID No. 42 (CCR7_MOUSE_N term), o) Preferably, at least two or all of Y3, Y15 and Y17 are sulfated, such as SEQ ID NO: 43 (CCR8_HUMAN_TRD), SEQ ID NO: 44 (CCR8_MACFA_TRD), SEQ ID NO: 46 (C=X or S, CCR8_HUMAN_N term), or SEQ ID NO: 47 (C=X or S, CCR8_MACFA_N term), p) Preferably, at least two or all of Y3, Y14 and Y15 are sulfated, such as Sequence ID No. 45 (CCR8_MOUSE_TRD) or Sequence ID No. 48 (C=X or S, CCR8_MOUSE_N term), or q) Preferably at least Y28, preferably Y17 and / or Y37 are also sulfated, such as SEQ ID NO: 61 (CCR9_HUMAN_TRD), SEQ ID NO: 64 (CCR9_HUMAN_N term), SEQ ID NO: 62 (CCR9_MACFA_TRD) or SEQ ID NO: 65 (CCR9_MACFA_N term), or r) Preferably, at least Y28 is sulfated, and preferably Y19 is also sulfated, such as Sequence ID No. 63 (CCR9_MOUSE_TRD) or Sequence ID No. 66 (CCR9_MOUSE_N term), or s) Preferably, at least one or both of Y14 and Y22 are sulfated, such as SEQ ID NO: 67 (CCR10_HUMAN_TRD), SEQ ID NO: 70 (CCR10_HUMAN_N term), SEQ ID NO: 68 (CCR10_MACFA_TRD), or SEQ ID NO: 71 (CCR10_MACFA_N term), or t) Preferably, at least one, two or all of Y14, Y17 and Y22 are sulfated, such as Sequence ID No. 69 (CCR10_MOUSE_TRD) or Sequence ID No. 72 (CCR10_MOUSE_N term), u) Preferably Y27 is sulfated, such as sequence number 73 (CXCR1_HUMAN_TRD) or sequence number 76 (CXCR1_HUMAN_N term), or v) Preferably, at least one of Y14 and Y28 is sulfated, such as sequence number 74 (CXCR1_MACFA_TRD) or sequence number 77 (CXCR1_MACFA_N term), or w) Preferably, at least Y6 is sulfated, such as sequence number 75 (CXCR1_MOUSE_TRD) or sequence number 78 (CXCR1_MOUSE_N term), or x) Preferably Y23 and / or Y25 are sulfated, such as sequence number 79 (CXCR2_HUMAN_TRD) or sequence number 82 (CXCR2_HUMAN_N term), or y) Preferably Y20 and / or Y22 are sulfated, such as sequence number 80 (CXCR2_MACFA_TRD) or sequence number 83 (CXCR2_MACFA_N term), or z) Preferably, Y24 is sulfated, such as sequence number 81 (CXCR2_MOUSE_TRD) or sequence number 84 (CXCR2_MOUSE_N term), or aa) Preferably, at least one or both of Y27 and Y29 are sulfated, such as SEQ ID NO: 85 (CXCR3_HUMAN_TRD), SEQ ID NO: 88 (CXCR3_HUMAN_N term), SEQ ID NO: 86 (CXCR3_MACFA_TRD), SEQ ID NO: 89 (CXCR3_MACFA_N term), SEQ ID NO: 87 (CXCR3_MOUSE_TRD), or SEQ ID NO: 90 (CXCR3_MOUSE_N term), or bb) Preferably, at least Y12 and / or Y21 are sulfated, such as SEQ ID NO: 91 (CXCR4_HUMAN_TRD), SEQ ID NO: 94 (CXCR4_HUMAN_N term), SEQ ID NO: 92 (CXCR4_MACFA_TRD), or SEQ ID NO: 95 (CXCR4_MACFA_N term), or cc) Preferably, at least Y23 and / or Y14 are sulfated, such as SEQ ID NO: 93 (CXCR4_MOUSE_TRD) or SEQ ID NO: 96 (CXCR4_MOUSE_N term), or dd) Preferably, at least one of Y3 and Y27 is sulfated, such as SEQ ID NO: 97 (CXCR5_HUMAN_TRD), SEQ ID NO: 100 (CXCR5_HUMAN_N term), SEQ ID NO: 98 (CXCR5_MACFA_TRD), or SEQ ID NO: 101 (CXCR5_MACFA_N term), or ee) Preferably, at least Y3 and / or Y14 and / or Y20 and / or Y26 are sulfated, such as Sequence ID No. 99 (CXCR5_MOUSE_TRD) or Sequence ID No. 102 (CXCR5_MOUSE_N term), or ff) Preferably, at least one or both of Y6 and Y10 are sulfated, such as SEQ ID NO: 103 (CXCR6_HUMAN_TRD) or SEQ ID NO: 106 (CXCR6_HUMAN_N term), or gg) Preferably, at least two or all of Y4, Y7 and Y39 are sulfated, such as SEQ ID NO: 104 (CXCR6_MACFA_TRD) or SEQ ID NO: 107 (CXCR6_MACFA_N term), or hh) Preferably, at least one or both of Y11 and Y15 are sulfated, such as SEQ ID NO: 105 (CXCR6_MOUSE_TRD) or SEQ ID NO: 108 (CXCR6_MOUSE_N term), or ii) Preferably, at least Y14 is sulfated, such as sequence number 157 (CX3CR1_HUMAN_TRD) or sequence number 160 (CX3CR1_HUMAN_N term), or jj) Preferably, at least Y20 is sulfated, SEQ ID NO: 158 (CX3CR1_MACFA_TRD), or kk) Preferably, at least Y20 or Y22 is sulfated, SEQ ID NO: 161 (CX3CR1_MACFA_N term), or ll) Preferably, at least Y15 is sulfated, such as SEQ ID NO: 159 (CX3CR1_MOUSE_TRD) or SEQ ID NO: 162 (CX3CR1_MOUSE_N term), or (mm) Preferably, at least Y27 is sulfated, such as sequence number 163 (CXCR1_HUMAN_TRD) or sequence number 166 (CXCR1_HUMAN_N term), or nn) Preferably, SEQ ID NO: 164 (CXCR1_MACMU_TRD) in which at least Y14 is sulfated, or oo) Preferably, at least Y14 or Y28 is sulfated, SEQ ID NO: 167 (CXCR1_MACMU_N term), or pp) Preferably, at least Y6 is sulfated, such as SEQ ID NO: 165 (CXCR1_MOUSE_TRD) or SEQ ID NO: 168 (CXCR1_MOUSE_N term).

[0261] According to some embodiments A of the third embodiment of the sixth aspect, the (first) isolated sulfated polypeptide comprises or consists of the sequence described below: a. Preferably, at least Y10 and / or Y18 are sulfated, such as in SEQ ID NO: 1 (CCR1_HUMAN_TRD), or b. Preferably, at least Y26 is sulfated, such as in SEQ ID NO: 7 (CCR2_HUMAN_TRD), or c. Preferably Y16 and / or Y17 are sulfated, such as SEQ ID NO: 13 (CCR3_HUMAN_TRD), or d. Preferably, at least Y22 is sulfated, and more preferably Y16, Y19 and / or Y20 is sulfated, such as SEQ ID NO: 19 (CCR4_HUMAN_TRD), or e. Preferably, two, three or all of Y3, Y10, Y14, and Y15 are sulfated, such as SEQ ID NO: 25 (CCR5_HUMAN_TRD), or f. Preferably, at least two or three of Y18, Y26, and Y27 are sulfated, such as SEQ ID NO: 31 (CCR6_HUMAN_TRD), or g. Preferably, one or both of Y8 and Y17 are sulfated, such as SEQ ID NO: 37 (CCR7_HUMAN_TRD), or h. Preferably, at least two or all of Y3, Y15 and Y17 are sulfated, such as in SEQ ID NO: 43 (CCR8_HUMAN_TRD), or i. Preferably Y17 and / or Y37 are also sulfated, SEQ ID NO: 61 (CCR9_HUMAN_TRD), or j. Preferably, at least one or both of Y14 and Y22 are sulfated, such as in SEQ ID NO: 67 (CCR10_HUMAN_TRD), or k. Preferably, Y27 is sulfated, such as in SEQ ID NO: 73 (CXCR1_HUMAN_TRD), or l. Preferably, Y23 and / or Y25 are sulfated, such as in Sequence ID No. 79 (CXCR2_HUMAN_TRD), or m. Preferably, at least one or both of Y27 and Y29 are sulfated, such as in SEQ ID NO: 85 (CXCR3_HUMAN_TRD), or n. Preferably, at least Y12 and / or Y21 are sulfated, such as in SEQ ID NO: 91 (CXCR4_HUMAN_TRD), or o. Preferably, at least one of Y3 and Y27 is sulfated, such as SEQ ID NO: 97 (CXCR5_HUMAN_TRD), or p. Preferably, at least one or both of Y6 and Y10 are sulfated, such as SEQ ID NO: 103 (CXCR6_HUMAN_TRD), or q. Preferably, at least Y14 is sulfated, such as SEQ ID NO: 157 (CX3CR1_HUMAN_TRD), or r. Preferably, at least Y27 is sulfated, such as SEQ ID NO: 163 (CXCR1_HUMAN_TRD).

[0262] According to some embodiments B of the third embodiment of the sixth aspect, the (first) isolated sulfated polypeptide comprises or consists of the sequence described below: a) Preferably, at least Y10 and / or Y18 are sulfated, SEQ ID NO: 3 (CCR1_MOUSE_TRD), or b) Preferably, at least Y37 and / or Y39 are sulfated, such as in SEQ ID NO: 9 (CCR2_MOUSE_TRD), or c) Preferably Y20 and / or Y22 are sulfated, such as in Sequence ID No. 15 (CCR3_MOUSE_TRD), or d) Preferably at least Y22 is sulfated, and more preferably Y16, Y19 and / or Y20 is sulfated, such as SEQ ID NO: 21 (CCR4_MOUSE_TRD), or e) Preferably two or three of Y10, Y12 and Y16 are sulfated, such as SEQ ID NO: 27 (CCR5_MOUSE_TRD), or f) Preferably, at least two or three of Y13, Y18 and Y19 are sulfated, SEQ ID NO: 33 (CCR6_MOUSE_TRD), g) Preferably, one or both of Y8 and Y17 are sulfated, and Y20 may also be sulfated, such as SEQ ID NO: 39 (CCR7_MOUSE_TRD), or h) Preferably, at least two or all of Y3, Y14 and Y15 are sulfated, such as SEQ ID NO: 45 (CCR8_MOUSE_TRD), or i) Preferably, at least Y28 is sulfated, and preferably Y19 is also sulfated, such as SEQ ID NO: 63 (CCR9_MOUSE_TRD), or j) Preferably, at least one, two or all of Y14, Y17 and Y22 are sulfated, such as sequence number 69 (CCR10_MOUSE_TRD), or k) Preferably, at least Y6 is sulfated, such as SEQ ID NO: 75 (CXCR1_MOUSE_TRD), or l) Preferably, Y24 is sulfated, such as in SEQ ID NO: 81 (CXCR2_MOUSE_TRD), or m) Preferably, at least one or both of Y27 and Y29 are sulfated, such as in SEQ ID NO: 87 (CXCR3_MOUSE_TRD), or n) Preferably, at least Y23 and / or Y14 are sulfated, such as SEQ ID NO: 93 (CXCR4_MOUSE_TRD), or o) Preferably, at least Y3 and / or Y14 and / or Y20 and / or Y26 are sulfated, such as SEQ ID NO: 99 (CXCR5_MOUSE_TRD), or p) Preferably, at least one or both of Y11 and Y15 are sulfated, such as SEQ ID NO: 105 (CXCR6_MOUSE_TRD), or q) Preferably, at least Y15 is sulfated, such as SEQ ID NO: 159 (CX3CR1_MOUSE_TRD), or r) Preferably, at least Y6 is sulfated, such as SEQ ID NO: 165 (CXCR1_MOUSE_TRD).

[0263] According to some embodiments C of the third embodiment of the sixth aspect, the (first) isolated sulfated polypeptide comprises or consists of the sequence described below: a) Preferably, at least Y10 and / or Y18 are sulfated, such as in SEQ ID NO: 2 (CCR1_MACFA_TRD), or b) Preferably, at least Y26 is sulfated, such as in SEQ ID NO: 8 (CCR2_MACMU_TRD), or c) Preferably, Y16 is sulfated, such as SEQ ID NO: 14 (CCR3_MACFA_TRD), or d) Preferably, at least Y22 is sulfated, and more preferably Y16, Y19 and / or Y20 is sulfated, such as SEQ ID NO: 20 (CCR4_MACFA_TRD), or e) Preferably two, three or all of Y3, Y10, Y14 and Y15 are sulfated, such as SEQ ID NO: 26 (CCR5_MACMU_TRD), or f) Preferably, at least two or three of Y23, Y31 and Y32 are sulfated, such as SEQ ID NO: 32 (CCR6_MACFA_TRD), or g) Preferably, one or both of Y8 and Y17 are sulfated, such as SEQ ID NO: 38 (CCR7_MACFA_TRD), or h) Preferably, at least two or all of Y3, Y15 and Y17 are sulfated, such as SEQ ID NO: 44 (CCR8_MACFA_TRD), or i) Preferably at least Y28, preferably Y17 and / or Y37 are also sulfated, SEQ ID NO: 62 (CCR9_MACFA_TRD), or j) Preferably, at least one or both of Y14 and Y22 are sulfated, such as SEQ ID NO: 68 (CCR10_MACFA_TRD), or k) Preferably, at least one of Y14 and Y28 is sulfated, such as SEQ ID NO: 74 (CXCR1_MACFA_TRD), or l) Preferably Y20 and / or Y22 are sulfated, such as in SEQ ID NO: 80 (CXCR2_MACFA_TRD), or m) Preferably, at least one or both of Y27 and Y29 are sulfated, such as in SEQ ID NO: 86 (CXCR3_MACFA_TRD), or n) Preferably, at least Y12 and / or Y21 are sulfated, such as in SEQ ID NO: 92 (CXCR4_MACFA_TRD), or o) Preferably, at least one of Y3 and Y27 is sulfated, such as SEQ ID NO: 98 (CXCR5_MACFA_TRD), or p) Preferably, at least two or all of Y4, Y7 and Y39 are sulfated, such as in SEQ ID NO: 104 (CXCR6_MACFA_TRD), or q) Preferably, at least Y20 is sulfated, such as SEQ ID NO: 158 (CX3CR1_MACFA_TRD), or r) Preferably, SEQ ID NO: 164 (CXCR1_MACMU_TRD) in which at least Y14 is sulfated.

[0264] According to some fourth embodiments of the sixth embodiment, which may be the same as or different from the first, second and / or third embodiments of the sixth embodiment, the first isolated sulfated polypeptide comprises the N-terminus of a seven-transmembrane receptor including a tyrosine-rich domain (TRD), preferably a LID domain, wherein at least 25%, at least 50%, or at least 75% of the tyrosine residues of the TRD are sulfated, preferably at least one / cysteine ​​between the TRD and the LID domain is removed or replaced with a different amino acid.

[0265] According to some embodiments A of the fourth embodiment of the sixth aspect, the (first) isolated sulfated polypeptide comprises or consists of the sequence described below: a) Preferably, at least Y10 and / or Y18 are sulfated, SEQ ID NO: 4 (CCR1_HUMAN_N term), b) Preferably, at least Y26 is sulfated, SEQ ID NO: 10 (CCR2_HUMAN_N term), c) Preferably Y16 and / or Y17 are sulfated, SEQ ID NO: 16 (CCR3_HUMAN_N-terminus), d) Preferably at least Y22 is sulfated, and more preferably Y16, Y19 and / or Y20 is sulfated, SEQ ID NO: 22 (CCR4_HUMAN_N term), e) Preferably, two, three or all of Y3, Y10, Y14, and Y15 are sulfated, SEQ ID NO: 28 (CCR5_HUMAN_N term), f) Preferably, at least two or three of Y18, Y26, and Y27 are sulfated, SEQ ID NO: 34 (CCR6_HUMAN_N term), g) Preferably, one or both of Y8 and Y17 are sulfated, such as SEQ ID NO: 40 (CCR7_HUMAN_N term), h) Preferably, at least two or all of Y3, Y15 and Y17 are sulfated, SEQ ID NO: 46 (C=X or S, CCR8_HUMAN_N term), i) Preferably at least Y28, preferably Y17 and / or Y37 are also sulfated, SEQ ID NO: 64 (CCR9_HUMAN_N term), j) Preferably, at least one or both of Y14 and Y22 are sulfated, SEQ ID NO: 70 (CCR10_HUMAN_N term), k) Preferably Y27 is sulfated, SEQ ID NO: 76 (CXCR1_HUMAN_N term), l) Preferably Y23 and / or Y25 are sulfated, SEQ ID NO: 82 (CXCR2_HUMAN_N term), m) Preferably, at least one or both of Y27 and Y29 are sulfated, SEQ ID NO: 88 (CXCR3_HUMAN_N term), n) Preferably, at least Y12 and / or Y21 are sulfated, SEQ ID NO: 94 (CXCR4_HUMAN_N term), o) Preferably, at least one of Y3 and Y27 is sulfated, such as SEQ ID NO: 100 (CXCR5_HUMAN_N term), or p) Preferably, at least one or both of Y6 and Y10 are sulfated, such as SEQ ID NO: 106 (CXCR6_HUMAN_N term), q) Preferably, at least Y14 is sulfated, such as SEQ ID NO: 160 (CX3CR1_HUMAN_N term), or r) Preferably, at least Y27 is sulfated, such as SEQ ID NO: 166 (CXCR1_HUMAN_N term).

[0266] According to some embodiments B of the fourth embodiment of the sixth aspect, the (first) isolated sulfated polypeptide comprises or consists of the sequence described below: a) Preferably, at least Y10 and / or Y18 are sulfated, SEQ ID NO: 6 (CCR1_MOUSE_N term), or b) Preferably, at least Y37 and / or Y39 are sulfated, such as in SEQ ID NO: 12 (CCR2_MOUSE_N term), or c) Preferably Y20 and / or Y22 are sulfated, such as in Sequence ID No. 18 (CCR3_MOUSE_N term), or d) Preferably at least Y22 is sulfated, and more preferably Y16, Y19 and / or Y20 is sulfated, as in SEQ ID NO: 24 (CCR4_MOUSE_N term), or e) Preferably two or three of Y10, Y12, and Y16 are sulfated, such as SEQ ID NO: 30 (CCR5_MOUSE_N term), or f) Preferably, at least two or three of Y13, Y18 and Y19 are sulfated, SEQ ID NO: 36 (CCR6_MOUSE_N term), g) Preferably, one or both of Y8 and Y17 are sulfated, and Y20 may also be sulfated, such as SEQ ID NO: 42 (CCR7_MOUSE_N term), or h) Preferably, at least two or all of Y3, Y14 and Y15 are sulfated, SEQ ID NO: 48 (C=X or S, CCR8_MOUSE_N term), or i) Preferably, at least Y28 is sulfated, and preferably Y19 is also sulfated, such as SEQ ID NO: 66 (CCR9_MOUSE_N term), or j) Preferably, at least one, two or all of Y14, Y17 and Y22 are sulfated, such as SEQ ID NO: 72 (CCR10_MOUSE_N term), or k) Preferably, at least Y6 is sulfated, such as in SEQ ID NO: 78 (CXCR1_MOUSE_N term), or l) Preferably, Y24 is sulfated, such as in SEQ ID NO: 84 (CXCR2_MOUSE_N term), or m) Preferably, at least one or both of Y27 and Y29 are sulfated, such as in SEQ ID NO: 90 (CXCR3_MOUSE_N term), or n) Preferably, at least Y13 and / or Y14 are sulfated, such as in SEQ ID NO: 96 (CXCR4_MOUSE_N term), or o) Preferably, at least Y3 and / or Y14 and / or Y20 and / or Y26 are sulfated, such as SEQ ID NO: 102 (CXCR5_MOUSE_N term), or p) Preferably, at least one or both of Y11 and Y15 are sulfated, such as SEQ ID NO: 108 (CXCR6_MOUSE_N term), or q) Preferably, at least Y15 is sulfated, such as SEQ ID NO: 162 (CX3CR1_MOUSE_N term), or r) Preferably, at least Y6 is sulfated, such as SEQ ID NO: 168 (CXCR1_MOUSE_N term).

[0267] According to some embodiments C of the fourth embodiment of the sixth aspect, the (first) isolated sulfated polypeptide comprises or consists of the sequence described below: a) Preferably, at least Y10 and / or Y18 are sulfated, SEQ ID NO: 5 (CCR1_MACFA_N term), or b) Preferably, at least Y26 is sulfated, such as in SEQ ID NO: 11 (CCR2_MACMU_N term), or c) Preferably, Y16 is sulfated, such as SEQ ID NO: 17 (CCR3_MACFA_N term), or d) Preferably at least Y22 is sulfated, and more preferably Y16, Y19 and / or Y20 is sulfated, such as SEQ ID NO: 23 (CCR4_MACFA_N term), or e) Preferably two, three or all of Y3, Y10, Y14 and Y15 are sulfated, such as SEQ ID NO: 29 (CCR5_MACMU_N term), or f) Preferably, at least two or three of Y23, Y31 and Y32 are sulfated, such as SEQ ID NO: 35 (CCR6_MACFA_N term), or g) Preferably, one or both of Y8 and Y17 are sulfated, such as SEQ ID NO: 41 (CCR7_MACFA_N term), or h) Preferably, at least two or all of Y3, Y15 and Y17 are sulfated, SEQ ID NO: 47 (C=X or S, CCR8_MACFA_N term), or i) Preferably at least Y28, preferably Y17 and / or Y37 are also sulfated, SEQ ID NO: 65 (CCR9_MACFA_N term), or j) Preferably, at least one or both of Y14 and Y22 are sulfated, such as SEQ ID NO: 71 (CCR10_MACFA_N term), or k) Preferably, at least one of Y14 and Y28 is sulfated, such as in SEQ ID NO: 77 (CXCR1_MACFA_N term), or l) Preferably Y20 and / or Y22 are sulfated, such as in Sequence ID No. 83 (CXCR2_MACFA_N term), or m) Preferably, at least one or both of Y27 and Y29 are sulfated, such as in SEQ ID NO: 89 (CXCR3_MACFA_N term), or n) Preferably, at least Y12 and / or Y21 are sulfated, such as in SEQ ID NO: 95 (CXCR4_MACFA_N term), or o) Preferably, at least one of Y3 and Y27 is sulfated, such as SEQ ID NO: 101 (CXCR5_MACFA_N term), or p) Preferably, at least two or all of Y4, Y7 and Y39 are sulfated, such as SEQ ID NO: 107 (CXCR6_MACFA_N term), or q) Preferably, at least Y20 or Y22 is sulfated, such as SEQ ID NO: 161 (CX3CR1_MACFA_N term), or r) Preferably, at least Y14 or Y28 is sulfated, such as SEQ ID NO: 167 (CXCR1_MACMU_N term).

[0268] According to some fifth embodiments of the sixth embodiment, which may or may not be the same as the first, second, third and / or fourth embodiments according to the sixth embodiment, an isolated antibody or its antigen-binding fragment binds (specifically) to a second isolated sulfated polypeptide containing the tyrosine-rich domain (TRD) of a seven-transmembrane receptor.

[0269] Preferably, the seven-transmembrane receptor of TRD contained in the second isolated sulfated polypeptide is a) Different from the seven-transmembrane receptor of TRD contained in the first isolated sulfated polypeptide, or b) The first isolated sulfated polypeptide contains the corresponding seven-transmembrane receptor for TRDs derived from different species.

[0270] According to some preferred embodiments, a second isolated sulfated polypeptide containing a tyrosine-rich domain (TRD) of a seven-transmembrane receptor is an isolated polypeptide having the sequence described in the third and / or fourth embodiment of the sixth embodiment, unlike the first isolated sulfated polypeptide containing a tyrosine-rich domain (TRD) of a seven-transmembrane receptor. For example, the first isolated sulfated polypeptide may contain a TRD of a first species of seven-transmembrane receptor, and the second isolated sulfated polypeptide may contain a TRD of a second species of seven-transmembrane receptor, preferably the species being human and cynomolgus monkey, or human and mouse, or human and rat.

[0271] According to some embodiments A of the fifth embodiment of the sixth aspect, the antibody or fragment is a) A first isolated sulfated polypeptide comprising SEQ ID NO: 1 (CCR1_HUMAN_TRD), preferably having at least Y10 and / or Y18 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 2 (CCR1_MACFA_TRD), preferably having at least Y10 and / or Y18 sulfated, or b) A first isolated sulfated polypeptide comprising SEQ ID NO: 7 (CCR2_HUMAN_TRD), preferably having at least Y26 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 8 (CCR2_MACMU_TRD), preferably having at least Y26 sulfated, or c) A first isolated sulfated polypeptide comprising SEQ ID NO: 13 (CCR3_HUMAN_TRD), preferably with Y16 and / or Y17 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 14 (CCR3_MACFA_TRD), preferably with Y16 sulfated, or d) A first isolated sulfated polypeptide comprising SEQ ID NO: 19 (CCR4_HUMAN_TRD), preferably having at least Y22 sulfated, and more preferably having Y16, Y19 and / or Y20 sulfated; and a second isolated sulfated polypeptide comprising SEQ ID NO: 20 (CCR4_MACFA_TRD), preferably having at least Y22 sulfated, and more preferably having Y16, Y19 and / or Y20 sulfated; or e) A first isolated sulfated polypeptide comprising SEQ ID NO: 25 (CCR5_HUMAN_TRD), preferably in which two, three or all of Y3, Y10, Y14, and Y15 are sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 26 (CCR5_MACMU_TRD), preferably in which two, three or all of Y3, Y10, Y14, and Y15 are sulfated, or f) A first isolated sulfated polypeptide comprising SEQ ID NO: 31 (CCR6_HUMAN_TRD), preferably having at least two or three of Y18, Y26, and Y27 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 32 (CCR6_MACFA_TRD), preferably having at least two or three of Y23, Y31, and Y32 sulfated, or g) A first isolated sulfated polypeptide comprising SEQ ID NO: 37 (CCR7_HUMAN_TRD), preferably in which one or both of Y8 and Y17 are sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 38 (CCR7_MACFA_TRD), preferably in which one or both of Y8 and Y17 are sulfated, or h) A first isolated sulfated polypeptide comprising SEQ ID NO: 43 (CCR8_HUMAN_TRD), preferably having at least two or all of Y3, Y15, and Y17 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 44 (CCR8_MACFA_TRD), preferably having at least two or all of Y3, Y15, and Y17 sulfated, or i) A first isolated sulfated polypeptide comprising SEQ ID NO: 61 (CCR9_HUMAN_TRD), preferably with Y17 and / or Y37 also sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 62 (CCR9_MACFA_TRD), preferably with at least Y28, preferably with Y17 and / or Y37 also sulfated, or j) A first isolated sulfated polypeptide comprising SEQ ID NO: 67 (CCR10_HUMAN_TRD), preferably having at least one or both of Y14 and Y22 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 68 (CCR10_MACFA_TRD), preferably having at least one or both of Y14 and Y22 sulfated, or k) A first isolated sulfated polypeptide comprising SEQ ID NO: 73 (CXCR1_HUMAN_TRD), preferably in which Y27 is sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 74 (CXCR1_MACFA_TRD), preferably in which at least one of Y14 and Y28 is sulfated, or l) A first isolated sulfated polypeptide comprising SEQ ID NO: 79 (CXCR2_HUMAN_TRD), preferably with Y23 and / or Y25 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 80 (CXCR2_MACFA_TRD), preferably with Y20 and / or Y22 sulfated, or m) A first isolated sulfated polypeptide comprising SEQ ID NO: 85 (CXCR3_HUMAN_TRD), preferably having at least one or both of Y27 and Y29 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 86 (CXCR3_MACFA_TRD), preferably having at least one or both of Y27 and Y29 sulfated, or n) A first isolated sulfated polypeptide comprising SEQ ID NO: 91 (CXCR4_HUMAN_TRD), preferably having at least Y12 and / or Y21 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 92 (CXCR4_MACFA_TRD), preferably having at least Y12 and / or Y21 sulfated, or o) A first isolated sulfated polypeptide comprising SEQ ID NO: 97 (CXCR5_HUMAN_TRD), preferably having at least one of Y3 and Y27 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 98 (CXCR5_MACFA_TRD), preferably having at least one of Y3 and Y27 sulfated, or p) A first isolated sulfated polypeptide comprising SEQ ID NO: 103 (CXCR6_HUMAN_TRD), preferably in which at least one or both of Y6 and Y10 are sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 104 (CXCR6_MACFA_TRD), preferably in which at least two or all of Y4, Y7, and Y39 are sulfated. q) A first isolated sulfated polypeptide comprising SEQ ID NO: 157 (CX3CR1_HUMAN_TRD), preferably having at least Y14 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 158 (CX3CR1_MACFA_TRD), preferably having at least Y20 sulfated, or r) A first isolated sulfated polypeptide comprising SEQ ID NO: 163 (CXCR1_HUMAN_TRD), preferably having at least Y27 sulfated, is bound to a second isolated sulfated polypeptide comprising SEQ ID NO: 164 (CXCR1_MACMU_TRD), preferably having at least Y14 sulfated.

[0272] According to some embodiments B of the fifth embodiment of the sixth aspect, the antibody or fragment is a) A first isolated sulfated polypeptide comprising SEQ ID NO: 4 (CCR1_HUMAN_N term), preferably having at least Y10 and / or Y18 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 5 (CCR1_MACFA_N term), preferably having at least Y10 and / or Y18 sulfated. b) A first isolated sulfated polypeptide comprising SEQ ID NO: 10 (CCR2_HUMAN_N term), preferably having at least Y26 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 11 (CCR2_MACMU_N term), preferably having at least Y26 sulfated. c) A first isolated sulfated polypeptide comprising SEQ ID NO: 16 (CCR3_HUMAN_N term), preferably having Y16 and / or Y17 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 17 (CCR3_MACFA_N term), preferably having Y16 sulfated. d) A first isolated sulfated polypeptide comprising SEQ ID NO: 22 (CCR4_HUMAN_N term), preferably having at least Y22 sulfated, and more preferably having Y16, Y19 and / or Y20 sulfated; and a second isolated sulfated polypeptide comprising SEQ ID NO: 23 (CCR4_MACFA_N term), preferably having at least Y22 sulfated, and more preferably having Y16, Y19 and / or Y20 sulfated. e) A first isolated sulfated polypeptide comprising SEQ ID NO: 28 (CCR5_HUMAN_N term), preferably in which two, three or all of Y3, Y10, Y14, and Y15 are sulfated; and a second isolated sulfated polypeptide comprising SEQ ID NO: 29 (CCR5_MACMU_N term), preferably in which two, three or all of Y3, Y10, Y14, and Y15 are sulfated. f) A first isolated sulfated polypeptide comprising SEQ ID NO: 34 (CCR6_HUMAN_N term), preferably having at least two or three of Y18, Y26, and Y27 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 35 (CCR6_MACFA_N term), preferably having at least two or three of Y23, Y31, and Y32 sulfated. g) A first isolated sulfated polypeptide comprising SEQ ID NO: 40 (CCR7_HUMAN_N term), preferably having one or both of Y8 and Y17 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 41 (CCR7_MACFA_N term), preferably having one or both of Y8 and Y17 sulfated. h) A first isolated sulfated polypeptide comprising SEQ ID NO: 46 (C=X or S, CCR8_HUMAN_N term), preferably having at least two or all of Y3, Y15, and Y17 sulfated; and a second isolated sulfated polypeptide comprising SEQ ID NO: 47 (C=X or S, CCR8_MACFA_N term), preferably having at least two or all of Y3, Y15, and Y17 sulfated. i) A first isolated sulfated polypeptide comprising SEQ ID NO: 64 (CCR9_HUMAN_N term), preferably with at least Y28, preferably Y17 and / or Y37 also sulfated; and a second isolated sulfated polypeptide comprising SEQ ID NO: 65 (CCR9_MACFA_N term), preferably with at least Y28, preferably Y17 and / or Y37 also sulfated. j) A first isolated sulfated polypeptide comprising SEQ ID NO: 70 (CCR10_HUMAN_N term), preferably having at least one or both of Y14 and Y22 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 71 (CCR10_MACFA_N term), preferably having at least one or both of Y14 and Y22 sulfated. k) A first isolated sulfated polypeptide comprising SEQ ID NO: 76 (CXCR1_HUMAN_N term), preferably in which Y27 is sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 77 (CXCR1_MACFA_N term), preferably in which at least one of Y14 and Y28 is sulfated. l) A first isolated sulfated polypeptide comprising SEQ ID NO: 82 (CXCR2_HUMAN_N term), preferably having Y23 and / or Y25 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 83 (CXCR2_MACFA_N term), preferably having Y20 and / or Y22 sulfated. m) A first isolated sulfated polypeptide comprising SEQ ID NO: 88 (CXCR3_HUMAN_N term), preferably having at least one or both of Y27 and Y29 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 89 (CXCR3_MACFA_N term), preferably having at least one or both of Y27 and Y29 sulfated. n) A first isolated sulfated polypeptide comprising SEQ ID NO: 94 (CXCR4_HUMAN_N term), preferably having at least Y12 and / or Y21 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 95 (CXCR4_MACFA_N term), preferably having at least Y12 and / or Y21 sulfated; o) A first isolated sulfated polypeptide comprising SEQ ID NO: 100 (CXCR5_HUMAN_N term), preferably having at least one of Y3 and Y27 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 101 (CXCR5_MACFA_N term), preferably having at least one of Y3 and Y27 sulfated; or p) A first isolated sulfated polypeptide comprising SEQ ID NO: 106 (CXCR6_HUMAN_N term), preferably having at least one or both of Y6 and Y10 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 107 (CXCR6_MACFA_N term), preferably having at least two or all of Y4, Y7 and Y39 sulfated, or q) A first isolated sulfated polypeptide comprising SEQ ID NO: 160 (CX3CR1_HUMAN_N term), preferably having at least Y14 sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 161 (CX3CR1_MACFA_N term), preferably having at least Y20 or Y22 sulfated, or r) Preferably, a first isolated sulfated polypeptide comprising SEQ ID NO: 166 (CXCR1_HUMAN_N term), in which at least Y27 is sulfated, is bound to a second isolated sulfated polypeptide comprising SEQ ID NO: 167 (CXCR1_MACMU_N term), in which at least Y14 or Y28 is sulfated.

[0273] According to some embodiment C1 of the fifth embodiment of the sixth aspect, the antibody or fragment specifically binds to a first isolated sulfated polypeptide containing a sequence according to some embodiment A of the third embodiment of the sixth aspect, and to a second isolated sulfated polypeptide containing a sequence according to some embodiment B or C of the third embodiment of the sixth aspect, preferably the first and second polypeptides contain TRDs of the same receptor but of different species.

[0274] According to some embodiment C2 of the fifth embodiment of the sixth aspect, the antibody or fragment specifically binds to a first isolated sulfated polypeptide containing a sequence according to some embodiment A of the fourth embodiment of the sixth aspect, and to a second isolated sulfated polypeptide containing a sequence according to some embodiment B or C of the fourth embodiment of the sixth aspect, preferably the first and second polypeptides contain TRDs of the same receptor but of different species.

[0275] According to some sixth embodiments of the sixth embodiment, which may be the same as or different from the first, second, third, fourth and / or fifth embodiments according to the sixth embodiment, the dissociation constant or EC50 of the antibody or antigen-binding fragment for binding the first isolated sulfated polypeptide and / or the seven-transmembrane receptor is less than 200 nM, 150 nM, 100 nM, 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM, or 0.25 nM.

[0276] According to some embodiments A of the sixth embodiment of the sixth aspect, the dissociation constant or EC50 of the antibody or antigen-binding fragment for binding the first isolated sulfated polypeptide and / or the seven transmembrane receptor is 200nM, 199nM, 198nM, 197nM, 196nM, 195nM, 194nM, 193nM, 192nM, 191nM, 190nM, 189nM, 188nM, 187nM, 186nM, 185nM, 184nM, 183nM, 182nM, 181nM, 180nM, 179nM, 178nM, 177nM, 176 nM, 175nM, 174nM, 173nM, 172nM, 171nM, 170nM, 169nM, 168nM, 167nM, 166nM , 165nM, 164nM, 163nM, 162nM, 161nM, 160nM, 159nM, 158nM, 157nM, 156nM, 15 5nM, 154nM, 153nM, 152nM, 151nM, 150nM, 149nM, 148nM, 147nM, 146nM, 145n M, 144nM, 143nM, 142nM, 141nM, 140nM, 139nM, 138nM, 137nM, 136nM, 135nM, 1 34nM, 133nM, 132nM, 131nM, 130nM, 129nM, 128nM, 127nM, 126nM, 125nM, 124 nM, 123nM, 122nM, 121nM, 120nM, 119nM, 118nM, 117nM, 116nM, 115nM, 114nM , 113nM, 112nM, 111nM, 110nM, 109nM, 108nM, 107nM, 106nM, 105nM, 104nM, 1 03nM, 102nM, 101nM, 100nM, 99nM, 98nM, 97nM, 96nM, 95nM, 94nM, 93nM, 92nM, 91nM, 90nM, 89nM, 88nM, 87nM, 86nM, 85nM, 84nM, 83nM, 82nM, 81nM, 80nM, 79 nM, 78nM, 77nM, 76nM, 75nM, 74nM, 73nM, 72nM, 71nM, 70nM, 69nM, 68nM, 67nM, 66nM, 65nM, 64nM, 63nM, 62nM, 61nM, 60nM, 59nM, 58nM, 57nM, 56nM, 55nM, 54 nM, 53nM, 52nM, 51nM, 50nM, 49nM, 48nM, 47nM, 46nM, 45nM, 44nM, 43nM, 42nM,41nM, 40nM, 39nM, 38nM, 37nM, 36nM, 35nM, 34nM, 33nM, 32nM, 31nM, 30nM, 29nM, 28 nM, 27nM, 26nM, 25nM, 24nM, 23nM, 22nM, 21nM, 20nM, 19nM, 18nM, 17nM, 16nM, 15nM, The values ​​are 14nM, 13nM, 12nM, 11nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.25nM, 0.2nM, 0.15nM, or less than 0.1nM.

[0277] Preferably, the dissociation constant or EC50 of the antibody or antigen-binding fragment for binding to the first isolated sulfated polypeptide and / or for the seven-transmembrane receptor is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM, 0.25 nM, 0.2 nM, 0.15 nM, or 0.1 nM.

[0278] Preferably, EC50 can be determined in CHO cells overexpressing the target. Antibodies obtained by the methods disclosed herein, for example as disclosed in Example 10.1.1, have excellent affinity for their respective targets. For example, TPP-21181, TPP-17578, TPP-19546, TPP-18206, TPP-21360, and TPP-23411 bound to human CCR8 with EC50s of 4.8 nM, 1.7 nM, 0.8 nM, 0.6 nM, about 0.9 nM, or 1.7 nM in CHO cells engineered to overexpress CCR8. Furthermore, TPP-21181, TPP-17578, TPP-19546, TPP-18206, TPP-21360, and TPP-23411 bound to cynomolgus monkey CCR8 cells with EC50 values ​​of 1.8 nM, 1 nM, 0.5 nM, 0.7 nM, approximately 0.55 nM, or 0.9 nM. In addition, TPP-17578, TPP-19546, TPP-18206, and TPP-21360 bound to human regulatory T cells with EC50 values ​​of 25 nM, 15 nM, 23 nM, or 10 nM. Moreover, the anti-mouse CCR8 antibody TPP-14099 bound to mouse CCR8-expressing CHO cells with an EC50 of 3 nM and to mouse iTreg cells with an EC50 of 13.2 nM (see Table 10.1.1.5).

[0279] According to some Embodiment B of the Sixth Embodiment of the Sixth Embodiment, which may or may not be the same as Embodiment A of the Sixth Embodiment, the dissociation constant or EC50 of the antibody or antigen-binding fragment for binding a second isolated sulfated polypeptide and / or a second seven-transmembrane receptor is 200nM, 199nM, 198nM, 197nM, 196nM, 195nM, 194nM, 193nM, 192nM, 191nM, 190nM, 189nM, 18 8nM, 187nM, 186nM, 185nM, 184nM, 183nM, 182nM, 181nM, 180nM, 179nM, 178nM, 177nM, 176nM, 175nM, 174nM, 173nM, 172nM, 171nM, 17 0nM, 169nM, 168nM, 167nM, 166nM, 165nM, 164nM, 163nM, 162nM, 161nM, 160nM, 159nM, 158nM, 157nM, 156nM, 155nM, 154nM, 153nM, 15 2nM, 151nM, 150nM, 149nM, 148nM, 147nM, 146nM, 145nM, 144nM, 143nM, 142nM, 141nM, 140nM, 139nM, 138nM, 137nM, 136nM, 135nM, 1 34nM, 133nM, 132nM, 131nM, 130nM, 129nM, 128nM, 127nM, 126nM, 125nM, 124nM, 123nM, 122nM, 121nM, 120nM, 119nM, 118nM, 117nM, 1 16nM, 115nM, 114nM, 113nM, 112nM, 111nM, 110nM, 109nM, 108nM, 107nM, 106nM, 105nM, 104nM, 103nM, 102nM, 101nM, 100nM, 99nM, 98 nM, 97nM, 96nM, 95nM, 94nM, 93nM, 92nM, 91nM, 90nM, 89nM, 88nM, 87nM, 86nM, 85nM, 84nM, 83nM, 82nM, 81nM, 80nM, 79nM, 78nM, 77nM,76nM, 75nM, 74nM, 73nM, 72nM, 71nM, 70nM, 69nM, 68nM, 67nM, 66nM, 65nM, 64nM, 63nM, 62nM, 61nM, 60nM, 59nM, 58nM, 57nM, 56nM, 55nM, 5 4nM, 53nM, 52nM, 51nM, 50nM, 49nM, 48nM, 47nM, 46nM, 45nM, 44nM, 43nM, 42nM, 41nM, 40nM, 39nM, 38nM, 37nM, 36nM, 35nM, 34nM, 33nM, 32n M, 31nM, 30nM, 29nM, 28nM, 27nM, 26nM, 25nM, 24nM, 23nM, 22nM, 21nM, 20nM, 19nM, 18nM, 17nM, 16nM, 15nM, 14nM, 13nM, 12nM, 11nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.25nM, 0.2nM, 0.15nM, or less than 0.1nM.

[0280] Preferably, the dissociation constant (KD) or EC50 of the antibody or antigen-binding fragment for binding the second isolated sulfated polypeptide and / or the second seven-transmembrane receptor is less than 10 nM, less than 5 nM, less than 2.5 nM, less than 1 nM, less than 0.5 nM, or less than 0.25 nM.

[0281] According to some implementations of Chia AB of the sixth embodiment of the sixth aspect, the dissociation constant (KD) or EC50 of the first isolated sulfated polypeptide and / or antibody or antigen-binding fragment for binding to the first seven-transmembrane receptor is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM, or 0.25 nM, and the dissociation constant (KD) or EC50 of the second isolated sulfated polypeptide and / or antibody or antigen-binding fragment for binding to the second seven-transmembrane receptor is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM, or 0.25 nM.

[0282] According to some seventh embodiments of the sixth embodiment, which may or may not be the same as the first, second, third, fourth, fifth and / or sixth embodiments according to the sixth embodiment, the kD of the antibody for binding to the first isolated sulfated polypeptide is lower than the kD of the antibody for binding to the first isolated non-sulfated polypeptide having the same sequence as the first isolated sulfated polypeptide. Preferably, the antibody does not substantially bind to the first isolated non-sulfated polypeptide having the same sequence as the first isolated sulfated polypeptide.

[0283] According to some embodiments A of the seventh embodiment of the sixth aspect, the dissociation constant or EC50 of the antibody for binding to the first isolated non-sulfated polypeptide is higher than 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 1.25 μM, 1.5 μM, 1.75 μM, 2 μM, 2.25 μM, 2.5 μM, 2.75 μM, or 3 μM, or is undetectable. Preferably, the dissociation constant or EC50 of the antibody for binding to the first isolated non-sulfated polypeptide is higher than 100 nM, 250 nM, 500 nM, 1 μM, 2 μM, or 3 μM, or is undetectable.

[0284] According to some Embodiment B of the Seventh Embodiment of the Sixth Embodiment, which may be the same as Embodiment A of the Sixth Embodiment, the dissociation constant or EC50 of the antibody or fragment for conjugating the first isolated sulfated polypeptide is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM, or 0.25 nM, and the dissociation constant or EC50 of the antibody or fragment for conjugating the first isolated non-sulfated polypeptide is higher than 10 nM, 25 nM, 50 nM, 100 nM, 250 nM, or 500 nM, or is undetectable.

[0285] The antibody according to the sixth embodiment may include CDRs derived from humans, monkeys, cynomolgus macaques, rhesus macaques, rodents, mice, rats, horses, cattle, pigs, dogs, cats, and camels. Preferably, the antibody according to the sixth embodiment includes CDRs derived from humans, rats, or mice.

[0286] According to several eighth embodiments of the sixth embodiment, which may be combined with, and are suggested to be combined with, the first, second, third, fourth, fifth, sixth, and / or seventh embodiments of the sixth embodiment, the isolated antibody or antigen-binding fragment comprises a human-derived CDR. According to several very preferred embodiments, the human-derived CDR has no deviation from the nearest human germline, or includes deviations of less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The nearest human germline can be determined in silico using IgBLAST (Ye, Jian, et al. “IgBLAST: an immunoglobulin variable domain sequence analysis tool.” Nucleic acids research 41.W1(2013):W34-W40.) with data retrieved from the IMGT human germline database, as is known in the art. Antibodies according to these eighth embodiments can be obtained, for example, as described in Example 6 or 8, or as described elsewhere in this specification.

[0287] According to some ninth embodiments of the sixth embodiment, which may be combined with, and are suggested to be combined with, the first, second, third, fourth, fifth, sixth, seventh, or eighth embodiments of the sixth embodiment, the isolated antibody or antigen-binding fragment is cross-reactive to humans and cynomolgus monkeys (see Example 10.1.1). Preferably, the dissociation constant (KD) or EC50 of the antibody or antigen-binding fragment for binding to the human chemokine receptor is less than 200 nM, 150 nM, 100 nM, 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5, or 0.25 nM, for example, less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5, or 0.25 nM. Preferably, the dissociation constant (KD) or EC50 of the antibody or antigen-binding fragment for binding to the cynomolgus monkey chemokine receptor is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5, or 0.25 nM.

[0288] According to some tenth embodiments of the sixth embodiment, which may be combined with, and are suggested to be combined with, the first, second, third, fourth, fifth, sixth, seventh, eighth and / or ninth embodiments of the sixth embodiment, the isolated antibody or antigen-binding fragment is characterized by an HCDR3 region whose composition deviates from the mean HCDR3 region. Preferably, the isolated antibody or antigen-binding fragment is characterized by an HCDR3 region containing 10-34% tyrosine and / or at least one histidine, preferably 2-20% histidine, most preferably 7-20% histidine.

[0289] In some embodiments A of the 10th embodiment of the 6th aspect, the isolated antibody or antigen-binding fragment is a) Having tyrosine (Y) residues >0 and <35%, >8 and <34%, 10 to 34%, or >15 and <31%, and / or b) Having histidine (H) residues > 0 and ≤ 16%, ≥ 2 and ≤ 20%, or ≥ 7 and ≤ 20%, and c) Preferably arginine(R) residues >0 and ≤18%, ≥7 and ≤10%, or ≥0 and ≤7%, and / or d) Preferably having aspartic acid (D) residues >0 and ≤25%, ≥7 and ≤16%, or ≥7 and ≤13%, and / or e) Preferably free of lysine (K) residues, and / or f) Preferably, the HCDR3 contains no glutamic acid (E) residue.

[0290] In some embodiments B of the 10th embodiment of the 6th embodiment, which may be the same as or different from Embodiment A, the isolated antibody or antigen-binding fragment is a) Having charged amino acids >0 and <47%, >22 and <50%, >10 and <34%, or >15 and <47%, and / or b) Positively charged amino acids >0 and <32%, >8 and <30%, or >10 and <37%, and / or c) Contains HCDR3 having negatively charged amino acids > 0 and < 26%, ≥ 7 and < 16%, or > 7 and < 14%.

[0291] In some embodiments C of the tenth embodiment of the sixth embodiment, which may be the same as or different from embodiments A and / or B, the isolated antibody or antigen-binding fragment comprises HCDR3 having a total of >0 and ≤42%, ≥10 and ≤42%, or ≥36 and ≤43% histidine and tyrosine residues.

[0292] Each of the embodiments and descriptions disclosed for CCR8 according to Embodiment 10 is generally disclosed herein for chemokine receptor antibodies, for example, with necessary modifications. The inventors believe that sulfated TRD motifs, rather than specific CCR8 sequences, promote the increased frequency of tyrosine and / or histidine.

[0293] According to some eleventh embodiments of the sixth embodiment, which may be, and is suggested to be, combined with the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and / or tenth embodiments of the sixth embodiment, the isolated antibody or antigen-binding fragment modulates CCR8 signaling at least partially.

[0294] For example, the antibody according to the present invention is a) Can block G protein-independent signaling, and / or b) It can block G protein-dependent signaling, and / or c) It can block G protein-dependent and G protein-independent signaling, and / or d) Can increase G protein-independent signaling, and / or e) Can increase G protein-dependent signaling, and / or f) It can increase G protein-dependent and G protein-independent signaling.

[0295] In certain contexts, regulation refers to the blockade of ligand-induced G protein-independent signaling and the induction of G protein-independent signaling.

[0296] According to some preferred embodiments, the antibody or fragment does not modulate G protein-independent signaling of the 7-transmembrane receptor. According to some preferred embodiments, the antibody or fragment does not block ligand-induced G protein-independent signaling of the target protein. According to some preferred embodiments, the antibody or fragment does not induce G protein-independent signaling.

[0297] According to some preferred embodiments, the antibody or fragment blocks G protein-dependent signaling. G protein-dependent signaling can be analyzed by chemotactic assays, or preferably by calcium flux assays, as described elsewhere in this specification.

[0298] According to some preferred embodiments, the antibody or fragment blocks G protein-dependent signaling of the chemokine receptor but not G protein-independent signaling of the chemokine receptor (see Example 10.4). G protein-independent signaling of the chemokine receptor is any signaling activity that is not G protein-dependent.

[0299] According to some twelfth embodiments of the sixth embodiment, which may be combined with, and are suggested to be combined with, the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, and eleventh embodiments of the sixth embodiment, the isolated antibody or antigen-binding fragment is a low-internalized or non-internalized antibody or antigen-binding fragment.

[0300] Overexpression can affect internalization behavior and is not well-suited for modeling internalization in a therapeutic environment; therefore, internalization is preferably determined using a model cell line having endogenous expression of the target chemokine receptor (see Example 10.5). For example, internalization can be determined over a time frame or at a specific point in time. Preferably, internalization can be determined at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, or 48 hours in cells endogenously expressing the target.

[0301] According to some embodiments A of the twelfth embodiment of the sixth aspect, the antibody or antigen-binding fragment has an internalization rate comparable to that of the isotype control.

[0302] According to some embodiments B of the twelfth embodiment of the sixth aspect, the antibody or antigen-binding fragment is characterized by internalization into cells having endogenous target expression lower than 1.5, 2, 3, 4, 5, 6, 7, or 10 times that of the isotype control. The antibody isotype control can be selected as known in the art, so as closely as possible to the antibody isotype but so that it does not bind to the target.

[0303] According to some embodiments C of the twelfth embodiment of the sixth aspect, the antibody or antigen-binding fragment is characterized by internalization into cells having endogenous target expression, which is less than 150%, 175%, 200%, 300%, 400%, or 500% of the internalization of the isotype control after, for example, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, or 48 hours.

[0304] According to some thirteenth embodiments of the sixth embodiment, which may be, and is suggested to be, combined with the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh and / or twelfth embodiments of the sixth embodiment, an isolated antibody or antigen-binding fragment induces ADCC and / or ADCP in cells expressing the antibody's target receptor.

[0305] In some preferred embodiments of the 13th embodiment of the sixth aspect, the antibody or antigen-binding fragment is afucosylated.

[0306] In some embodiments A1 of the 13th embodiment of the sixth aspect, the antibody or antigen-binding fragment binds to the human Fc gamma receptor IIIA variant V176 (CD16a) with a dissociation constant (KD) of 530 nM, 500 nM, 450 nM, 400 nM, 300 nM, or less than 200 nM. Preferably, the KD can be determined using surface plasmon resonance.

[0307] In some embodiments B1 of the 13th embodiment of the sixth embodiment, which may be the same as or different from Embodiment A1, an antibody or antigen-binding fragment induces antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells expressing the target receptor via human effector cells such as human NK cells. ADCC induction can be analyzed by assays known in the art, e.g., those described according to Example 10.3.3ff, or assays described elsewhere in this specification. Preferably, the assay is performed using Treg cells in which at least 80% or 85% of the Treg cells express CCR8.

[0308] In some embodiment C1 of the thirteenth embodiment of Embodiment 6, which may be the same as or different from Embodiment A1 or B1, the maximum ADCC-inducible depletion of cells expressing the target receptor is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%.

[0309] In some embodiment D1 of the 13th embodiment of the 6th embodiment, which may be the same as or different from embodiment A1, B1, or C1, the EC50 of ADCC-induced depletion of target expression cells is less than 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 12.5 pM, 10 pM, 5 pM, or 2.5 pM.

[0310] In some embodiments A2 of the 13th embodiment of the 6th aspect, which may or may not be the same as embodiments A1, B1, C1 and / or D1, the antibody or antigen-binding fragment binds to human Fc gamma RIIA (CD32a) with a dissociation constant (KD) of 30 μM, 20 μM, 10 μM, 5 μM, or less than 1 μM.

[0311] In some embodiments B2 of the 13th embodiment of the sixth embodiment, which may be the same as embodiments A1, B1, C1 and / or D1 and may be the same as embodiment A2, an antibody or antigen-binding fragment induces antibody-dependent cell-mediated phagocytosis (ADCP) in cells expressing a target receptor via human effector cells, such as human macrophages. For example, the human macrophages may be M2 or M1 macrophages.

[0312] In some embodiments C2 of the thirteenth embodiment of the sixth aspect, which may be the same as embodiments A1, B1, C1 and / or D1 and may be the same as embodiments A2 and / or B2, the ADCP-induced maximum depletion of cells expressing the target receptor is at least 5, 10, 15, 20, 25, 30, 40 or 50%.

[0313] In some embodiment D2 of the 13th embodiment of the 6th embodiment, which may be the same as embodiments A1, B1, C1 and / or D1, and may be the same as embodiments A2, B2 and / or C2, the EC50 for ADCP-induced depletion of activated human regulatory T cells is 1500 pM, 1000 pM, 500 pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM, 25 pM, or less than 10 pM.

[0314] In some preferred embodiments of the 13th embodiment of the sixth aspect, an isolated antibody or antigen-binding fragment thereof that specifically binds to a chemokine receptor is provided, and the antibody or antigen-binding fragment is a) Binds to human Fc gamma receptor IIIA variant V176 (CD16a) with dissociation constants (KD) of 530 nM, 500 nM, 450 nM, 400 nM, 300 nM or less than 200 nM, and / or b) Binds to human Fc gamma RIIA (CD32a) with dissociation constants (KD) of 30 μM, 20 μM, 10 μM, 5 μM, or less than 1 μM.

[0315] In some preferred embodiments of the 13th embodiment of the sixth aspect, an isolated antibody or antigen-binding fragment thereof that specifically binds to a chemokine receptor is provided, and the antibody or antigen-binding fragment is a) Induce antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells expressing human chemokine receptors via human effector cells such as human NK cells, and / or b) Induce antibody-dependent cell-mediated phagocytosis (ADCP) in target cells expressing human chemokine receptors via human effector cells such as human macrophages.

[0316] In some preferred embodiments of the 13th embodiment of the sixth aspect, an isolated antibody or antigen-binding fragment thereof that specifically binds to a chemokine receptor is provided. a) The maximum ADCC-induced depletion in target cells expressing human chemokine receptors is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%, and / or b) The maximum ADCP-induced depletion in target cells expressing human chemokine receptors is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50%, and / or c) Maximum depletion of target cells expressing human chemokine receptors is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 99%.

[0317] In some preferred embodiments of the 13th embodiment of the sixth aspect, an isolated antibody or antigen-binding fragment thereof that specifically binds to a chemokine receptor is provided. a) The EC50 of ADCC-induced depletion of target cells expressing human chemokine receptors is 200 pM, 100 pM, 50 pM, 25 pM, 12.5 pM, 10 pM, or less than 5 pM, and / or b) The EC50 of ADCP-induced depletion in target cells expressing human chemokine receptors is 500 pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM, or less than 25 pM.

[0318] According to some 14th embodiments of the 6th embodiment, which may be combined with, and are suggested to be combined with, the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and / or thirteenth embodiments of the 6th embodiment, the isolated antibody or antigen-binding fragment comprises a human, rat, or mouse IgG antibody, preferably a human IgG1 antibody or a mouse IgG2a antibody (see Examples 6 and 8).

[0319] According to some 15th embodiments of the sixth embodiment, which may be combined with, and are suggested to be combined with, the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth and / or fourteenth embodiments of the sixth embodiment, the isolated antibody or antigen-binding fragment is an scFv, Fab, Fab', or F(ab')2 fragment (see Example 6).

[0320] The antibody according to this embodiment can be conjugated, for example, as discussed elsewhere in this specification. The antibody according to this embodiment can be used to treat tumors or diseases characterized by the involvement of cells expressing a seven-transmembrane receptor, for example, as discussed elsewhere in this specification. The antibody according to this embodiment can be used as a diagnostic agent in vivo or in vitro, for example, as discussed elsewhere in this specification. Furthermore, a kit containing the antibody according to this embodiment with instructions for use is provided.

[0321] Preferred combination according to the sixth aspect The following embodiments disclose preferred combinations of the sixth embodiment, thereby highlighting the modularity of the present invention. According to Preferred Embodiment I, an isolated antibody or antigen-binding fragment thereof is provided that specifically binds to a first isolated sulfated polypeptide which may include the tyrosine-rich domain (TRD) and its LID domain of a seven-transmembrane receptor, wherein at least 25%, at least 50%, or at least 75% of the tyrosine residues of the TRD are sulfated. According to Preferred Embodiment II, an isolated antibody or antigen-binding fragment according to Preferred Embodiment I is provided, wherein the cysteine ​​between the TRD domain and the LID domain is removed or replaced with a different amino acid. According to Preferred Embodiment III, an isolated antibody or antigen-binding fragment according to Preferred Embodiment I or II is provided, wherein the seven-transmembrane receptor is a human, cynomolgus monkey, or mouse seven-transmembrane receptor. According to Preferred Embodiment IV, an isolated antibody or antigen-binding fragment according to any of Preferred Embodiments I, II, or III is provided, wherein the seven-transmembrane receptor is a) CC chemokine receptor, preferably CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, or CCR10, b) CXC chemokine receptor, preferably CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, or CXCR6, or c) CX3CR1 or CXCR1. According to Preferred Embodiment V, an isolated antibody or antigen-binding fragment according to any of Preferred Embodiments I, II, III, or IV is provided, wherein the first isolated sulfated polypeptide comprises or consists of the following sequence: a. Preferably, at least Y10 and / or Y18 are sulfated, such as SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 3 or SEQ ID NO: 6, b. Preferably, at least Y26 is sulfated, such as SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 8 or SEQ ID NO: 11, c. Preferably, at least Y37 and / or Y39 are sulfated, such as in SEQ ID NO: 9 or SEQ ID NO: 12, or d. Preferably Y16 and / or Y17 are sulfated, such as SEQ ID NO: 13 or SEQ ID NO: 16, e. Preferably Y16 is sulfated, SEQ ID NO: 14 or SEQ ID NO: 17, or f. Preferably Y20 and / or Y22 is sulfated, SEQ ID NO: 15 or SEQ ID NO: 18, or g. Preferably at least Y22 is sulfated, and preferably Y16, Y19 and / or Y20 is further sulfated, such as SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 21 or SEQ ID NO: 24, h. Preferably, two, three or all of Y3, Y10, Y14 and Y15 are sulfated, such as SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 26 or SEQ ID NO: 29, i. Preferably, two or three of Y10, Y12, and Y16 are sulfated, such as SEQ ID NO: 27 or SEQ ID NO: 30. or j. Preferably, at least two or three of Y18, Y26, and Y27 are sulfated, such as SEQ ID NO: 31 or SEQ ID NO: 34, or k. Preferably, at least two or three of Y23, Y31, and Y32 are sulfated, such as SEQ ID NO: 32 or SEQ ID NO: 35, or l. Preferably, at least two or three of Y13, Y18 and Y19 are sulfated, such as SEQ ID NO: 33 or SEQ ID NO: 36, or m. Preferably, one or both of Y8 and Y17 are sulfated, such as SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 38 or SEQ ID NO: 41, n. Preferably one or both of Y8 and Y17, and Y20 may be sulfated, as shown in SEQ ID NO: 39 or SEQ ID NO: 42 or o. Preferably, at least two or all of Y3, Y15 and Y17 are sulfated, such as SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46 or SEQ ID NO: 47, p. Preferably, at least two or all of Y3, Y14, and Y15 are sulfated, such as SEQ ID NO: 45 or SEQ ID NO: 48, or q. Preferably at least Y28, preferably Y17 and / or Y37 are also sulfated, such as SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 62 or SEQ ID NO: 65, r. Preferably, at least Y28 is sulfated, and preferably Y19 is also sulfated, such as SEQ ID NO: 63 or SEQ ID NO: 66, or s. Preferably, at least one or both of Y14 and Y22 are sulfated, such as SEQ ID NO: 67, SEQ ID NO: 70, SEQ ID NO: 68 or SEQ ID NO: 71, t. Preferably, at least one, two, or all of Y14, Y17, and Y22 are sulfated, such as SEQ ID NO: 69 or SEQ ID NO: 72, or u. Preferably Y27 is sulfated, as in SEQ ID NO: 73 or SEQ ID NO: 76, or v. Preferably at least one of Y14 and Y28 is sulfated, as in SEQ ID NO: 74 or SEQ ID NO: 77, or w. Preferably, at least Y6 is sulfated, such as SEQ ID NO: 75 or SEQ ID NO: 78, or x. Preferably Y23 and / or Y25 are sulfated, such as SEQ ID NO: 79 or SEQ ID NO: 82, or y. Preferably Y20 and / or Y22 are sulfated, such as SEQ ID NO: 80 or SEQ ID NO: 83, z. Preferably Y24 is sulfated, SEQ ID NO: 81 or SEQ ID NO: 84, or aa. Preferably at least one or both of Y27 and Y29 are sulfated, SEQ ID NO: 85, SEQ ID NO: 88, SEQ ID NO: 86, SEQ ID NO: 89, SEQ ID NO: 87 or SEQ ID NO: 90, or bb. Preferably, at least Y12 and / or Y21 are sulfated, such as SEQ ID NO: 91, SEQ ID NO: 94, SEQ ID NO: 92 or SEQ ID NO: 95, cc. Preferably, at least Y23 and / or Y14 are sulfated, such as SEQ ID NO: 93 or SEQ ID NO: 96, or dd. Preferably, at least one of Y3 and Y27 is sulfated, such as SEQ ID NO: 97, SEQ ID NO: 100, SEQ ID NO: 98 or SEQ ID NO: 101, ee. Preferably, at least Y3 and / or Y14 and / or Y20 and / or Y26 are sulfated, such as SEQ ID NO: 99 or SEQ ID NO: 102, ff. Preferably, at least one or both of Y6 and Y10 are sulfated, such as SEQ ID NO: 103 or SEQ ID NO: 106, Preferably, at least two or all of Y4, Y7 and Y39 are sulfated, such as SEQ ID NO: 104 or SEQ ID NO: 107, or hh. Preferably, at least one or both of Y11 and Y15 are sulfated, such as SEQ ID NO: 105 or SEQ ID NO: 108, ii. Preferably, at least Y14 is sulfated, such as SEQ ID NO: 157 or SEQ ID NO: 160, or Preferably, at least Y20 is sulfated in SEQ ID NO: 158, or kk. Preferably, at least Y20 or Y22 is sulfated, SEQ ID NO: 161 or ll. Preferably, at least Y15 is sulfated, such as SEQ ID NO: 159 or SEQ ID NO: 162, or mm. Preferably, at least Y27 is sulfated, such as SEQ ID NO: 163 or SEQ ID NO: 166, or Preferably, SEQ ID NO: 164, in which at least Y14 is sulfated, or ○○. Preferably, at least Y14 or Y28 is sulfated, such as SEQ ID NO: 167 or pp. Preferably, at least Y6 is sulfated, such as SEQ ID NO: 165 or SEQ ID NO: 168.

[0322] According to Preferred Embodiment VI, an isolated antibody or antigen-binding fragment according to any of Preferred Embodiments I, II, III, IV, or V is provided, wherein the dissociation constant or EC50 of the antibody for binding to a first isolated sulfated polypeptide and / or for the seven-transmembrane receptor is less than 150 nM, 100 nM, 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5, or 0.25 nM. According to Preferred Embodiment VII, an isolated antibody or antigen-binding fragment according to any of Preferred Embodiments I to VI is provided, wherein the isolated antibody or antigen-binding fragment specifically binds to a second isolated sulfated polypeptide containing the TRD of the seven-transmembrane receptor, preferably the seven-transmembrane receptor of the TRD contained in the second isolated sulfated polypeptide, a. Different from the seven-transmembrane receptor of TRD contained in the first isolated sulfated polypeptide, or b. The first isolated sulfated polypeptide contains the corresponding seven-transmembrane receptor for TRDs derived from different species.

[0323] According to Preferred Embodiment VIII, an isolated antibody or antigen-binding fragment according to Preferred Embodiment VII is provided, wherein the dissociation constant or EC50 of the antibody for binding to a second isolated sulfated polypeptide and / or to a second seven-transmembrane receptor is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5, or 0.25 nM. According to Preferred Embodiment IX, an isolated antibody or antigen-binding fragment according to any of Preferred Embodiments I to VIII is provided, wherein the dissociation constant (KD) of the antibody for binding to a first isolated sulfated polypeptide is lower than the dissociation constant (KD) of the antibody for binding to a first isolated non-sulfated polypeptide having the same sequence as the first isolated sulfated polypeptide. According to Preferred Embodiment X, an isolated antibody or antigen-binding fragment according to Preferred Embodiment IX is provided, wherein the dissociation constant and / or EC50 of the antibody for binding to a first isolated non-sulfated polypeptide is greater than 150 nM, 250 nM, 500 nM, 1 μM, 2 μM, or 3 μM, or is undetectable. According to Preferred Embodiment XI, an isolated antibody or antigen-binding fragment according to Preferred Embodiment IX or X is provided, wherein the dissociation constant or EC50 of the antibody or fragment for binding to a first isolated sulfated polypeptide is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM, or 0.25 nM, and the dissociation constant of the antibody or fragment for binding to a first isolated non-sulfated polypeptide is higher than 10 nM, 25 nM, 50 nM, 100 nM, 250 nM, or 500 nM, or undetectable. According to Preferred Embodiment XII, an isolated antibody or antigen-binding fragment according to any of Preferred Embodiments I to XI is provided, wherein the antibody comprises a CDR derived from human, rat, or mouse. According to Preferred Embodiment XIII, an isolated antibody or antigen-binding fragment according to any of Preferred Embodiments I to XII is provided, wherein the antibody is a. Contains human-derived CDRs and / or b. Cross-reactive to humans and cynomolgus monkeys, and / or c. Characterized by an HCDR3 region containing 10-34% tyrosine and / or 2-20% histidine, and / or d. Does not regulate G protein-independent signaling of 7-transmembrane receptors, and / or e. Characterized by non-internalized antibodies or internalization into cells having endogenous target expression lower than 1.5, 2, 3, 4, 5, 6, 7, or 10 times that of the isotype control, and / or f. Induce ADCC and / or ADCP, g. Human, rat, or mouse IgG antibody, preferably human IgG1 antibody or mouse IgG2a antibody, and / or This is a fragment of h.scFv, Fab, Fab', or F(ab')2.

[0324] According to preferred embodiment XIV, a conjugate comprising an antibody or antigen-binding fragment according to any of preferred embodiments I to XIII is provided, preferably the conjugate comprising a. a radioactive element, b. Cytotoxic agents such as auristatin, maytansinoids, kinesin spindle protein inhibitors, nicotinamide phosphoribosyltransferase inhibitors, or pyrrolobenzodiazepine derivatives. c. Further antibody or antigen-binding fragments, or d. Includes chimeric antigen receptors.

[0325] According to Preferred Embodiment XV, an antibody or antigen-binding fragment according to any of Preferred Embodiments I to XIII, or a conjugate according to Preferred Embodiment XIV, is provided for use in treating tumors or diseases characterized by the involvement of cells expressing a seven-transmembrane receptor, which may be combined with an antibody targeting a checkpoint inhibitor. According to Preferred Embodiment XVI, an antibody or antigen-binding fragment according to any of Preferred Embodiments I to XIII, or a conjugate according to Preferred Embodiment XIV, is provided for use as a diagnostic agent in vivo or in vitro. According to Preferred Embodiment XVII, a kit is provided comprising an antibody or antigen-binding fragment according to any of Preferred Embodiments I to XIII, or a conjugate according to Preferred Embodiment XIV, along with instructions for use.

[0326] Antibody that binds CCR8 The following embodiments 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18 relate to antibodies that specifically bind to CCR8. Although each embodiment is described individually, different structural or functional embodiments can be combined and are proposed to be combined with each other, unless they are obviously incompatible. Furthermore, each embodiment of each embodiment can be combined with each embodiment of the same or different embodiments, unless they are obviously incompatible.

[0327] Unless otherwise specified, CCR8 may be derived from any species, such as humans, monkeys, cynomolgus monkeys (macaca fascicularis), rhesus macaques (macaca mulatta), rodents, mice, rats, horses, cattle, pigs, dogs, cats, and camels. Antibodies that bind to CCR8 derived from at least two species (one of which is human) are very preferred and are suggested to be combined with the embodiments disclosed for these aspects. Antibodies having human-derived CDRs are very preferred and are suggested to be combined with the embodiments disclosed for these aspects. According to this aspect, antibodies having an HCDR3 domain characterized by at least 21% tyrosine residues and / or at least 2%, 7%, or 10% histidines are very preferred and are suggested to be combined with the embodiments disclosed for this aspect. According to this embodiment, antibodies that induce both ADCC and ADCP, such as afucosylated antibodies, are very preferred and are suggested to be combined with each embodiment disclosed for these embodiments. According to this embodiment, low-internalized antibodies or non-internalized antibodies or fragments are very preferred and are suggested to be combined with each embodiment disclosed for these embodiments. Several very preferred features applicable to each of the embodiments described are described in the section “Preferred Combinations by ‘All Embodiments’”.

[0328] Aspect 7 - CCR8 antibody-conjugated sulfated TRD The extracellular domain of human CCR8 can be structured into the following four regions: (i) An N-terminal domain, a) Membrane distal tyrosine-rich domain (TRD) formed by amino acids 1-24 (SEQ ID NO: 43) b) Cysteine ​​at amino acid position 25, and c) LID domain formed by amino acids 26-35 (SEQ ID NO: 49) (iii) Extracellular domain 1 (ECL1) as described in Sequence ID No. 52, (iii) Extracellular domain 2 (ECL2) as described in Sequence ID No. 55, and (iv) Extracellular domain 3 (ECL3) as described in Sequence ID No. 58.

[0329] According to a seventh embodiment, which may or may not be the same as the sixth embodiment, an isolated antibody or an antigen-binding fragment thereof that specifically binds to the tyrosine sulfate-rich domain of CCR8 is provided.

[0330] For example, an antibody or antigen-binding fragment is bound to the first isolated sulfated polypeptide, which is, a) The tyrosine-rich domain (TRD) of CCR8, or b) Containing the N-terminus of CCR8 including TRD, At least the cysteine ​​between the TRD domain and the LID domain may be removed or replaced with a different amino acid. The sulfated polypeptide according to this embodiment is preferably a polypeptide in which at least 25%, at least 50%, or at least 75% of the tyrosine residues of TRD are sulfated. If the polypeptide contains the N-terminus of CCR8 containing TRD, preferably the cysteine ​​between TRD and the LID domain is replaced with serine or removed.

[0331] While not bound by theory, the specific recognition of the sulfated pattern provided for CCR8 appears to influence when the antibody competes with CCL1, the natural ligand for CCR8, and when and how the antibody agonizes or antagonizes CCR8 signaling, as described, for example, in the eleventh aspect.

[0332] In some preferred embodiments according to this model, the antibodies of the present invention are <5E-8M, <4E-8M, <3E-8M, <2E-8M, <1E-8M, <9E-9M, <8E-9M, <7E-9M, <6E-9M, <5E-9M, <4E-9M, <3E-9M, <2.5E-9M, <2E-9M, <1.5E-9M, < The antibodies bind to the sulfated TRD of human and / or cynomolgus monkey CCR8 with KD values ​​of 1E-9M, <9E-10M, <8E-10M, <7E-10M, <6E-10M, <5E-10M, <4E-10M, <3E-10M, <2.5E-10M, <2E-10M, <1.5E-10M, <1E-10M, or <9E-11M. For example, the antibodies of the present invention can bind to the sulfated TRD of human and / or cynomolgus monkey CCR8 with KD values ​​of <8E-9M to >4E-10M. For example, the antibodies of the present invention can bind to the sulfated TRD of human and / or cynomolgus monkey CCR8 with KD values ​​of <8E-9M to >5.5E-10M. In some more preferred embodiments of these embodiments, the antibody of the present invention binds to the sulfated N-terminus (i.e., the aforementioned sulfated TRD) of human and / or cynomolgus monkey CCR8 with substantially the same KD value. In some of the most preferred embodiments of these embodiments, the antibody of the present invention does not substantially bind to the unsulfated TRD of human and / or cynomolgus monkey CCR8.

[0333] In some preferred embodiments according to this model, the antibody of the present invention has the sulfated N-terminus of human and / or cynomolgus monkey CCR8 modified to <5E-8M, <4E-8M, <3E-8M, <2E-8M, <1E-8M, <9E-9M, <8E-9M, <7E-9M, <6E-9M, <5E-9M, <4E-9M, <3E-9M, <2.5E-9M, <2E-9M, <1.5E-9M, <1E-9M, <9E-10M, <8E-10M , binding with KD values ​​of <7E-10M, <6E-10M, <5E-10M, <4E-10M, <3E-10M, <2.5E-10M, <2E-10M, <1.5E-10M, <1E-10M, <9E-11M, <8E-11M, <7E-11M, <6E-11M, or <5E-11M. For example, the antibody of the present invention can bind to the sulfated N-terminus of human and / or cynomolgus monkey CCR8 with a KD value of <8E-9M to >8E-11M. For example, the antibody of the present invention can bind to the sulfated N-terminus of human and / or cynomolgus monkey CCR8 with a KD value of <8E-9M to >7.5E-11M. In some more preferred embodiments of these embodiments, the antibody of the present invention binds to the sulfated TRD (i.e., the aforementioned sulfated N-terminal subsequence) of human and / or cynomolgus monkey CCR8 with substantially the same or similar KD values. In some of the most preferred embodiments of these embodiments, the antibody of the present invention does not substantially bind to the unsulfated TRD of human and / or cynomolgus monkey CCR8.

[0334] According to some first embodiments of the seventh aspect, the first isolated sulfated polypeptide is a) Preferably, at least two or all of Y3, Y15 and Y17 are sulfated, SEQ ID NO: 43 (CCR8_HUMAN_TRD), b) Preferably, at least two or all of Y3, Y15 and Y17 are sulfated, such as SEQ ID NO: 44 (CCR8_MACFA_TRD), and / or c) Preferably containing SEQ ID NO: 45 (CCR8_MOUSE_TRD), in which at least two or all of Y3, Y14, and Y15 are sulfated.

[0335] According to some second embodiments of the seventh aspect, the first isolated sulfated polypeptide is a) Preferably, at least two or all of Y3, Y15 and Y17 are sulfated, SEQ ID NO: 46 (C=X or S, CCR8_HUMAN_N term), b) Preferably, at least two or all of Y3, Y15 and Y17 are sulfated, SEQ ID NO: 47 (C=X or S, CCR8_MACFA_N term), and / or c) Preferably containing SEQ ID NO: 48 (C=X or S, CCR8_MOUSE_N term), in which at least two or all of Y3, Y14, and Y15 are sulfated.

[0336] According to some third embodiments of the seventh embodiment, which may be the same as or different from the first and / or second embodiments, the antibody or antigen-binding fragment specifically binds to the first isolated sulfated polypeptide with a dissociation constant or EC50 of <15 nM, <10 nM, <5 nM, <1 nM, or <0.6 nM.

[0337] According to some preferred embodiments of these embodiments, the isolated antibody or its antigen-binding fragment has a dissociation constant or EC50 of <15 nM, <10 nM, <5 nM, <1 nM, or <0.6 nM. a) A human CCR8 or isolated polypeptide described in SEQ ID NO: 46, wherein at least two or all of Y3, Y15, and Y17 are sulfated. b) Polypeptides isolated by cynomolgus monkey CCR8 or SEQ ID NO: 47, in which at least two or all of Y3, Y15, and Y17 are sulfated, and / or c) Specifically binds to the isolated polypeptide described in mouse CCR8 or SEQ ID NO: 48, in which at least two or all of Y3,...

Claims

1. Isolated anti-CCR8 antibody or its antigen-binding fragment, a. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, sequence numbers 420, 421, 422, 424, 425, and 426, b. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, sequence numbers 474, 475, 476, 478, 479, and 480, c. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, sequence numbers 492, 493, 494, 496, 497, and 498, d. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, SEQ ID NOs: 510, 511, 512, 514, 515, and 516, e. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, sequence numbers 600, 601, 602, 604, 605, and 606, f. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, SEQ ID NOs: 618, 619, 620, 622, 623, and 624, g. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, SEQ ID NOs: 661, 662, 663, 665, 666, and 667, h. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, SEQ ID NOs: 681, 682, 683, 685, 686, and 687, i. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, SEQ ID NOs: 703, 704, 705, 707, 708, and 709, j. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, SEQ ID NOs: 723, 724, 725, 727, 728, and 729, k. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, sequence numbers 743, 744, 745, 747, 748, and 749, l. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, SEQ ID NOs: 763, 764, 765, 767, 768, and 769, m. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, sequence numbers 783, 784, 785, 787, 788, and 789, n. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, SEQ ID NOs: 803, 804, 805, 807, 808, and 809, o. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, SEQ ID NOs: 827, 828, 829, 831, 832, and 833, p. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, SEQ ID NOs: 847, 848, 849, 851, 852, and 853, q. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, SEQ ID NOs: 867, 868, 869, 871, 872, and 873, r. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, SEQ ID NOs: 887, 888, 889, 891, 892, and 893, s. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, sequence numbers 907, 908, 909, 911, 912, and 913, t. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, sequence numbers 927, 928, 929, 931, 932, and 933, or u. Represented as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, sequence numbers 947, 948, 949, 951, 952, and 953 An isolated anti-CCR8 antibody or its antigen-binding fragment containing the sequence.

2. An isolated anti-CCR8 antibody or its antigen-binding fragment according to Claim 1, a. Variable heavy chain sequence described in Sequence ID No. 419 and variable light chain sequence described in Sequence ID No. 423, b. Variable heavy chain sequence described in Sequence ID No. 473 and variable light chain sequence described in Sequence ID No. 477, c. Variable heavy chain sequence described in Sequence ID No. 491 and variable light chain sequence described in Sequence ID No. 495, d. Variable heavy chain sequence described in Sequence ID No. 509 and variable light chain sequence described in Sequence ID No. 513, e. Variable heavy chain sequence described in Sequence ID No. 599 and variable light chain sequence described in Sequence ID No. 603, f. Variable heavy chain sequence described in Sequence ID No. 617 and variable light chain sequence described in Sequence ID No. 621, g. Variable heavy chain sequence described in Sequence ID No. 660 and variable light chain sequence described in Sequence ID No. 664, h. Variable heavy chain sequence described in Sequence ID No. 680 and variable light chain sequence described in Sequence ID No. 684, i. Variable heavy chain sequence described in Sequence ID No. 702 and variable light chain sequence described in Sequence ID No. 706, j. Variable heavy chain sequence described in Sequence ID No. 722 and variable light chain sequence described in Sequence ID No. 726, k. Variable heavy chain sequence described in Sequence ID No. 742 and variable light chain sequence described in Sequence ID No. 746, l. Variable heavy chain sequence described in Sequence ID No. 762 and variable light chain sequence described in Sequence ID No. 766, m. Variable heavy chain sequence described in Sequence ID No. 782 and variable light chain sequence described in Sequence ID No. 786, n. Variable heavy chain sequence described in Sequence ID No. 802 and variable light chain sequence described in Sequence ID No. 806, o. Variable heavy chain sequence described in Sequence ID No. 826 and variable light chain sequence described in Sequence ID No. 830, p. Variable heavy chain sequence described in Sequence ID No. 846 and variable light chain sequence described in Sequence ID No. 850, q. Variable heavy chain sequence described in Sequence ID No. 866 and variable light chain sequence described in Sequence ID No. 870, r. Variable heavy chain sequence described in Sequence ID No. 886 and variable light chain sequence described in Sequence ID No. 890, s. Variable heavy chain sequence described in Sequence ID No. 906 and variable light chain sequence described in Sequence ID No. 910, t. Variable heavy chain sequence described in Sequence ID No. 926 and variable light chain sequence described in Sequence ID No. 930, or u. Variable heavy chain sequence described in Sequence ID No. 946 and variable light chain sequence described in Sequence ID No. 950 An isolated anti-CCR8 antibody or its antigen-binding fragment comprising a variable heavy chain sequence and a variable light chain sequence having at least 95%, 98%, or 100% sequence identity.

3. An isolated anti-CCR8 antibody or its antigen-binding fragment according to Claim 1, a. The heavy chain described in Sequence ID No. 435 and the light chain described in Sequence ID No. 436, b. The heavy chain described in Sequence ID No. 489 and the light chain described in Sequence ID No. 490, c. The heavy chain described in Sequence ID No. 507 and the light chain described in Sequence ID No. 508, d. The heavy chain described in Sequence ID No. 525 and the light chain described in Sequence ID No. 526, e. The heavy chain described in Sequence ID No. 615 and the light chain described in Sequence ID No. 616, f. The heavy chain described in Sequence ID No. 633 and the light chain described in Sequence ID No. 634, g. The heavy chain described in Sequence ID No. 676 and the light chain described in Sequence ID No. 677, h. The heavy chain described in Sequence ID No. 696 and the light chain described in Sequence ID No. 697, i. The heavy chain described in Sequence ID No. 718 and the light chain described in Sequence ID No. 719, j. The heavy chain described in Sequence ID No. 738 and the light chain described in Sequence ID No. 739, k. The heavy chain described in Sequence ID No. 758 and the light chain described in Sequence ID No. 759, l. The heavy chain described in Sequence ID No. 778 and the light chain described in Sequence ID No. 779, m. The heavy chain described in Sequence ID No. 798 and the light chain described in Sequence ID No. 799, n. The heavy chain described in Sequence ID No. 818 and the light chain described in Sequence ID No. 819, o. The heavy chain described in Sequence ID No. 842 and the light chain described in Sequence ID No. 843, p. The heavy chain described in Sequence ID No. 862 and the light chain described in Sequence ID No. 863, q. The heavy chain described in Sequence ID No. 882 and the light chain described in Sequence ID No. 883, r. The heavy chain described in Sequence ID No. 902 and the light chain described in Sequence ID No. 903, s. The heavy chain described in Sequence ID No. 922 and the light chain described in Sequence ID No. 923, t. The heavy chain described in Sequence ID No. 942 and the light chain described in Sequence ID No. 943 or u. Heavy chain described in Sequence ID No. 962 and light chain described in Sequence ID No. 963 An isolated anti-CCR8 antibody or its antigen-binding fragment comprising a heavy chain sequence and a light chain sequence having at least 95%, 98%, or 100% sequence identity.

4. The isolated antibody or antigen-binding fragment according to claims 1 to 3, wherein the antibody or antigen-binding fragment is afucosylated.

5. The isolated antibody or antigen-binding fragment thereof according to claims 1 to 4, wherein the antibody induces ADCC and / or ADCP.

6. A conjugate comprising an antibody or antigen-binding fragment according to any one of claims 1 to 5, wherein the conjugate is a. radioactive elements, b. Cytotoxic agents, c. Further antibody or antigen-binding fragments, or d. Chimeric antigen receptor, A conjugate containing an antibody or antigen-binding fragment.

7. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 5 or a conjugate according to claim 6, and one or more further therapeutically active compounds.

8. The aforementioned further therapeutically active compound a. Antibodies or small molecules that target checkpoint proteins, b. Antibodies that target further chemokine receptors, c. Antibodies that target proteins specifically expressed by tumor cells. d. Antibodies or small molecules targeting HER2 and / or EGFR, e. Chemotherapy agents, and / or f. Targeted kinase inhibitors A pharmaceutical composition according to claim 7, selected from the above.

9. A pharmaceutical product comprising an antibody or antigen-binding fragment according to any one of claims 1 to 5, a conjugate according to claim 6, or a pharmaceutical composition according to claim 7 or 8.

10. A pharmaceutical composition for use in the treatment of a tumor or disease characterized by CCR8-positive tumor cells or CCR8-positive regulatory T cells, comprising an antibody or antigen-binding fragment according to any one of claims 1 to 5 or a conjugate according to claim 6, or a pharmaceutical composition according to claim 7 or 8.

11. In the treatment of tumors or diseases characterized by CCR8-positive regulatory T cells, (i) one or more further therapeutic compounds and / or (ii) radiotherapy and / or (iii) Depletion of intratumor B cells, A pharmaceutical composition for use simultaneously, separately, or in sequential combination, comprising an antibody or antigen-binding fragment according to any one of claims 1 to 5 or a conjugate according to claim 6, or a pharmaceutical composition according to claim 7 or 8.

12. The aforementioned further therapeutically active compound a. Antibodies or small molecules that target checkpoint proteins, b. Antibodies that target further chemokine receptors, c. Antibodies that target proteins specifically expressed by tumor cells. d. Antibodies or small molecules targeting HER2 and / or EGFR, e. Chemotherapy agents, and / or f. Targeted kinase inhibitors A pharmaceutical composition for use according to claim 11, selected from the above.

13. A pharmaceutical composition for use according to any one of claims 10 to 12, wherein the tumor is selected from the group of T-cell acute lymphoblastic leukemia, breast cancer, triple-negative breast cancer, triple-positive breast cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), testicular cancer, gastric cancer, head and neck squamous cell carcinoma, thymoma, esophageal adenocarcinoma, colorectal cancer, pancreatic adenocarcinoma, ovarian cancer or cervical cancer, acute myeloid leukemia, kidney cancer, bladder cancer, skin cancer, melanoma, thyroid cancer, mesothelioma, sarcoma and prostate cancer, B-cell lymphoma, T-cell lymphoma, or any other cancers associated with CCR8-expressing cells.

14. A polynucleotide encoding an antibody or antigen-binding fragment according to any one of claims 1 to 5.

15. A vector comprising a polynucleotide as described in claim 14.

16. Isolated cells configured for the production of an antibody or antigen-binding fragment according to any one of claims 1 to 5.

17. A method for producing an antibody or antigen-binding fragment according to any one of claims 1 to 5, or a conjugate according to claim 6, comprising culturing the cells according to claim 16 and purifying the antibody or antigen-binding fragment.

18. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 5, or a conjugate according to claim 6, for use as a diagnostic agent in vivo.

19. A kit comprising, together with instructions for use, an antibody or antigen-binding fragment according to any one of claims 1 to 5, a conjugate according to claim 6, or a pharmaceutical composition according to claim 7.

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