Anti-TIRC7 antigen-binding protein

Abstract

The present invention provides novel human T-cell immune response cDNA7 (TIRC7) antigen-binding proteins, e.g., antibodies with improved binding affinity and / or activity to TIRC7. The TIRC7 antibodies of the present invention were generated by mutation of a parent TIRC7 antibody and tested in various cellular experiments. The present invention also relates to methods for producing the antigen-binding proteins of the present invention, nucleic acids encoding them, and vectors and host cells for expressing them. The present invention further relates to methods for treating or diagnosing diseases, such as autoimmune diseases, cancer diseases, or other immune-related diseases, e.g., diseases involving a cellular immune response, using the anti-TIRC7 antigen-binding proteins (ABPs) of the present invention.

Description

Technical Field

[0001] The present invention provides novel human T cell immune response cDNA7 (TIRC7) antigen-binding proteins, such as antibodies, with improved binding affinity and / or activity to TIRC7. The TIRC7 antibodies of the present invention were generated by mutation of parental TIRC7 antibodies and tested in various cell experiments. The present invention also relates to methods for generating the antigen-binding proteins of the present invention, nucleic acids encoding them, and vectors and host cells for expressing them. The present invention further relates to methods for treating or diagnosing diseases, such as autoimmune diseases, cancer diseases or other immune-related diseases, for example, diseases involving cellular immune responses, using the anti-TIRC7 antigen-binding proteins (ABPs) of the present invention.

Background Art

[0002] T cell immune response cDNA7 (TIRC7), a membrane protein, was identified by differential display in human peripheral blood lymphocytes activated by allogeneic antigen and was transiently upregulated in the early stage of its activation process [1, 2, 3, 4]. Since anti-TIRC7 antibodies have been shown to prevent organ rejection and prolong the survival time of kidney allografts [1], it has been suggested that TIRC7 could be a novel target for suppressing unwanted (cellular) immune responses in the context of organ transplantation and other autoimmune diseases.

[0003] The TIRC7 gene is composed of 15 exons, with a full length of 7.9 kb, and is localized on chromosome 11q13.4 to q13.5 [5]. No amino acid homology has been associated with any known protein involved in T cell activation. TIRC7 contains an immunoreceptor tyrosine-based inhibitory motif, which may indicate an inhibitory role in the immune system [6]. Most of the currently identified costimulatory molecules expressed in lymphocytes belong to the immunoglobulin superfamily or the TNF receptor superfamily. Therefore, the predicted multiple transmembrane structure and the unconventional extracellular C-terminus of TIRC7 are considered unique.

[0004] TIRC7 is present in the cell membrane and within the intracellular compartment of subsets of unstimulated T and B lymphocytes (5 - 10% and 10 - 40% respectively)

[14] , as well as monocytes (30 - 90%). After activation of T cells, the cell surface expression of TIRC7 increases within 30 minutes and reaches its highest level within 2 hours

[15] . Before cell activation, the expression of TIRC7 is approximately 100-fold higher in the intracellular compartment compared to the cell surface of lymphocytes. After cell stimulation, simultaneously with the maximum protein expression on the cell surface, the intracellular level of TIRC7 decreases, and the expression in both the intracellular and extracellular compartments returns to the control level within 6 hours. These results indicate that TIRC7 exists in the intracellular compartment as a pre-formed protein, translocates to the cell surface when T cells are activated, and then returns to the cytoplasmic storage site. Clathrin-coated vesicles have been identified as potential vehicles for TIRC7 transport

[15] . Similar to other T cell activation-related molecules localized at the immunological synapse [16 - 20], TIRC7 concentrates at the activated protein C binding site upon antigen activation, and the directed movement of intracellular TIRC7 towards the antigen binding site occurs in parallel with the reorientation of TIRC7 on the cell surface

[15] . These data suggest that TIRC7 exists in the intracellular compartment in a pre-formed state and moves to the cell surface within minutes of immune activation, implicating it in the initial signaling events that control further downstream signals.

[0005] Along with the early expression at the cell membrane upon activation, the distribution pattern of TIRC7 among lymphocyte subsets is different from that of other costimulatory molecules used in therapeutic interventions such as CD40L, CD3, CD4 or cytotoxic T lymphocyte antigen (CTLA)-4. These proteins are expressed exclusively and constitutively either on T cells (CD3, CD4, CD40L) or B cells (CD20, CD40), or are found on the cell surface 24 hours after immune activation (CTLA-4)

[20] . Thus, TIRC7 may be the first negative regulatory signal provided to immune cells after activation. Anti-TIRC7 antibodies rapidly induced CTLA-4 on the surface of T cells, showed significant inhibition of the proliferation of human peripheral blood lymphocytes stimulated with mitogens or allogeneic antigens, and caused potent inhibition of IFNγ, IL6, IL12 and IL-2 (corresponding to T helper cell type 1 [Th1] and [Th17] cytokines) following tbet in a dose-dependent manner (Frischer et al. 2016), whereas suppression of IL-4 or IL-10 was not observed. Thus, targeting of TIRC7 antibodies results in selective inhibition of inflammatory cytokines, as well as t-bet as a major transcription factor controlling Th1 and Th17. When recombinant IL-2 was added to the culture, the inhibition of proliferation was reversed. Therefore, targeting of TIRC7 brought about anergy when cells responded to restimulation. However, since IFNγ expression only partially disappeared, it was shown that TIRC7 mAb could induce stable and distinct cytokine inhibition.

[0006] Data generated from studies using agonistic anti-TIRC7 antibodies have assigned a negative regulatory role for TIRC7 in lymphocyte function, due to its anti-proliferative and anti-inflammatory effects on TIRC7

[14] . This antibody was shown to specifically decrease the proliferation of CD4+ cells. The IC50 value of the agonistic anti-TIRC7 mAb was significantly decreased by crosslinking the mAb prior to incubation with lymphocytes. Furthermore, the anti-TIRC7 mAb exhibited agonist properties, as it lost its negative signaling properties when the Fab fragment was used in the proliferation assay. Additionally, the hypothesis of a negative regulatory role for TIRC7 in lymphocyte function was supported by the observation that TIRC7-deficient cells showed significantly hyperproliferative T and B cell responses to various antigens

[23] . More importantly, the population of effector memory T cells was significantly increased in TIRC7 knockout mice, while CTLA-4 expression was decreased. Thus, TIRC7 acts as a checkpoint inhibitor that induces negative signals via its binding to HLA-DR alpha2, its ligand, on the cell surface upstream of other known checkpoint inhibitors.

[0007] Millions of people worldwide suffer from autoimmune diseases. Over 80 autoimmune diseases are known, including multiple sclerosis, rheumatoid arthritis, psoriasis, lupus, Crohn's disease, and type I diabetes, among many others. Multiple sclerosis (MS) affects approximately 1 in 1000 people in Western populations and is thought to result from the expansion of autoreactive T cells against myelin proteins such as myelin basic protein (MBP), proteolipid protein (PLP), or myelin oligodendrocyte glycoprotein (MOG) (Stern et al. 2008 Proc. Natl. Acad. Sci. USA 105:5172 - 5176). Cells that play a role in the regulation of the immune response include regulatory T cells. Several autoimmune diseases, including both multiple sclerosis and type 1 diabetes, are associated with a deficiency in the number or function of regulatory T cells (Astier, A.L. and Hafler, D.A. 2007, J. Neuroimmunol. 191(1 - 2):70 - 78), so proteins expressed on T cells are interesting therapeutic targets for various immunological disorders.

[0008] Based on the above, there is a need in the art for therapeutic antibodies that specifically address immune diseases associated with cellular immunity, such as preferably T cell - mediated immune responses, as well as B cell - mediated and monocyte - mediated immune responses. Thus, there is a need for improved therapeutic ABP that addresses at least one of the problems outlined above, and thus a humanized TIRC7 protein that targets ABP. To date, the provision of humanized variants of anti - TIRC7 antibodies has not been addressed and is needed. SUMMARY OF THE INVENTION

[0009] Generally, and as a simple explanation, the main aspects of the present invention can be described as follows.

[0010] In a first aspect, the present invention relates to an antigen - binding protein (ABP) capable of binding to human T cell immune response cDNA7 (TIRC7), and this antigen - binding protein (i) comprising the CDRH1 (GYTFTTYV) of SEQ ID NO: 01, the CDRH2 (INPYNDGT) of SEQ ID NO: 02, and the CDRH3 (AEFITKTVGGSNWYLDV) of SEQ ID NO: 03, or, independently in each case, having a CDRH1, CDRH2, and / or CDRH3 that each contain a sequence having 3 or fewer, preferably 1 or fewer, amino acid substitutions, deletions, or insertions compared to SEQ ID NO: 01, SEQ ID NO: 02, or SEQ ID NO: 03, one, preferably two, heavy chain variable regions, and (ii) comprising the CDRL1 (SSISY) of SEQ ID NO: 05, the CDRL2 (DTS) of SEQ ID NO: 06, and the CDRL3 (HQRSSYTWT) of SEQ ID NO: 07, or, independently in each case, having a CDRL1, CDRL2, and / or CDRL3 that each contain a sequence having 3 or fewer, preferably 1 or fewer, amino acid substitutions, deletions, or insertions compared to SEQ ID NO: 05, SEQ ID NO: 06, or SEQ ID NO: 07, one, preferably two, light chain variable regions comprising (i) each of its one, preferably two, heavy chain variable regions and its one, preferably two, light chain variable regions comprises an antibody framework region having at least a portion of a human antibody consensus framework sequence, and / or (ii) the ABP of the first aspect is characterized in that its heavy chain variable domain comprises a sequence having 10 or fewer amino acid substitutions, insertions, or deletions compared to the sequence shown in SEQ ID NO: 29

[0011] In a second aspect, the present invention relates to an antigen-binding protein (ABP) or an antigen-binding fragment thereof that can bind to TIRC7 and can compete with the binding of the ABP of the first aspect to TIRC7.

[0012] In a third aspect, the present invention relates to an isolated nucleic acid comprising a sequence encoding the ABP or an antigen-binding fragment or monomer (e.g., heavy chain or light chain) of the ABP of the first or second aspect, or a sequence encoding the bispecific ABP described in the third aspect.

[0013] In a fourth aspect, the invention relates to a nucleic acid construct (NAC) comprising a nucleic acid of the fourth aspect and one or more additional sequence features enabling the expression in a cell of an antigen-binding protein (ABP) encoded thereby or a component of said ABP (e.g., a heavy or light chain of an antibody).

[0014] In a fifth aspect, the invention relates to a recombinant host cell comprising a nucleic acid of the third aspect or a nucleic acid construct (NAC) as described in the second aspect.

[0015] In a sixth aspect, the invention relates to a pharmaceutical composition comprising (i) an antigen-binding protein (ABP) of the first or second aspect, or (ii) a nucleic acid of the third aspect or an NAC of the fourth aspect, or (iii) a recombinant host cell as described in the fifth aspect, and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

[0016] In a seventh aspect, the invention relates to a component for use in a medicament, said component being selected from the list consisting of (i) an antigen-binding protein (ABP) of the first or second aspect, or (ii) a nucleic acid of the third aspect or an NAC of the fourth aspect, or (iii) a recombinant host cell as described in the fifth aspect and a pharmaceutical composition as described in the sixth aspect.

[0017] In a ninth aspect, the invention relates to a method of modulating a cellular immune response in a human cell expressing human TIRC7, said method comprising contacting said cell with a component as described in the seventh aspect in the presence of immune cells such as T cells, thereby modulating, preferably inhibiting, the cellular immune response.

[0018] In a tenth aspect, the invention relates to a method for preventing and / or treating a disorder associated with a pathological immune response in a subject, said method comprising administering to the subject a therapeutically effective amount of a component as described in the seventh aspect, wherein the disorder associated with the pathological immune response is characterized by the expression of TIRC7 in cells associated with the disorder.

DETAILED DESCRIPTION OF THE INVENTION

[0019] In the following, the elements of the present invention will be described. Although these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to form further embodiments. The various examples and preferred embodiments described should not be construed as limiting the invention to only the explicitly described embodiments. This description is to be understood as supporting and encompassing embodiments that combine two or more of the explicitly described embodiments, or embodiments that combine one or more of the explicitly described embodiments with any number of disclosed and / or preferred elements. Further, unless the context indicates otherwise, any permutation and combination of all the elements described in this application should be considered to be disclosed by the description of this application.

[0020] Compound In a first aspect, the present invention provides an antigen-binding protein (ABP) that can bind to human T cell immune response cDNA7 (TIRC7), comprising CDRH1 (GYTFTTYV) of SEQ ID NO: 01, CDRH2 (INPYNDGT) of SEQ ID NO: 02, and CDRH3 (AEFITKTVGGSNWYLDV) of SEQ ID NO: 03, or, independently in each case, having a CDRH1, CDRH2, and / or CDRH3 that each contain a sequence having 3 or fewer, preferably 1 or fewer, amino acid substitutions, deletions, or insertions compared to SEQ ID NO: 01, SEQ ID NO: 02, or SEQ ID NO: 03, respectively, and one, preferably two, heavy chain variable regions, and CDRL1 (SSISY) of SEQ ID NO: 05, CDRL2 (DTS) of SEQ ID NO: 06, and CDRL3 (HQRSSYTWT) of SEQ ID NO: 07, or, independently in each case, having a CDRL1, CDRL2, and / or CDRL3 that each contain a sequence having 3 or fewer, preferably 1 or fewer, amino acid substitutions, deletions, or insertions compared to SEQ ID NO: 05, SEQ ID NO: 06, or SEQ ID NO: 07, respectively, and one, preferably two, light chain variable regions. Preferably, (i) the ABP of the present invention may be characterized in that its one, preferably two, heavy chain variable regions and its one, preferably two, light chain variable regions each contain an antibody framework region having at least a part of a human antibody consensus framework sequence, and / or (ii) the ABP of the first aspect is characterized in that the heavy chain variable domain contains a sequence having 10 or fewer amino acid substitutions, insertions, or deletions compared to the sequence shown in SEQ ID NO: 29.

[0021] The present invention also provides a novel and effective antibody construct with high affinity derived from a humanized version of an anti-TIRC antibody (mouse anti-TIRC7 antibody according to the sequence of the cAb1457 antibody disclosed in Table 1 below), which was first humanized herein by CDR grafting, which means inserting the CDR regions of a murine parental antibody into the framework regions of the heavy and light chains of a human antibody. However, in order to obtain the ABP of the present invention, the humanized antibody had to be significantly altered in the sequence of the variable region. To overcome various problems during the humanization process, various changes were required, including non-intuitive human-to-mouse back mutations in the framework 3 region. Most surprisingly, the antibodies of the present invention, although humanized, show an improvement in target binding affinity of more than at least one order of magnitude. In principle, any variable human light chain and / or variable heavy chain can serve as a framework for CDR grafting. In an exemplary example of the humanized antibody of the present invention, the CDRs of the light chain of the antibody (meaning the CDR loops of SEQ ID NO: 5 to SEQ ID NO: 7) can be inserted into the (variable domain) of the human κ light chain sequences IGKV1-9 (accession number Z00013), IGKV3-11 (accession number X01668), IGKV6-21*01 or IGKV1-17*03 deposited in the IMGT / LIGM database. See also Ichiyoshi Y., Zhou M., Casali P. A human anti-insulin IgG autoantibody apparently arises through clonal selection from an insulin-specific ‘germ-line’ natural antibody template. Analysis by V gene segment reassortment and site-directed mutagenesis J. Immunol. 154(1):226-238(1995).In another illustrative example of the humanized antibody of the present invention, the CDRs of the heavy chain of the antibody (meaning the CDR loops of SEQ ID NO: 1 to SEQ ID NO: 3) may be included in the (variable domain) of the heavy chain sequences IGHV1-2 (accession number X07448), IGHV7-4-1*02 or IGHV1-46*01 deposited in the IMGT / LIGM database (see also Watson C.T., et al. Complete haplotype sequence of the human immunoglobulin heavy-chain variable, diversity, and joining genes and characterization of allelic and copy-number variation. Am. J. Hum. Genet. 92(4):530-546(2013)).

[0022] As used herein, the term "antigen-binding protein" or "ABP" means a protein that specifically binds to a target antigen, such as one or more epitopes presented by or present on the target antigen. The antigen of the ABP of the present invention is TIRC7. Usually, the antigen-binding protein is an antibody (or a fragment thereof), however, other forms of antigen-binding proteins are also contemplated by the present invention. For example, the ABP may have a binding function by, for example, using another (non-antibody) receptor protein derived from a small robust non-immunoglobulin "scaffold", such as a method for designing combinatorial proteins (Gebauer & Skerra, 2009; Curr Opin Chem Biol, 13:245).Specific examples of such non-antibody ABP include affibody molecules based on the Z domain of protein A (Nygren, 2008; FEBS J 275:2668), affilins based on crystallizable gamma-B and / or ubiquitin (Ebersbach et al, 2007; J Mo Biol, 372:172), affimers based on cystatin (Johnson et al, 2012; Anal Chem 84:6553), affilins based on Sac7d from Sulfolobus acidcaldarius (Krehenbrink et al, 2008; J Mol Biol 383:1058), alphabodies based on triple-stranded coiled coils (Desmet et al, 2014; Nature Comms 5:5237), anticalins based on lipocalin (Skerra, 2008; FEBS J 275:2677), avimers based on the A domain of various membrane receptors (Silverman et al, 2005; Nat Biotechnol 23:1556), DARPins based on the ankyrin repeat motif (Strumpp et al, 2008; Drug Discov Today, 13:695), finomers based on the SH3 domain of Fyn (Grabulovski et al, 2007; J Biol Chem 282:3196), knotted domain peptides based on the knotted domains of various protease inhibitors (Nixon et al, Curr opin Drug Discov Devel, 9:261), and centyrins and monobodies based on the tenth type III domain of fibronectin (Diem et al., 2014; Protein Eng Des Sel 27:419 doi:10.1093 / protein / gzuo16; Koide & Koide, 2007; Methods Mol Biol 352:95).

[0023] As used herein, the term "complementary determining region" (or "CDR" or "hypervariable region") broadly refers to one or more of the hypervariable or complementary determining regions (CDRs) found in the variable regions of the light or heavy chains of an antibody. See, for example, "IMGT", Lefranc et al, 2003, Dev Comp Immunol 27:55; Honegger & Pluckthun, 2001, J Mol Biol 309:657, Abhinandan & Martin, 2008, Mol Immunol 45:3832, Kabat, et al. (1987): Sequences of Proteins of Immunological Interest National Institutes of Health, Bethesda, Md. These expressions include hypervariable regions as defined by Kabat et al (1983) Sequences of Proteins of Immunological Interest, US Dept of Health and Human Services, or hypervariable loops in the three-dimensional structure of an antibody (Chothia and Lesk, 1987; J Mol Biol 196:901). The CDRs in each chain are proximally held by framework regions and, together with the CDRs of the other chains, contribute to the formation of the antigen-binding site. Within the CDRs are selected amino acids that have been described as selectivity determining regions (SDRs) corresponding to important contact residues used by the CDR in antibody-antigen interactions (Kashmiri, 2005; Methods 36:25).

[0024] As used herein, "ABP" can have one or more domains having a sequence with at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the corresponding naturally occurring domain of immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin D or immunoglobulin E. In this regard, it should be noted that the term "about" or "approximately" as used herein means within 20% deviation of a given value or range, for example, within 10% deviation or within 5%.

[0025] The term "antibody" generally refers to a proteinaceous binding molecule based on immunoglobulin. Typical examples of such antibodies are derivatives or functional fragments of immunoglobulins having binding specificity. Techniques for making antibodies and antibody fragments are well known in the art. The term "antibody" also includes immunoglobulins (Ig) of various classes (i.e., IgA, IgG, IgM, IgD and IgE) and subclasses (e.g., IgG1, IgG2, etc.). As mentioned above, exemplary examples of antibody derivatives or antibody molecules include Fab fragments, F(ab’)2, Fv fragments, single-chain Fv fragments (scFv), diabodies or domain antibodies (Holt LJ et al, Trends Biotechnol. 21(11), 2003, 484-490). Thus, the definition of the term "antibody" also includes embodiments such as chimeric antibodies, single-chain antibodies and humanized antibodies.

[0026] As used herein, "percent (%) sequence identity" means the percentage of paired identical residues after a homology alignment of two sequences, based on the number of residues in the longer of the sequences of a polypeptide of the invention and a sequence of interest. Alignments for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve the maximum alignment over the full length of the sequences being compared. The same applies to the nucleotide sequences disclosed herein.

[0027] As used herein, "immunoglobulin" is generally a glycosylated tetrameric protein composed of two light (L) chains, each approximately 25 kDa, and two heavy (H) chains, each approximately 50 kDa. Two types of light chains, called lambda and kappa, can be found in immunoglobulins. Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to five major classes: A, D, E, G, and M, some of which can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgM immunoglobulins consist of five basic heterotetrameric units in addition to an additional polypeptide called the J chain and contain ten antigen-binding sites. IgA immunoglobulins contain two to five basic four-chain units that can polymerize to form a multivalent aggregate together with the J chain. In the case of IgG, the four-chain unit is generally about 150,000 daltons.

[0028] In the IgG class of immunoglobulins, there are several immunoglobulin domains in the heavy chain. The "immunoglobulin (Ig) domain" in this specification means a region of an immunoglobulin having a different tertiary structure. In the context of IgG antibodies, each IgG isotype has three CH regions. "CH1" refers to positions 118 to 220 according to the EU index as in Kabat et al., "CH2" refers to positions 237 to 340, and "CH3" refers to positions 341 to 447. The "hinge" or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" or "H" in this specification means a flexible polypeptide containing the amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU position 220, and the IgG CH2 domain starts from the residue at EU position 237. Thus, for IgG, the hinge is defined herein as including positions 221 (D221 in IgG1) to 236 (G236 in IgG1), where the numbering follows the EU index as in Kabat et al. The constant heavy chain as defined herein refers to the region from the N-terminus of the CH1 domain to the C-terminus of the CH3 domain, and thus includes positions 118 to 447, where the numbering follows the EU index.

[0029] The term "variable" refers to the portion of the immunoglobulin domain (i.e., the "variable domain") that exhibits diversity in its sequence and is involved in determining the specificity and binding affinity of a particular antibody. The diversity is not uniformly distributed throughout the entire variable domain of the antibody but is concentrated in each of the subdomains of the heavy chain variable region and the light chain variable region. These subdomains are called "hypervariable regions", "HVRs", "HVs", or "complementary determining regions" (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domain are called "framework" regions (FRs). The naturally occurring variable domains of the heavy and light chains each contain four FR regions (usually in a β-sheet conformation) connected by three hypervariable regions, and the three hypervariable regions form loops that connect and in some cases form part of the β-sheet structure. The hypervariable regions in each chain are held in proximity by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site (see Kabat et al., see below). Generally, naturally occurring immunoglobulins contain six CDRs (see below), i.e., three in VH (CDRH1, CDRH2, CDRH3) and three in VL (CDRL1, CDRL2, CDRL3). In naturally occurring immunoglobulins, CDRH3 and CDRL3 exhibit the greatest diversity among the six CDRs, and in particular, CDRH3 is thought to play a unique role in conferring excellent specificity on the immunoglobulin. The constant domains are not directly involved in antigen binding but exhibit various effector functions, such as antibody-dependent cell-mediated cytotoxicity and complement activation.

[0030] The terms "VH" (also referred to as VH) and "VL" (also referred to as VL) are used herein to refer to the heavy chain variable domain and the light chain variable domain of an immunoglobulin, respectively. The variable region of the light or heavy chain of an immunoglobulin consists of "framework" regions interrupted by three hypervariable regions. Thus, the term "hypervariable region" refers to the amino acid residues of an antibody that are involved in antigen binding. The hypervariable regions include the amino acid residues of "complementary determining regions" or "CDRs". There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, as used herein, "CDR" refers to all three heavy chain CDRs (CDRH1, CDRH2, and CDRH3) or all three light chain CDRs (CDRL1, CDRL2, and CDRL3), or, where appropriate, both all heavy chain CDRs and all light chain CDRs. The three CDRs constitute the binding properties of the light chain variable region and three constitute the binding properties of the heavy chain variable region. CDRs determine the antigen specificity of an immunoglobulin molecule and are interrupted by amino acid sequences that include the scaffold or framework regions. The exact boundaries and lengths that define CDRs depend on various classification and numbering systems. The structure and protein folding of an antibody may mean that other residues are considered part of the antigen binding region and will be so understood by those skilled in the art. CDRs provide most of the contact residues for an immunoglobulin to bind to an antigen or epitope.

[0031] CDR3 is typically the most molecularly diverse origin within the antibody binding site. For example, H3 can be as short as 2 amino acid residues or longer than 26 amino acids. The subunit structures and three-dimensional arrangements of various classes of immunoglobulins are well known in the art. For an overview of antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al, 1988. One of ordinary skill in the art will recognize that each subunit structure, e.g., CH, VH, CL, VL, CDR, FR structures, includes a part of an active fragment, e.g., a VH, VL or CDR subunit that binds to an antigen, i.e., an antigen-binding fragment, or a part of a CH subunit that binds to and / or activates, e.g., Fc receptors and / or complement. CDR typically refers to Kabat CDR as described in Sequences of Proteins of immunological Interest, US Department of Health and Human Services (1991), eds. Kabat et al. Another standard for characterizing the antigen-binding site refers to hypervariable loops as reported by Chothia. See, e.g., Chothia, et al. (1992; J. Mol. Biol. 227:799-817; and Tomlinson et al. (1995) EMBO J. 14:4628-4638. Yet another standard is the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg) for a broad reference. Embodiments described with respect to Kabat CDR can alternatively be implemented using similar relationships described with respect to Chothia hypervariable loops or AbM-defined loops.In the context of the present invention, specific positions within the variable domain of an antibody are referred to according to the nomenclature of the IMGT database.

[0032] The corresponding immunoglobulin mu heavy chain, gamma heavy chain, alpha heavy chain, delta heavy chain, epsilon heavy chain, lambda light chain or kappa light chain can be of any species, for example, mammalian species including rodent species, amphibians such as Lissamphibia (including, for example, frogs, Xenopus laevis, toads or newts), or invertebrate species. Examples of mammals include, but are not limited to, rats, mice, rabbits, guinea pigs, monkeys, hamsters, gerbils, camels, American rabbits, armadillos, dogs, cynomolgus monkeys, goats, pigs, cows, opossums, horses, bats, woodchucks, orangutans, rhesus monkeys, woolly monkeys, macaques, chimpanzees, tamarins (cotton-top tamarins), marmosets or humans.

[0033] As referred to herein, an immunoglobulin is typically a glycoprotein or an antigen-binding portion thereof comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain has a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In some embodiments, the heavy chain constant region comprises three domains CH1, CH2 and CH3. Each light chain has a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) inserted into more conserved regions called framework regions (FRs). The CDRs contain most of the residues involved in the specific interaction of the antibody with the antigen. Each VH and each VL have three CDRs and four FRs arranged in the following order, FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus towards the carboxy terminus. The variable regions of the heavy and light chains comprise a binding domain that interacts with an epitope of an antigen.

[0034] The residues of the "framework region" or "FR" are variable domain residues other than the hypervariable regions. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. Thus, a "human framework region" is a framework region that is substantially identical (about 85% or more, usually 90-95% or more) to the framework regions of naturally occurring human immunoglobulins. The framework region of an antibody, i.e., the combination of the framework regions of the component light and heavy chains, serves to define the positions of, and to align, the CDRs. The CDRs are mainly involved in binding to the epitopes of an antigen.

[0035] The terms "Fab", "Fab region", "Fab portion" or "Fab fragment" are understood to define a polypeptide containing the VH, CH1, VL and CL immunoglobulin domains. Fab can refer to this region alone or in the context of an ABP and a full-length immunoglobulin or immunoglobulin fragment. Typically, the Fab region includes the entire light chain of the antibody. The Fab region can be interpreted as defining the "arm" of the immunoglobulin molecule. It contains the epitope-binding portion of that Ig. The Fab region of a naturally occurring immunoglobulin can be obtained as a proteolytic fragment by papain digestion. The "F(ab’)2 portion" is a proteolytic fragment of an immunoglobulin digested with pepsin. The "Fab’ portion" is a product resulting from the reduction of the disulfide bonds of the F(ab’)2 portion. As used herein, the terms "Fab", "Fab region", "Fab portion" or "Fab fragment" may further include the hinge region that defines the C-terminus of the antibody arm. This hinge region corresponds to the hinge region found at the C-terminus of the CH1 domain in a full-length immunoglobulin that can be interpreted such that the arm of the ABP defines a Y. The term hinge region is used in the art because immunoglobulins have some mobility in this region. As used herein, "Fab heavy chain" is understood as a portion or polypeptide of a Fab fragment containing VH and CH1, whereas "Fab light chain" as used herein is understood as a portion or polypeptide of a Fab fragment containing VL and CL.

[0036] The terms "Fc region" or "Fc fragment" are used herein to define the C-terminal region of an immunoglobulin heavy chain, including both the native sequence Fc region and variant Fc regions. The Fc portion mediates the effector functions of an antibody, such as activation of the complement system and immune effector cells (e.g., NK cells) that have Fc receptors. In human IgG molecules, the Fc region is generated by papain cleavage N-terminal to Cys226. The boundary of the Fc region of an immunoglobulin heavy chain can vary, but the human IgG heavy chain Fc region is typically defined as extending from the amino acid residue at position Cys226 or Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during the production or purification of an ABP, or by recombinant manipulation of the nucleic acid encoding the heavy chain of the ABP. Native sequence Fc regions include IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4 from mammals, such as humans or mice. The Fc region contains two or three constant domains depending on the class of the antibody. In embodiments where the immunoglobulin is IgG, the Fc region has CH2 and CH3 domains. In certain preferred embodiments of the invention, the Fc region of the ABP of the invention is mutated to reduce Fc receptor binding and thereby reduce ADCC. Such preferred ABPs of the invention are "Fc-attenuated".

[0037] The term "single-chain variable fragment" (scFv) is used herein to define an antibody fragment in which the variable region of the heavy chain (VH) of an immunoglobulin is fused to the variable region of the light chain (VL) by a short linker peptide of 10 to about 25 amino acids. The linker is typically glycine-rich for flexibility and serine-rich or threonine-rich for solubility, and can connect the N-terminus of VH to the C-terminus of VL or the N-terminus of VL to the C-terminus of VH. The scFv fragment has a specific antigen-binding site but does not have the constant domains of an immunoglobulin.

[0038] The term "epitope", also known as "antigenic determinant", refers to a part of an antigen to which an antibody or a T cell receptor specifically binds, thereby forming a complex. Thus, the term "epitope" includes any molecular or protein determinant capable of specific binding to an immunoglobulin or a T cell receptor. The binding site (paratope) of the ABP described herein can specifically bind to / interact with a conformational epitope or a continuous epitope that is unique to the target structure. Epitope determinants usually consist of the chemically active surface of a molecule, for example, amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. In some embodiments, epitope determinants include the chemically active surface of a molecule, for example, amino acids, sugar side chains, phosphoryl or sulfonyl. In certain embodiments, epitope determinants can have specific three-dimensional structural characteristics and / or specific charge characteristics. With respect to polypeptide antigens, conformational epitopes or discontinuous epitopes are characterized in that two or more separate amino acid residues are separated in the primary sequence, but when the polypeptide is folded into a native protein / antigen, they are assembled into a consistent structure on the surface of the molecule (Sela, M., Science (1969) 166, 1365 - 1374; Laver, W.G., et al. Cell (1990) 61, 553 - 556). Those two or more separate amino acid residues contributing to the epitope can be present in separate compartments of one or more polypeptide chains. These residues come together on the surface of the molecule when the polypeptide chain is folded into a three-dimensional structure to form the epitope. In contrast, continuous epitopes or linear epitopes consist of two or more separate amino acid residues present as a single linear segment of a polypeptide chain.

[0039] As used in this context, the term "specific" or "specifically binds to" which is also used as "targeting" means, according to the present invention, that the antibody or immunoreceptor fragment can specifically interact with and / or bind to a specific antigen or ligand or a set of specific antigens or ligands, but does not essentially bind to other antigens or ligands. Such binding can be exemplified by the specificity of the "lock and key principle". Antibodies are said to "bind to the same epitope" when they compete with each other, and only one antibody can bind to that epitope at a given time, i.e., one antibody prevents the binding or regulatory effect of the other.

[0040] As used herein, the term "isolated ABP" refers to an ABP that has been identified and separated and / or recovered from the components of its natural environment. The contaminating components of its natural environment are substances that can interfere with the diagnostic or therapeutic use of the antibody, and these can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the ABP is purified to more than 95% by weight (e.g., more than 99% by weight) of the antibody as measured by the Lowry method. In some embodiments, the antibody is purified uniformly as determined by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. The isolated ABP can, in some embodiments, be present in recombinant cells in which one or more components of the natural environment of the antibody are absent. Usually, the isolated antibody is prepared by at least one purification step.

[0041] The (recombinant) ABP of the present invention that binds to TIRC7 and / or immune cells expressing TIRC7 as described herein can be in any suitable recombinant antibody format, for example, as an Fv fragment, scFv, a monovalent antibody lacking the hinge region, a minibody, a Fab fragment, a Fab’ fragment, an F(ab’)2 fragment. The recombinant ABP of the present invention can also include a constant domain (region), such as a human IgG constant region, a CH1 domain (as included in a Fab fragment) and / or the entire Fc region. Alternatively, the ABP of the present invention can also be a full-length (complete) antibody, preferably in a bispecific format.

[0042] There are several mechanisms by which antibodies can mediate cellular effects, including anti-proliferation via blockade of required signaling pathways, mimicking of receptor-ligand interactions, or prevention of ligand-receptor interactions, regulation of antigen turnover or internalization. The efficacy of TIRC7 antibodies may result from a combination of these mechanisms, and their relative importance in clinical treatment for autoimmune diseases is considered to be disease-dependent.

[0043] The ABP of the present invention can bind to human TIRC7. The terms “T cell immune response cDNA7” or “TIRC7” are used interchangeably herein and include variants, isoforms and species homologs of human TIRC7. The human TIRC7 protein has the UniProt (www.uniprot.org) accession number Q13488 (version of September 26, 2019). Further synonyms for this protein include vacuolar proton ATPase 116 kDa subunit a isoform 3. The gene for human TIRC7 (also named TCIRG1) is located at 11q13.2 and has the HGNC accession HGNC:11647 (www.genenames.org - HGNC version of September 26, 2019). However, the antibodies of the present invention may not cross-react with TIRC7 from non-human species in certain preferred cases.

[0044] To determine the epitope, standard epitope mapping methods known in the art can be used. For example, a soluble protein that is a fragment (peptide) of TIRC7 that binds to the above antibody (e.g., a synthetic peptide) can be used to determine whether the candidate antibody or its antigen-binding fragment binds to the same epitope. In the case of a linear epitope, overlapping peptides of a defined length (e.g., 8 or more amino acids) are synthesized. These peptides can be shifted one amino acid at a time to prepare a series of peptides that cover all 8-amino acid fragments of the TIRC7 protein sequence. Fewer peptides can be prepared by increasing the number of amino acids to be shifted, e.g., using 2 or 3 amino acids. Additionally, longer peptides (e.g., 9, 10, or 11mers) can also be synthesized. The binding of the peptides to the antibody or antigen-binding fragment can be measured using standard methods including surface plasmon resonance (BIACORE) and ELISA assays. When examining conformational epitopes, larger TIRC7 fragments can be used. Other methods using mass spectrometry to define conformational epitopes have already been reported and may exist (see, e.g., Baerga-Ortiz et al, Protein Science 11:1300-1308, 2002 and references cited therein). Still other methods for determining epitopes are provided in standard laboratory references such as Current Protocols in Immunology, Coligan et al, eds., John Wiley & Sons, Unit 6.8 (“Phage Display Selection and Analysis of B-cell Epitopes”) and Unit 9.8 (“Identification of Antigenic Determinants Using Synthetic Peptide Combinatorial Libraries”).Epitopes can be confirmed by introducing point mutations or deletions into known epitopes and then testing their binding to one or more antibodies or antigen-binding fragments to determine which mutations reduce binding of the antibody or antigen-binding fragment.

[0045] In other embodiments, an ABP that binds to TIRC7, which is a regulator of TIRC7 function, alternatively or additionally, · can inhibit, impair, reduce, or reverse a cellular immune response (e.g., in an in vitro assay or in a subject (e.g., a subject in need thereof)), and / or · can inhibit, impair, reduce, or reverse a humoral immunity (e.g., in an in vitro assay or in a subject (e.g., a subject in need thereof)).

[0046] As used herein, the term "cellular immune response" includes the maturation, proliferation, activation, migration, infiltration and / or differentiation of T cells, and / or the activation / regulation / migration / infiltration of macrophages, natural killer cells, T lymphocytes (or T cells), helper T lymphocytes, memory T lymphocytes and / or cytotoxic T lymphocytes (CTLs), and / or the production, release and / or effect of one or more factors that can be secreted by cells or one or more factors secreted by cells, such as cytokines or autacoids (particularly pro-inflammatory cytokines), and / or any one or more components of such processes (such as cytokines or autacoids, particularly pro-inflammatory cytokines), and responses in a host organism that involve, use and / or promote any one or a combination thereof, but are not limited thereto. As used herein, the term "cellular immune response" may include cellular responses involving genetically engineered, in vitro cultured, autologous, heterologous, modified and / or transplanted T lymphocytes, or factors that can be secreted by cells or factors secreted by cells (such as cytokines or autacoids, particularly pro-inflammatory cytokines) produced by genetic engineering. The cellular immune response is preferably a pathological immune response that may harm the host.

[0047] In certain embodiments, the cells mediating the cellular immune response can be mediated by cells (such as immune cells) that can secrete (e.g., secrete) pro-inflammatory cytokines (such as those selected from the group consisting of interleukin-1 (IL-1), IL-2, IL-12, IL-17 and IL-18, tumor necrosis factor (TNF)[alpha], interferon gamma (IFN-gamma) and granulocyte macrophage colony-stimulating factor).

[0048] In certain embodiments, the cellular immune response can be mediated by cells that secrete pro-inflammatory cytokines, such as lymphocytes (e.g., T cells), particularly cytotoxic T lymphocytes (CTLs).

[0049] In certain embodiments, the cellular immune response can induce the killing of self-cells in the context of an autoimmune disease.

[0050] The term "humoral immunity" (or "humoral immune response") is also readily understood by those skilled in the art and includes aspects of the immune response mediated by macromolecules found in extracellular fluids such as secreted antibodies, complement proteins, and certain antimicrobial peptides. Humoral immunity is so named because substances found in the humors, i.e., body fluids, are involved. The aspect involving antibodies can be called antibody-mediated immunity.

[0051] In some preferred embodiments, the ABP of the present invention includes, in its first antigen-binding domain, a heavy-chain variable region comprising a human framework region of allele IGHV1-2 (accession number X07448), IGHV7-4-1*02, or IGHV1-46*01, preferably IGHV1-2. The ABP of the present invention preferably includes, within the light chain, a variable region having a framework of IGKV1-9 (accession number Z00013), IGKV3-11 (accession number X01668), IGKV6-21*01, or IGKV1-17*03. However, it is one of the achievements of the present invention to provide mutant variants of humanized anti-TIRC7 antibodies with improved activity against binding affinity, avidity, and / or lymphocyte mobilization and activation of lymphocyte-mediated anti-tumor cytotoxicity, particularly activation of T cell-mediated immunity. Thus, according to the present invention, the ABP which is the ABP of the present invention in the first antigen-binding domain specific for TIRC7 preferably includes a heavy-chain variable region having mutations at one or more positions selected from positions 78, 80, 82, and / or 85 according to IMGT numbering. Most preferably, those mutations are any one or any combination or all of 78L, 80S, 82K, and / or 85S according to IMGT numbering.

[0052] In some preferred embodiments of the present invention, the heavy chain variable region has a sequence identity of at least 95% with an amino acid sequence selected from SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 or 85, or independently in each case, optionally, 10, 9, 8, 7, 6, 5, 4 or fewer, preferably 3, 2 or 1 or fewer amino acid substitutions, insertions or deletions compared to these sequences, and / or the light chain variable region has a sequence identity of at least 95% with an amino acid sequence selected from SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 or 86, or independently in each case, optionally, 10, 9, 8, 7, 6, 5, 4 or fewer, preferably 3, 2 or 1 or fewer amino acid substitutions, insertions or deletions compared to these sequences, an ABP is provided.

[0053] In some embodiments of the present invention, the heavy chain variable region of the ABP comprises an amino acid sequence having a sequence identity of at least 85%, at least 90% or at least 95% with the amino acid sequence shown in SEQ ID NO: 11, and the light chain variable region comprises an amino acid sequence having a sequence identity of at least 95% with the amino acid sequence shown in SEQ ID NO: 12. Preferably, the heavy chain variable region of the ABP comprises the amino acid sequence shown in SEQ ID NO: 11, or optionally, 10, 9, 8, 7, 6, 5, 4 or fewer, preferably 3, 2 or 1 or fewer amino acid substitutions, insertions or deletions compared to this sequence, and the light chain variable region comprises the amino acid shown in SEQ ID NO: 12, or optionally 10, 9, 8, 7, 6, 5, 4 or fewer, preferably 3, 2 or 1 or fewer amino acid substitutions, insertions or deletions compared to this sequence.

[0054] In some preferred embodiments of the present invention, the heavy chain variable region has a sequence identity of at least 95%, preferably 100%, to the amino acid sequence selected from SEQ ID NO: 25, or, optionally, 10, 9, 8, 7, 6, 5, 4 or fewer, preferably 3, 2 or 1 or fewer amino acid substitutions, insertions or deletions compared to this sequence, and the light chain variable region has a sequence identity of at least 95% to the amino acid sequence selected from SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 or 86, or, in each case independently, optionally, 10, 9, 8, 7, 6, 5, 4 or fewer, preferably 3, 2 or 1 or fewer amino acid substitutions, insertions or deletions compared to these sequences. An ABP is provided that includes an amino acid sequence.

[0055] In some embodiments of the present invention, the heavy chain variable region of the ABP includes an amino acid sequence having a sequence identity of at least 85%, at least 90% or at least 95% to the amino acid sequence shown in SEQ ID NO: 25, and the light chain variable region includes an amino acid sequence having a sequence identity of at least 95% to the amino acid sequence shown in SEQ ID NO: 26. Preferably, the heavy chain variable region of the ABP includes the amino acid sequence shown in SEQ ID NO: 25, or, optionally, 10, 9, 8, 7, 6, 5, 4 or fewer, preferably 3, 2 or 1 or fewer amino acid substitutions, insertions or deletions compared to this sequence, and the light chain variable region includes the amino acid shown in SEQ ID NO: 26, or, optionally, 10, 9, 8, 7, 6, 5, 4 or fewer, preferably 3, 2 or 1 or fewer amino acid substitutions, insertions or deletions compared to this sequence.

[0056] In some embodiments of the present invention, the heavy chain variable region of ABP comprises an amino acid sequence having at least 85%, at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 28. Preferably, the heavy chain variable region of ABP comprises the amino acid sequence shown in SEQ ID NO: 27, or, optionally, an amino acid sequence comprising 10, 9, 8, 7, 6, 5, 4 or fewer, preferably 3, 2 or 1 or fewer amino acid substitutions, insertions or deletions compared to this sequence, and the light chain variable region comprises the amino acids shown in SEQ ID NO: 28, or, optionally, an amino acid comprising 10, 9, 8, 7, 6, 5, 4 or fewer, preferably 3, 2 or 1 or fewer amino acid substitutions, insertions or deletions compared to this sequence.

[0057] In some embodiments of the present invention, the heavy chain variable region of ABP comprises an amino acid sequence having at least 85%, at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 29, and the light chain variable region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 30. Preferably, the heavy chain variable region of ABP comprises the amino acid sequence shown in SEQ ID NO: 29, or, optionally, an amino acid sequence comprising 10, 9, 8, 7, 6, 5, 4 or fewer, preferably 3, 2 or 1 or fewer amino acid substitutions, insertions or deletions compared to this sequence, and the light chain variable region comprises the amino acids shown in SEQ ID NO: 30, or, optionally, an amino acid comprising 10, 9, 8, 7, 6, 5, 4 or fewer, preferably 3, 2 or 1 or fewer amino acid substitutions, insertions or deletions compared to this sequence.

[0058] In some embodiments of the present invention, the heavy chain variable region of ABP comprises an amino acid sequence having at least 85%, at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 31, and the light chain variable region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 32. Preferably, the heavy chain variable region of ABP comprises the amino acid sequence shown in SEQ ID NO: 31, or, optionally, an amino acid sequence comprising 10, 9, 8, 7, 6, 5, 4 or fewer, preferably 3, 2 or 1 or fewer amino acid substitutions, insertions or deletions compared to this sequence, and the light chain variable region comprises the amino acid shown in SEQ ID NO: 32, or, optionally, an amino acid comprising 10, 9, 8, 7, 6, 5, 4 or fewer, preferably 3, 2 or 1 or fewer amino acid substitutions, insertions or deletions compared to this sequence.

[0059] In some specific embodiments of the present invention, an ABP comprising 78L in the heavy chain variable region is preferred, where the numbering follows the IMGT system.

[0060] In some embodiments, the ABP according to the present invention is an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100% sequence identity to the human antibody heavy chain constant sequence, or an amino acid sequence having 20, 15, 10, 9, 8, 7, 6, 4 or fewer, preferably 3 or 2 or fewer, preferably 1 or fewer amino acid substitutions, deletions or insertions compared to the human antibody heavy chain constant sequence, and may be an ABP comprising at least one antibody heavy chain constant sequence. Such an antibody may have one or more of the CH1, CH2 or CH3 regions.

[0061] In some embodiments, the ABP is an antibody or an antigen-binding fragment thereof composed of at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences, wherein the antibody heavy chain sequence and the antibody light chain sequence each comprise one variable region sequence of one of the following combinations. JPEG0007691977000001.jpg20276

[0062] Furthermore, some embodiments of the present invention relate to an ABP that competes with the ABP of the present invention for binding to TIRC7, for example, competitively inhibiting the binding of an antibody of the present invention to TIRC7. Various assays known to those skilled in the art can be used to measure competitive inhibition. For example, by using a cross-competition assay, it can be determined whether an antibody or an antigen-binding fragment thereof competitively inhibits the binding of another antibody or an antigen-binding fragment thereof to TIRC7. These include cell-based methods using flow cytometry or solid-phase binding assays. Other assays can also be used to evaluate the ability of an antibody or an antigen-binding fragment thereof to cross-compete for TIRC7 molecules not expressed on the cell surface, either in solid phase or liquid phase.

[0063] In some embodiments, the ABP of the present invention can bind to TIRC7 or its paralog, ortholog, or other variant (e.g., via one or more epitopes presented by one or more extracellular domains thereof), or in particular, bind to the extracellular domain of TIRC7 with a K of less than 20 nM (e.g., less than about 10 nM, 5 nM, or 2 nM (in particular, less than about 1 nM)) D Preferably, in a preferred embodiment, the ABP of the present invention binds to TIRC7 or a domain or its variant (e.g., its epitope) with a K of less than 100 pM D In a more preferred embodiment, the ABP of the present invention binds to TIRC7 or an extracellular domain or its variant with a K of less than 50 pM D In the most preferred embodiment, the ABP of the present invention binds to TIRC7 or a domain or its variant with a K of less than 30 pM D and in certain embodiments, with a K of less than 10 pM D K D is preferably measured by surface plasmon resonance.

[0064] As used herein, the term "extracellular domain" ("ECD" or "EC" domain) generally refers to the region of a protein that is exposed to the extracellular space and is involved in the binding of a ligand or antibody. TIRC7 is a seven-transmembrane domain protein that includes an intracellular N-terminus, a larger extracellular loop between transmembrane domains 5 and 6, and a mobile extracellular C-terminus. The preferred extracellular domain to which the ABP of the present invention binds is the region located in the protein loop between the fifth and sixth transmembrane domains of TIRC7.

[0065] As used herein, the term "K D " is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). The K D value of an antibody can be measured using methods established in the art such as surface plasmon resonance (BIAcore®), ELISA, and KINEXA. The preferred method for measuring the K D of an antibody is a method by using surface plasmon resonance, preferably a method by using a biosensor system such as a BIAcore® system or a method by ELISA. As used herein, "K a " (or "K-assoc") broadly refers to the association rate of a specific antibody-antigen interaction, whereas the term "K d " (or "K-diss") used herein refers to the dissociation rate of a specific antibody-antigen interaction.

[0066] The ABP according to the present invention may have two chains, namely, a short chain that can be a light chain in some embodiments and a main chain that can also be treated as a heavy chain in some embodiments. The ABP is typically a dimer of these two chains.

[0067] The ABP of the present invention can preferably be a bispecific ABP. The bispecific ABP has: (i) a variable region containing a heavy chain variable domain and a light chain variable domain as defined in any one of the preceding claims, wherein the variable region contains a first antigen-binding domain capable of binding to human TIRC7; and (ii) a heavy chain variable region and a light chain variable region of the ABP that can contain a second antigen-binding domain. The binding site for TIRC7 is preferably understood to be the binding site of the TIRC7-binding antibody of the present invention described herein.

[0068] A "bispecific" or "bifunctional" ABP is an ABP that has two different epitope / antigen-binding domains (or "sites"), and thus has binding specificity for two different target epitopes. These two epitopes may be epitopes of the same antigen, or, in the case preferred in the present invention, may be epitopes of different antigens, such as different antigens TIRC7 and CD3 or TIRC7 and TCR, and preferably these antigens are human antigens.

[0069] "Bispecific ABP" can be an ABP that binds to one antigen or epitope using one of two or more binding arms defined by a first pair of a heavy chain and a light chain or a main chain and a short / mini chain, and binds to a different antigen or epitope on a second arm defined by a second pair of a heavy chain and a light chain or a main chain and a small chain. Such embodiments of bispecific ABP have two antigen-binding arms with different specificities and CDR sequences. Usually, bispecific ABP is monovalent for each antigen it binds to, i.e., binds to each antigen or epitope with only one arm. However, bispecific antibodies can also dimerize or multimerize, which is preferred in the context of the present invention. Bispecific antibodies can be hybrid ABPs that can have a first binding region defined by a first light chain variable region and a first heavy chain variable region and a second binding region defined by a second light chain variable region and a second heavy chain variable region. It is contemplated by the present invention that one of these binding regions can be defined by a heavy chain / light chain pair. In the context of the present invention, bispecific ABP can have a first binding site defined by the variable regions of a main chain and a small chain and a second different binding site defined by the variable regions of an scFv fragment contained in the main chain of the ABP.

[0070] Methods for making bispecific ABP are known in the art and include, for example, chemical conjugation of two different monoclonal antibodies, or chemical conjugation of, for example, two antibody fragments, such as two Fab fragments. Alternatively, bispecific ABP can be made by the quadroma technique by fusion of hybridomas producing the parental antibodies. Since the combination of H chain and L chain is random, a potential mixture of 10 different antibody structures is generated, and only one of them has the desired binding specificity.

[0071] The bispecific ABP of the present invention can act as a monoclonal antibody (mAb) against each target. In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a fully human antibody. The bispecific ABP can be, for example, a bispecific tandem single-chain Fv, a bispecific Fab2, or a bispecific diabody.

[0072] It should be noted that in this context, it is within the scope of the present invention that the ABP can contain one or more mutated amino acid residues. The terms "mutated", "mutant", and "mutation" with respect to a nucleic acid or polypeptide refer to an exchange, deletion, or insertion of one or more nucleotides or amino acids, respectively, compared to the "native" or "parent" (when a reference is provided) nucleic acid or polypeptide, i.e., a reference sequence that can be interpreted as defining the wild type. For example, the variable domain of the ABP of the present invention as described herein, obtained by extensive mutagenic modification of the parental 4G8 molecule, can be interpreted as the parental sequence.

[0073] In this regard, the term "position", when used in accordance with the present invention, is understood to mean the position of an amino acid within the amino acid sequence depicted herein. This position can be indicated with reference to a similar native sequence, for example, the sequence of a naturally occurring IgG domain or chain. The term "corresponding" as used herein also includes the fact that the position is not necessarily determined by the aforementioned nucleotide / amino acid numbers, i.e., not determined solely by the aforementioned nucleotide / amino acid numbers. Thus, the position of a given amino acid according to the present invention that can be substituted can vary due to a deletion or addition of an amino acid anywhere in the antibody chain.

[0074] Accordingly, it should be understood that under the "corresponding position" according to the present invention, amino acids may have different indicated numbers but may still have similar adjacent amino acids. Amino acids that can be exchanged, deleted or added are also encompassed by the term "corresponding position". To determine whether an amino acid residue in a given amino acid sequence corresponds to a particular position in the amino acid sequence of a naturally occurring immunoglobulin domain or chain, one of ordinary skill in the art can use means and methods well known in the art, such as manual alignment or a computer program (e.g., BLAST 2.0, which is an abbreviation for Basic Local Alignment Search Tool, or ClustalW or any other suitable program suitable for generating sequence alignments).

[0075] In some embodiments, the substitution (or replacement) is a conservative substitution. Conservative substitutions are generally the substitutions listed below, followed by one or more replacements that can be interpreted as conservative for each amino acid being mutated. Ala→Gly, Ser, Val; Arg→Lys; Asn→Gln, His; Asp→Glu; Cys→Ser; Gln→Asn; Glu→Asp; Gly→Ala; His→Arg, Asn, Gln; Ile→Leu, Val; Leu→Ile, Val; Lys→Arg, Gln, Glu; Met→Leu, Tyr, Ile; Phe→Met, Leu, Tyr; Ser→Thr; Thr→Ser; Trp→Tyr; Tyr→Trp, Phe; Val→Ile, Leu. Other substitutions are also acceptable and can be determined empirically or can be consistent with other known conservative or non-conservative substitutions. As a further orientation, each of the following eight groups contains amino acids that can usually be interpreted as defining conservative substitutions for each other. - Alanine (Ala), Glycine (Gly); - Aspartic acid (Asp), Glutamic acid (Glu); - Asparagine (Asn), Glutamine (Gln); - Arginine (Arg), Lysine (Lys); - Isoleucine (Ile), Leucine (Leu), Methionine (Met), Valine (Val); - Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp); - Serine (Ser), Threonine (Thr); and - Cysteine (Cys), Methionine (Met)

[0076] If such substitutions change the biological activity, more substantial changes can be introduced, such as those further described below or for the amino acid classes as described below, and the product can be screened for the desired characteristics. Examples of such more substantial changes are: Ala→Leu, Ile; Arg→Gln; Asn→Asp, Lys, Arg, His; Asp→Asn; Cys→Ala; Gln→Glu; Glu→Gln; His→Lys; Ile→Met, Ala, Phe; Leu→Ala, Met, norleucine; Lys→Asn; Met→Phe; Phe→Val, Ile, Ala; Trp→Phe; Tyr→Thr, Ser; Val→Met, Phe, Ala.

[0077] In some embodiments, the ABP according to the present invention comprises one or more amino acid residues (including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acid residues) that are mutated to prevent dimerization via cysteine residues or to modulate the Fc function (see above). In some of these embodiments, one or more amino acid residues in the CH2 domain and / or the hinge region capable of mediating binding to an Fc receptor are mutated. One or more amino acid residues capable of mediating binding to an Fc receptor, if present, can be amino acid residues that can activate antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-mediated cytotoxicity (CDC). In some embodiments, each amino acid residue capable of mediating binding to an Fc receptor is generally replaced by another amino acid when comparing the sequence to the sequence of the corresponding naturally occurring domain in an immunoglobulin such as IgG. In some embodiments, such amino acid residues capable of mediating binding to an Fc receptor are generally deleted relative to the sequence of the corresponding naturally occurring domain in an immunoglobulin such as IgG.

[0078] Optionally, as described above, substitutions or deletions of amino acid residues can be made for this effect. Suitable mutations can be obtained, for example, from Armour et al. (Eur. J. Immunol.

[1999] 29, 2613 - 2624). Further positions suitable for mutations in the sequence of the antibody chain can be obtained from the crystal structure data published for the complex of FcγRIII and the human IgG1 Fc fragment (Sondermann et al, Nature

[2000] 406, 267 - 273). In addition to the measurement of binding affinity as described above, it is also possible to functionally evaluate the ability (or lack thereof) to mediate binding to Fc receptors in order to assess the level of "Fc attenuation" or loss of binding affinity. In the case of an ABP that binds to CD3 as one target, for example, it is possible to evaluate the binding through the mitogenicity of such CD3 - binding ABP on the cell surface. Mitogenicity is mediated by the binding of a CD3 antibody to Fc receptors on accessory cells such as monocytes. While the ABP of the present invention having one binding site for CD3 does not show a cell - division promoting effect, whereas a parental monoclonal anti - CD3 antibody having a functional Fc portion induces strong mitosis in T cells, it is clear that due to the absence of mitosis, the ABP of the present invention lacks the ability to bind Fc and can thus be regarded as an "Fc knockout" molecule. Illustrative examples of methods for evaluating anti - CD3 - mediated mitogenicity are described by Davis, Vida & Lipsky (J. Immunol (1986) 137, 3758) and Ceuppens, JL, & van Vaeck, F (J. Immunol. (1987) 139, 4067 or Cell. Immunol. (1989) 118, 136, see also).Further exemplary and preferred examples of assays for evaluating the mitogenicity of antibodies are described by Rosenthal-Allieri et al. (Rosenthal-Allieri MA, Ticcioni M, Deckert M, Breittmeyer JP, Rochet N, Rouleaux M, and Senik A, Bernerd A, Cell Immunol. 1995 163(1):88-95) and Grosse-Hovest et al. (Grosse-Hovest L, Hartlapp I, Marwan W, Brem G, Rammensee H-G, and Jung G, Eur J Immunol.

[2003] May;33(5):1334-1340). Further, the lack of Fc binding can be evaluated by whether the ABP of the present invention can mediate one or more of the well-known effector functions of the Fc portion.

[0079] As described above, substitution or deletion of cysteine residues can be performed to introduce or remove one or more disulfide bonds (including introduction or removal of potential or pre-existing disulfide bonds). Thereby, the linkage between the backbone of the ABP according to the present invention and the lower molecular weight / shorter chain can be controlled (including establishment, strengthening or disappearance). By introducing or removing one or more cysteine residues, disulfide bridges can be introduced or removed. As an exemplary example, the tetrameric ABP according to the present invention generally has one or more disulfide bonds that link two dimeric ABPs. One such disulfide bond is usually defined by a cysteine in the backbone of the first dimeric ABP and a cysteine in the hinge region of the second dimeric ABP. In this regard, in some embodiments, the antibody according to the present invention may include amino acid substitution of the natural cysteine residues at positions 226 and / or 229 with another amino acid residue, based on the sequence of human IgG immunoglobulin according to Kabat numbering [EU index].

[0080] To modify the glycosylation pattern of an antibody, substitution or deletion of amino acid residues such as arginine, asparagine, serine, threonine or tyrosine residues may also be performed. As an illustrative example, an IgG molecule has a single N-linked biantennary carbohydrate at Asn297 of the CH2 domain. In the case of serum-derived IgG, or IgG produced ex vivo in hybridomas or engineered cells, the IgG is heterogeneous with respect to the carbohydrate linked to Asn297. In the case of human IgG, the core oligosaccharide usually consists of GlcNAc2Man3GlcNAc and the number of outer residues varies.

[0081] As shown, in addition to antigen / epitope binding, immunoglobulins have additional "effector functions", which are biological activities that can be attributed to the Fc region of the immunoglobulin (Fc region of the native sequence or Fc region of an amino acid sequence variant), and are known to vary depending on the immunoglobulin isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. The exertion of antibody effector functions generally requires the recruitment of effector cells. Some of the effector functions of immunoglobulins are mediated by Fc receptors (FcRs) that bind to the Fc region of the antibody. FcRs are defined by their specificity for immunoglobulin isotypes, the Fc receptor for IgG antibodies is called FcγR, the Fc receptor for IgE is called FcεR, and the Fc receptor for IgA is called FcαR, etc. To assess whether the ABP of the present invention lacks the ability to bind Fc, any of these effector functions (or loss of such effector functions) such as CDC or ADCC can be used.

[0082] In this context, it should be noted that the term "Fc receptor" or "FcR" generally defines a receptor, a protein, that can bind to the Fc region of an antibody. Fc receptors are found on the surface of certain cells of an organism's immune system, such as natural killer cells, macrophages, neutrophils, and mast cells. Fc receptors in vivo bind to immunoglobulins immobilized on infected cells or present on invading pathogens. Their activity stimulates phagocytic or cytotoxic cells to destroy microorganisms or infected cells by antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity. Some viruses, such as flaviviruses, use Fc receptors to help infect cells by a mechanism known as antibody-dependent enhancement of infection. FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al, Immunomethods 4:25-34 (1994); and de Haas et al, J. Lab. Clin. Med. 126:330-41 (1995).

[0083] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is triggered by the binding of the first component of the complement system (Clq) to an antibody (an antibody of the appropriate subclass) bound to a cognate antigen. To assess complement activation, a CDC assay, such as an assay as described in Gazzano-Santoro et al, J. Immunol. Methods 202:163 (1997), can be performed.

[0084] The term "complement system" is generally used in the art to refer to several low molecular weight proteins called complement factors found in the blood that circulate as inactive precursors (proproteins). This term refers to the ability of this inflexible and adaptable system to "complement" the ability of antibodies and phagocytic cells to remove pathogens such as bacteria and antigen-antibody complexes from the organism. Examples of complement factors are the C1 complex, which includes C1q and the two serine proteases C1r and C1s. This C1 complex is a component of the CDC pathway. C1q is a hexavalent molecule with a molecular weight of approximately 460,000 and has a structure resembling a bouquet of tulips in which six "stems" of collagen are connected to six globular head regions. To activate the complement cascade, C1q must bind to at least two molecules of IgG1, IgG2, or IgG3.

[0085] "Antibody-dependent cell-mediated cytotoxicity" or ADCC refers to a form of cell injury in which cytotoxic effector cells, which are present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), can specifically bind to target cells bearing an antigen via immunoglobulin molecules bound to Fc receptors (FcRs) on these cells, and then kill the target cells by cytotoxicity. Antibodies "arm" the cytotoxic cells and are required for the killing of target cells by this mechanism. NK cells, which are the major cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To evaluate the ADCC activity of a molecule of interest, an in vitro ADCC assay (e.g., the assays described in U.S. Patent Nos. 5,500,362 or 5,821,337) can be performed. Effector cells useful for such assays include, but are not limited to, peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. In some embodiments, the ADCC activity of the molecule of interest can be evaluated in vivo, e.g., in an animal model (e.g., the animal models disclosed in Clynes et al, PNAS USA 95:652-656 (1998)).

[0086] In certain preferred embodiments, the present invention encompasses all known strategies developed to reduce or eliminate the cytotoxicity of antibodies. One strategy effective in human antibodies is to eliminate N-linked glycosylation at residue Asn297, which is achieved by substituting Asn297 with alanine, glycine or aspartic acid, or by modifying the serine / threonine residue at position 299. Alternative strategies encompassed by the present invention for attenuating the effector function of antibodies include substitution of residues in the lower hinge of the antibody, such as L234A and L235A (LALA). These residues form part of the Fcγ receptor binding site on the CH2 domain, and their importance in ADCC has been revealed by the exchange of these residues between antibody isotypes with higher or lower effector function. Alanine substitutions at these sites are effective in reducing ADCC in both human and mouse antibodies, but these substitutions are less effective in reducing CDC activity. Another single variant, P329A, identified by a random mutagenesis approach for mapping the C1q binding site of Fc, has been shown to be highly effective in reducing CDC activity while retaining ADCC activity.

[0087] The ABP of the present invention can be produced by using any known established expression system and recombinant cell culture technology, for example, by expression in a bacterial host (prokaryotic cell line), or a eukaryotic cell line, such as yeast, fungi, insect cells or mammalian cells. The ABP of the present invention can also be produced in a transgenic organism, such as a goat, a plant, or a XENOMOUSE transgenic mouse, a engineered mouse strain that has a large fragment of the human immunoglobulin locus and is deficient in mouse antibody production. Antibodies can also be produced by chemical synthesis.

[0088] When producing the recombinant ABP of the present invention, usually, a polynucleotide encoding an antibody is isolated and inserted into a replicable vector such as a plasmid for further cloning (amplification) or expression. An exemplary example of a suitable expression system is the glutamate synthetase system (e.g., those sold by Lonza Biologics), where the host cell is, for example, CHO or NSo. The polynucleotide encoding the antibody is easily isolated and sequenced using conventional procedures. Vectors that can be used include plasmids, viruses, phages, transposons, mini-chromosomes, among which plasmids are typical embodiments. Generally, such vectors further include a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence operably linked to the light chain and / or heavy chain polynucleotides to facilitate expression. The polynucleotides encoding the light chain and heavy chain may be inserted into separate vectors and transfected into the same host cell, or if desired, both the heavy chain and the light chain may be inserted into the same vector and transfected into the host cell. Both chains can be placed under the control of, for example, a dicistronic operon and expressed as described in Skerra, A. (1994) Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli, Gene 151, 131-135 or Skerra, A. (1994) A general vector, pASK84, for cloning, bacterial production, and single-step purification of antibody Fab fragments, Gene 141, 79-8, resulting in a correctly folded and functional ABP.Accordingly, according to one aspect of the present invention, there is provided a process for constructing a vector encoding the light chain and / or heavy chain of the antibody or antigen-binding fragment thereof of the present invention, the method comprising inserting a polynucleotide encoding the light chain and / or heavy chain of the ABP of the present invention into a vector.

[0089] When recombinant techniques are used, the ABP can be produced intracellularly or in the periplasmic space, or secreted directly into the medium (see also Skerra 1994, supra). If the antibody is produced intracellularly as a first step, particulate debris, which is the host cell or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al, Bio / Technology 10:163-167 (1992) describes procedures for isolating antibodies secreted into the periplasmic space of E. coli. Antibodies can be produced in any oxidizing environment. Such an oxidizing environment can be provided in the periplasm of Gram-negative bacteria such as E. coli, the extracellular environment of Gram-positive bacteria, or the lumen of the endoplasmic reticulum of eukaryotic cells (including animal cells such as insect cells or mammalian cells), and is usually favorable for the formation of structural disulfide bonds. However, it is also possible to produce the ABP of the present invention in the cytosol of a host cell such as E. coli. In this case, the polypeptide can be obtained directly in a folded soluble state, or can be regenerated in vitro after being recovered in the form of inclusion bodies. A further option is to use a specific host strain that has an intracellular oxidizing environment and can therefore form disulfide bonds in the cytosol (Venturi M, Seifert C, Hunte C. (2002) “High level production of functional antibody Fab fragments in an oxidizing bacterial cytoplasm.” J. Mol. Biol. 315, 1-8).

[0090] The ABP produced by the above cells can be purified using any conventional purification technique, such as hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being one of the preferred purification methods. The ABP can be purified via affinity purification using a protein / ligand that specifically and reversibly binds to a constant domain such as the CH1 or CL domain. Examples of such proteins are bacterial proteins that bind to immunoglobulins, such as Protein A, Protein G, Protein A / G, or Protein L, where the binding of Protein L is limited to ABP containing a kappa light chain. An alternative method for purifying an antibody having a κ-light chain is to use an anti-kappa antibody (KappaSelect) bound to beads. The suitability of Protein A as an affinity ligand depends on the species and isotype of the immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies (Lindmark et al, J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human gamma 3 (Guss et al, EMBO J. 5:1567 - 1575 (1986)). The selection of the purification method to be used for a particular ABP of the present invention is within the knowledge of those skilled in the art.

[0091] It is also possible to equip one of the chains of the ABP of the present invention with one or more affinity tags. Affinity tags such as Strep-tag® or Strep-tag® II (Schmidt, T.G.M. et al. (1996) J. Mol. Biol. 255, 753 - 766), myc-tag, FLAG™-tag, His6-tag, or HA-tag enable easy detection of the recombinant ABP and also enable its facile purification.

[0092] Turning now to the nucleic acids of the present invention, the nucleic acid molecule encoding one or more chains of the antibody according to the present invention can be any nucleic acid in any possible arrangement, such as single-stranded, double-stranded, or a combination thereof. Examples of nucleic acids include DNA molecules, RNA molecules, nucleotide analogs, or DNA or RNA analogs produced using nucleic acid chemistry, locked nucleic acid molecules (LNAs), and peptide nucleic acid molecules (PNAs). The DNA or RNA may be of genomic origin or synthetic origin, and may be single-stranded or double-stranded. Such nucleic acids can be, for example, mRNA, cRNA, synthetic RNA, genomic DNA, cDNA synthetic DNA, copolymers of DNA and RNA, oligonucleotides, and the like. Each nucleic acid may further contain unnatural nucleotide analogs and / or may be linked to an affinity tag or label.

[0093] In some embodiments, nucleic acid sequences encoding chains such as the heavy chain and / or light chain of the antibody according to the present invention are included in a vector such as a plasmid. When substitutions or deletions are included in the antibody chain as compared to the naturally occurring domain or region of the antibody, the coding sequences of the respective natural domains / regions, for example, the coding sequences included in the immunoglobulin sequence, can be used as a starting point for mutagenesis. For mutagenesis of selected amino acid positions, those skilled in the art can freely utilize various standard methods established for site-directed mutagenesis. A commonly used technique is to introduce mutations using PCR (polymerase chain reaction) with a mixture of synthetic oligonucleotides having a degenerate base composition at the desired sequence position. For example, by using the codons NNK or NNS (where N = adenine, guanine, cytosine or thymine; K = guanine or thymine; S = adenine or cytosine), it becomes possible to incorporate all 20 amino acids and the amber stop codon in mutagenesis, whereas the codon VVS excludes the incorporation of the amino acids Cys, Ile, Leu, Met, Phe, Trp, Tyr, Val at the selected position of its polypeptide sequence, thus limiting the number of amino acids that can be incorporated to 12. By using the codon NMS (where M = adenine or cytosine), for example, the incorporation of the amino acids Arg, Cys, Gly, Ile, Leu, Met, Phe, Trp, Val at the selected sequence position is excluded, thus limiting the number of amino acids that can be present at the selected sequence position to 11. In this regard, it should be noted that codons for other amino acids such as selenocysteine or pyrrolysine (amino acids other than the 20 commonly occurring naturally occurring amino acids) can also be incorporated into the nucleic acid of the ABP.As described in Wang, L., et al. (2001) Science 292, 498 - 500 or Wang, L., and Schultz, P.G. (2002) Chem. Comm. 1, 1 - 11, it is also possible to use "artificial" codons such as UAG, which are normally recognized as stop codons, to insert other rare amino acids, for example, o - methyl - L - tyrosine or p - aminophenylalanine.

[0094] For example, the use of nucleotide building blocks with low base - pair specificity, such as inosine, 8 - oxo - 2'deoxyguanosine or 6(2 - deoxy - β - D - ribofuranosyl) - 3,4 - dihydro - 8H - pyrimido[1,2 - a]oxazin - 7 - one (Zaccolo et al. (1996) J. Mol. Biol. 255, 589 - 603), is another option for introducing mutations into a selected sequence segment. A further possibility is so - called triplet mutagenesis. In this method, a mixture of different nucleotide triplets (each encoding one amino acid for incorporation into the coding sequence) is used (Virnekas B, et al., 1994 Nucleic Acids Res 22, 5600 - 5607).

[0095] The nucleic acid molecules encoding chains such as the heavy chain and / or light chain of the antibody according to the present invention can be expressed using any suitable expression system, for example, in a suitable host cell or cell - free system. The resulting ABP can be concentrated by selection and / or isolation. Preferably, the nucleic acids of the present invention are provided as gene constructs such as vectors / plasmids.

[0096] A nucleic acid system, that is, a construct containing such nucleic acids of the present invention is further provided, where the system of the present invention contains at least two nucleic acids of the present invention, each of these nucleic acids encoding one monomer of the ABP of the present invention, for example, one nucleic acid encodes the heavy - chain sequence and the second nucleic acid encodes the light - chain sequence.

[0097] In some embodiments, the polypeptide of the ABP of the present invention can be encoded by a nucleic acid for expression in vivo or in vitro. Accordingly, in some embodiments, an isolated nucleic acid encoding the ABP of the present invention is provided. In some embodiments, the nucleic acid encodes a portion or monomer of the ABP of the present invention (e.g., one of the two chains (heavy chain and light chain) of an antibody), and / or another nucleic acid encodes another portion or monomer of the ABP of the present invention (e.g., the other of the two chains of an antibody). Such nucleic acids can be provided in combination or together as one system. In some embodiments, the nucleic acid encodes two or more ABP polypeptide chains, e.g., at least two antibody chains. The nucleic acid encoding multiple ABP chains can include a nucleic acid cleavage site between at least two chain sequences, can encode a transcription start site or a translation start site between two or more chain sequences, and / or can encode a proteolytic target site between two or more ABP chains.

[0098] In one related aspect, the present invention relates to a nucleic acid construct (NAC) comprising at least one nucleic acid of the present invention (e.g., as described above). Such a NAC can include one or more additional features that enable the expression of the encoded ABP or components of said ABP in a cell (e.g., a host cell). Examples of the NAC of the present invention include, but are not limited to, plasmid vectors, viral vectors, mRNA, non-episomal mammalian vectors, and expression vectors, e.g., recombinant expression vectors. The nucleic acid construct of the present invention can contain the nucleic acid of the present invention in a form suitable for its expression in a cell such as a host cell (see below). The nucleic acid construct of the present invention is typically a recombinant nucleic acid and / or can be isolated and / or substantially pure. A recombinant nucleic acid is typically non-natural, especially when it contains portions derived from different species and / or synthetic, in vitro or mutagenic methods.

[0099] In some embodiments, the NAC of the present invention includes one or more constructs, any of which contains a nucleic acid encoding the heavy or light chain of an antibody. In some embodiments, the NAC of the present invention includes two constructs, one of which contains a nucleic acid encoding the heavy chain of an antibody and the other contains a nucleic acid encoding the light chain of an antibody, such that a complete antibody molecule can be generated by expression from both constructs. In some embodiments, the NAC of the present invention includes a construct containing a nucleic acid encoding both the heavy and light chains of an antibody, such that a complete antibody molecule can be expressed from one construct. In other embodiments, the NAC of the present invention can include a single construct encoding a single strand sufficient to form the ABP of the present invention, for example, when the encoded ABP is a scFv or a single domain antibody (e.g., a camelid antibody).

[0100] Furthermore, one further aspect of the present invention provides a vector (e.g., an expression vector) containing a nucleic acid encoding an ABP or a part or monomer of an ABP as disclosed herein. For example, in some embodiments where the ABP is a multimeric protein, the nucleic acid encodes only one polypeptide chain of the antigen construct. Thus, to express such an antigen-binding construct, the expression vector of the present invention can contain two or more nucleic acids each encoding a distinct part or monomer of the ABP, which together express the whole ABP. Similarly, an expression vector of the present invention containing a nucleic acid encoding only a part or monomer of an antigen-binding construct can be used in combination with other distinct expression vectors of the present invention each encoding a distinct part or monomer of the ABP. In other embodiments, the nucleic acid encodes multiple polypeptide chains of the ABP of the present invention. In some embodiments, the expression vector includes pcDNA3.1 for expression in mammals TMThe / myc-His(-) version A vector (Invitrogen, Inc.) or a variant thereof is included. The pcDNA3.1 expression vector features a CMV promoter for expression in mammals and both a mammalian (neomycin) selection marker and a bacterial (ampicillin) selection marker. In some embodiments, the expression vector includes a plasmid. In some embodiments, the vector includes a viral vector, e.g., a retroviral vector or an adenoviral vector. In an embodiment, the vector includes a cosmid, YAC or BAC.

[0101] In another related aspect, the invention relates to a cell (e.g., a host cell and / or a recombinant host cell) comprising one or more nucleic acids of the invention. Preferably, such a cell can express the ABP (or a component thereof) encoded by the nucleic acid. For example, if the ABP of the invention comprises two separate polypeptide chains (e.g., the heavy and light chains of IgG), the cell of the invention may comprise a first nucleic acid encoding (and capable of expressing) the heavy chain of such an ABP and a second nucleic acid encoding (and capable of expressing) the light chain of such an ABP, or alternatively, the cell may comprise a single nucleic acid encoding both chains of such an ABP. Thus, such a cell of the invention will be able to express the functional ABP of the invention. The (host) cell of the invention can be one of the mammalian, prokaryotic or eukaryotic host cells as described elsewhere herein, and in particular, the cell is a Chinese hamster ovary (CHO) cell.

[0102] In certain embodiments of such aspects, the (host) cell is a human cell, and in particular, can be a human cell (e.g., autologous human cell) sampled from a particular individual. In such embodiments, such human cells can be grown and / or manipulated in vitro for introducing the nucleic acids of the invention. The utility of the engineered human cells derived from a particular individual can be, for example, in making the ABP of the invention, including reintroducing such a population of engineered human cells into a human subject, such as for use in therapy. In certain such uses, the engineered human cells can be introduced into the same human individual from whom they were initially sampled, e.g., as autologous human cells.

[0103] The human cells subjected to such manipulations can be of any germ cell type or somatic cell type in the body. For example, donor cells can be somatic or germ cells selected from the group consisting of fibroblasts, B cells, T cells, dendritic cells, keratinocytes, adipocytes, epithelial cells, epidermal cells, chondrocytes, cumulus cells, nerve cells, glial cells, astrocytes, heart cells, esophageal cells, muscle cells, melanocytes, hematopoietic cells, macrophages, monocytes, and mononuclear cells. Donor cells can be obtained from any organ or tissue in the body, and can be, for example, cells derived from an organ selected from the group consisting of the liver, stomach, intestine, lung, pancreas, cornea, skin, gallbladder, ovary, testis, kidney, heart, bladder, and urethra. Pharmaceutical composition

[0104] The present invention also provides a pharmaceutical composition comprising the ABP of the present invention, and optionally, a pharmaceutically acceptable excipient and / or carrier.

[0105] For use in therapy, the ABP, nucleic acid or NAC (or a cell such as a host cell) of the present invention can be formulated into a pharmaceutical composition suitable for facilitating administration to an animal or human. The term "pharmaceutical composition" means a mixture of substances containing a therapeutically active substance (e.g., the ABP of the present invention) for pharmaceutical use.

[0106] As an example, the pharmaceutical composition of the present invention may contain 0.1% to 100% (w / w) of an active ingredient (for example, the ABP of the present invention), for example, about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 8%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%, preferably about 1% to about 20%, about 10% to 50% or about 40% to 90%.

[0107] The ABP according to the present invention can be administered via any parenteral or non-parenteral (enteral) route that is therapeutically effective for protein drugs. Examples of parenteral application methods include intradermal, subcutaneous, intramuscular, intratracheal, intranasal, intravitreal or intravenous injection and infusion techniques in the form of injection solutions, infusion solutions or tinctures, etc., and aerosol injection and inhalation in the form of aerosol mixtures, sprays or powders, etc. An overview of drug delivery to the lungs, i.e., delivery via either inhalation of an aerosol (which can also be used for intranasal administration) or intratracheal injection, is provided, for example, in J.S. Patton et al. The lungs as a portal of entry for systemic drug delivery. Proc. Amer. Thoracic Soc. 2004 Vol.1 pages 338 - 344. Non-parenteral delivery modes are, for example, oral modes in the form of pills, tablets, capsules, solutions or suspensions, etc., or rectal modes in the form of suppositories. The ABP of the present invention can be administered systemically or locally as a formulation containing conventional non-toxic pharmaceutically acceptable excipients or carriers, additives and vehicles as desired.

[0108] In one embodiment of the present invention, the pharmaceutical is administered parenterally to a mammal, particularly a human. Corresponding administration methods include, for example, intradermal, subcutaneous, intramuscular, intratracheal or intravenous injection and infusion techniques in the form of an injection solution, an infusion solution or a tincture, and aerosol infusion and inhalation in the form of an aerosol mixture, a spray or a powder, but are not limited thereto. A combination of intravenous and subcutaneous infusion and / or injection may be most convenient in the case of compounds with a relatively short serum half-life. The pharmaceutical composition can be an aqueous solution, an oil-in-water emulsion or a water-in-oil emulsion.

[0109] In this regard, it should be noted that transdermal delivery techniques such as those described in Meidan VM and Michniak BB 2004 Am.J.Ther.11(4):312-316, for example, delivery enhanced by iontophoresis, sonophoresis or microneedles, can also be used for the transdermal delivery of ABP described herein. Non-parenteral delivery modes are, for example, oral modes in the form of tablets, pills, capsules, solutions or suspensions, or rectal administration in the form of suppositories. The ABP of the present invention can be administered systemically or locally as a formulation containing various conventional non-toxic pharmaceutically acceptable excipients or carriers, additives and vehicles.

[0110] The dosage of ABP applied can vary within a wide range to achieve the desired preventive effect or therapeutic response. It depends, for example, on the affinity of the ABP for the selected target, as well as the in vivo half-life of the complex of the ABP and the ligand. Furthermore, the optimal dosage depends on the in vivo distribution of the ABP or its conjugate, the mode of administration, the severity of the disease / disorder being treated, and the patient's medical condition. For example, when used as an ointment for topical application, a high concentration of ABP can be used. However, if required, the ABP can also be administered as a sustained-release formulation, such as a liposome dispersion or a hydrogel-based polymer microsphere like PolyActiveTM or OctoDEXTM (see Bos et al, Business Briefing: Pharmatech 2003: 1-6). Other available sustained-release formulations are, for example, PLGA-based polymers (PR pharmaceuticals), PLA-PEG-based hydrogels (Medincell), and PEA-based polymers (Medivas).

[0111] Accordingly, the ABP of the present invention can be formulated into a composition using pharmaceutically acceptable ingredients and established preparation methods (Gennaro, A.L. and Gennaro, A.R. (2000) Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wilkins, Philadelphia, PA). For preparing a pharmaceutical composition, pharmaceutically inert inorganic excipients or organic excipients can be used. For example, for preparing tablets, powders, gelatin capsules, or suppositories, lactose, talc, stearic acid and its salts, fats, waxes, solid or liquid polyols, natural oils, and hardened oils can be used. Suitable excipients for making solutions, suspensions, emulsions, aerosol mixtures, or powders that are reconstituted into a solution or aerosol mixture before use include water, alcohol, glycerol, polyols, and their suitable mixtures, and vegetable oils.

[0112] The pharmaceutical composition may also contain additives such as fillers, binders, wetting agents, lubricants, stabilizers, preservatives, emulsifying agents, and further solvents or solubilizing agents or agents for achieving a depot effect or the active substance. The latter are those in which the fusion protein can be incorporated into a sustained release system or a controlled release system or a targeted delivery system such as liposomes and microcapsules.

[0113] The above preparations can be sterilized by numerous means including filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterilized solid composition which can be dissolved or dispersed in sterile water or other sterile media immediately before use.

[0114] For ease of administration and for the uniformity of dosage, it is particularly advantageous to formulate oral, rectal or parenteral compositions in dosage unit form. As used herein, the dosage unit form includes physically discrete units suitable as unit doses for the subject to be treated. Each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect together with the required pharmaceutical carrier. The specification of the dosage unit forms of the present invention is defined by, and directly dependent on, the unique characteristics of the active compound and the particular therapeutic effect to be achieved and the limitations inherent in the art of compounding such active compounds for the treatment of individuals.

[0115] In some embodiments, a pharmaceutical composition comprising the ABP or other component (e.g., nucleic acid or NAC) of the present invention is in the form of a unit dose of 10 to 1000 mg of the ABP or other component of the present invention. In some embodiments, a pharmaceutical composition comprising the ABP or other component of the present invention is in the form of a unit dose of 10 to 200 mg of the ABP or other component. In some embodiments, a pharmaceutical composition comprising ABP is in the form of a unit dose of 200 to 400 mg of the ABP or other component. In some embodiments, a pharmaceutical composition comprising the ABP or other component is in the form of a unit dose of 400 to 600 mg of the ABP or other component. In some embodiments, a pharmaceutical composition comprising the ABP or other component is in the form of a unit dose of 600 to 800 mg of the ABP or other component. In some embodiments, a pharmaceutical composition comprising the ABP or other component is in the form of a unit dose of 800 to 1000 mg of the ABP or other component.

[0116] Exemplary unit dosage forms of a pharmaceutical composition comprising the ABP or other component are tablets, capsules (e.g., as powders, granules, minitablets or micropellets), suspensions or disposable pre-filled syringes. In certain embodiments, a kit for making a single dose administration unit is provided. The kit can include both a first container having the dried active ingredient and a second container having an aqueous formulation. Alternatively, the kit can include single chamber and multi-chamber pre-filled syringes.

[0117] The toxicity and therapeutic efficacy (e.g., effectiveness) of such active ingredients can be measured in cell cultures or experimental animals by standard pharmaceutical procedures, for example, to measure LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxicity and therapeutic efficacy is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Active substances showing a high therapeutic index are preferred. Compounds showing toxic side effects may also be used, but care should be taken to design a delivery system that targets such compounds to the diseased tissue site in order to minimize potential damage to non-infected cells and thereby reduce side effects.

[0118] Data obtained from cell culture assays and animal tests can be used, for example, in formulating an active ingredient (e.g., the ABP of the present invention or other components) in a range of dosages for use in humans. The dosage of such active ingredients is preferably within the range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration. For any active ingredient used in the therapeutic approach of the present invention, the (therapeutic) effective amount can first be estimated from cell culture assays. In animal models, the dosage can be formulated to achieve a range of circulating plasma concentrations that includes the IC50 (i.e., the concentration of the active ingredient that achieves 50% inhibition of the maximum symptoms) measured in cell culture. By using such information, a useful (e.g., effective) amount or dosage for administration to humans, for example, can be determined more accurately. The pharmaceutical composition can be included in a container, pack, or dispenser, together with instructions for administration.

[0119] In the context of the present invention, an effective amount of the ABP of the present invention or other components or pharmaceutical composition can be one that induces a biological, physiological, pharmacological, therapeutic or medical response in a cell, tissue, system, body, animal, individual, patient or human being being investigated by a researcher, scientist, pharmacologist, pharmacist, veterinarian, physician or other clinician, for example, alleviates the effects / symptoms of a disorder, disease or condition (e.g., a proliferative disorder, e.g., cancer or tumor), or kills or inhibits the growth of cells involved in a proliferative disorder (e.g., tumor cells). The effective amount thereof can be determined by standard procedures including the procedures described below.

[0120] According to any aspect and embodiment of the medical use and method of treatment provided herein, the effective amount administered at least once to a subject in need of treatment with the ABP of the present invention or other components is usually about 0.01 mg / kg to about 100 mg / kg per administration, for example, about 1 mg / kg to about 10 mg / kg per administration. In some embodiments, the effective amount of the ABP or other components administered at least once to a subject is about 0.01 mg / kg to about 0.1 mg / kg per administration, about 0.1 mg / kg to about 1 mg / kg per administration, about 1 mg / kg to about 5 mg / kg per administration, about 5 mg / kg to about 10 mg / kg per administration, about 10 mg / kg to about 50 mg / kg per administration or about 50 mg / kg to about 100 mg / kg per administration.

[0121] In the case of preventing or treating a disease, the appropriate dosage of ABP or other components (or a pharmaceutical composition containing them) depends on the type of disease being treated, the severity and course of the disease, whether ABP or other components and / or the pharmaceutical composition are administered for prophylactic purposes or for therapeutic purposes, previous treatments, the patient's medical history, age, size / weight and response to ABP or other components and / or the pharmaceutical composition, as well as the discretion of the attending physician. ABP or other components and / or the pharmaceutical composition are appropriately administered to the patient either once or over a series of treatments. When such ABP or other components and / or the pharmaceutical composition are administered over a series of treatments, the total number of administrations in a given treatment course can consist of about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than about 10 treatments. For example, a treatment can be administered once (or 2, 3 or 4 times a day) a day over a period of one week, one month or several months. In certain embodiments, the treatment course may continue indefinitely.

[0122] The amount of ABP or other component(s) administered and / or the pharmaceutical composition depends on variables such as the type and extent of the disease or indication being treated, the overall health status of the patient, age, size / weight, the in vivo efficacy of the ABP or other component(s) and / or the pharmaceutical composition, and the route of administration. The initial dose can be increased above the upper limit in order to rapidly achieve the desired blood or tissue level. Alternatively, the initial dose can be less than the optimal dose, and the daily dose can be gradually increased during the course of treatment. Human doses can be optimized, for example, in conventional Phase I dose escalation studies designed to be carried out from a relatively low initial amount, for example, from about 0.01 mg / kg to about 20 mg / kg of the active ingredient. The dosing frequency can vary depending on factors such as the route of administration, the dose, and the disease being treated. Exemplary dosing frequencies are once daily, once a week, and once every two weeks. The formulation of the ABP or other component(s) of the present invention (or for use with the present invention) is within the ordinary skill in the art. In some embodiments of the present invention, such ABP or other component(s) are lyophilized and reconstituted with buffered saline at the time of administration. The ABP or other component(s) and / or the pharmaceutical composition can further reduce the recurrence of the disease being treated, or can reduce the incidence of drug resistance, or can extend the time until drug resistance appears, and in the case of cancer, can extend the progression-free survival and / or overall survival. Treatment and Diagnosis of Diseases

[0123] The ABP of the present invention, as well as any nucleic acid, NAC, cell, or composition, can be suitable for the treatment and / or prevention of a disease in a subject and can be used therein. Preferably, such a disease is, for example, a pathological immune response of the subject in response to a self-antigen or a foreign (allogeneic) antigen. Thus, inflammatory and autoimmune diseases can likely be treated by the components of the present invention. In a related aspect, the present invention relates to a product or component for use in medicine, and to the use of a product for the manufacture of a medicament, the product being selected from the list consisting of the ABP, nucleic acid, NAC, or host cell of the present invention, or a pharmaceutical composition comprising any of the foregoing.

[0124] In other embodiments described elsewhere in this specification, methods for detecting and / or diagnosing a disease, disorder or condition in a mammalian subject are provided, preferably such methods comprising the step of detecting the binding of the ABP of the present invention to TIRC7 or TIRC7-positive cells.

[0125] The present invention also relates to recombinant cell lines or the ABP of the present invention, various methods for generating hybridomas or host cells capable of producing the ABP of the present invention, as well as various measurement methods and / or diagnostic methods, or uses, and kits useful for such measurement methods and / or diagnostic methods, and various methods for identifying and / or characterizing compounds and / or for identifying, preparing and / or generating the ABP, for example, methods suitable for use in medicaments.

[0126] As used herein, the term "treatment" is meant to include therapy, for example, therapeutic treatment, as well as prophylactic or suppressive measures against a disease (or disorder or condition). Thus, for example, successful administration of an ABP (or nucleic acid or NAC or cell, e.g., a host cell) prior to the onset of a disease results in treatment of that disease. "Treatment" also encompasses administration of an ABP after the disease has manifested in order to reverse or eradicate the disease (or its symptoms). Administration of an ABP after the onset and after clinical symptoms have appeared, where alleviation of clinical symptoms is expected and in some cases the disease may resolve, also constitutes treatment of the disease. Persons "in need of treatment" include subjects (e.g., human subjects) already having a disease, disorder or condition, as well as those who are likely to develop or are suspected of having a disease, disorder or condition (including those in whom the disease, disorder or condition is to be prevented).

[0127] Autoimmune diseases: The term "autoimmune disease" refers to a disease caused by the breakdown of self-tolerance in which the adaptive and innate immune systems respond to self-antigens, mediating cell and tissue damage. Autoimmune diseases are often characterized by the involvement of a single organ or single cell type or the involvement of multiple organs or tissue systems. Autoimmune diseases have also been referred to as "collagen" diseases or "collagen vascular" diseases or "connective tissue" diseases. Autoimmune disorders are often associated with hypersensitivity reactions. The components of the present invention may be useful for the treatment and / or prevention of various types of autoimmune diseases.Non-limiting specific examples of autoimmune disorders include systemic lupus erythematosus, insulin-dependent (type I) diabetes, inflammatory arthritis, rheumatoid arthritis, multiple sclerosis, autoimmune hepatitis, chronic aggressive hepatitis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, autoimmune atrophic gastritis of pernicious anemia, autoimmune encephalomyelitis, encephalitis, autoimmune orchitis, autoimmune pancreatitis, acquired hemophilia, ankylosing spondylitis, antiphospholipid antibody syndrome, alopecia areata, Lyme disease, lichen sclerosus, Behçet's syndrome, myocarditis, inclusion body myositis, autoimmune myocarditis, anti-GBM nephritis, Still's disease, bullous pemphigoid, polyarteritis nodosa, psoriatic arthritis, fibromyalgia, rheumatic fever, sarcoidosis, systemic sclerosis, chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, Hashimoto's thyroiditis, Takayasu arteritis, temporal arteritis / giant cell arteritis, Graves' disease, immune thrombocytopenic purpura, interstitial cystitis, juvenile arthritis, juvenile myositis, type 1 diabetes, thyroid eye disease, IgA nephropathy, subacute bacterial endocarditis, Goodpasture's syndrome, fibrosing alveolitis, relapsing polychondritis, eosinophilic fasciitis, esophagitis, endometriosis, primary sclerosing cholangitis, dermatitis, polymyalgia rheumatica, Kawasaki disease, cicatricial pemphigoid, cold agglutinin disease, polymyositis, dermatomyositis, discoid lupus, sympathetic ophthalmia, essential mixed cryoglobulinemia, fibromyalgia, fibromyositis, Guillain-Barré syndrome, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, juvenile arthritis, systemic sclerosis, polyarteritis nodosa, polychondritis, dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, high IgE, systemic progressive sclerosis, psoriasis, Reiter's syndrome, sarcoidosis, stiff-person syndrome, uveitis or uveitis associated with tubulointerstitial nephritis, vasculitis, pemphigus, optic neuritis, undifferentiated inflammatory connective tissue disease, transverse myelitis, Tolosa-Hunt syndrome, scleritis, sympathetic ophthalmia, vitiligo, Hashimoto's thyroiditis, Goodpasture's disease, pernicious anemia, Addison's disease, dermatomyositis, Sjögren's syndrome, dermatomyositis, myasthenia gravis, Graves' disease, allergic encephalomyelitis, glomerulonephritis, celiac disease, Chagas' disease, fibrotic diseases, etc. (N Engl J Med, Vol. 345, No. 5, August 2, 2001, P340-350).DNA or RNA released from DNA-containing or RNA-containing microorganisms may stimulate the production of autoantibodies specific to their own RNA-containing or DNA-containing complexes, and as a result, may lead to autoimmune diseases including but not limited to SLE.

[0128] The autoimmune diseases that can be treated by the ABP of the present invention or another component disclosed herein are diseases that can be treated by inhibiting and / or reducing the T cell-mediated immune response. For example, such diseases that can be treated and / or prevented using the ABP of the present invention are selected from the diseases that benefit from the immunosuppressive effects of the exemplary ABP shown in Figure 8 herein.

[0129] Preferred autoimmune diseases that can be treated or prevented by the components and methods of the present invention are selected from the group consisting of autoimmune hepatitis, IgG4-related autoimmune diseases, pulmonary fibrosis, Sjogren's syndrome, systemic lupus erythematosus, uveitis or uveitis associated with tubulointerstitial nephritis, vasculitis, chronic fatigue syndrome, and systemic sclerosis.

[0130] The ABP or other components of the present invention are further useful for treating immunological side effects caused by certain treatments, such as treatments that can induce an unwanted immune response (e.g., a cellular immune response, e.g., a T cell response) in a patient undergoing such treatment. Any treatment with a biological complex such as a CAR, antibody, T cell receptor, or any other large protein may cause the patient's immune system to "attack" the therapeutic agent as a potential foreign threat. For example, a typical side effect in cancer patients undergoing treatment with a therapeutic antibody, chimeric antigen receptor, or immune checkpoint inhibitor is the occurrence of a harmful immune response known as a cytokine storm. Therefore, preferably, autoimmune diseases are pathological immune responses caused by a first-choice treatment such as a cytokine storm.

[0131] Furthermore, in certain circumstances, B cell-mediated diseases such as Common Variable Immunodeficiency (CVID), an idiopathic immune deficiency disorder that is heterogeneous and poorly understood, characterized by humoral immunodeficiency and immunoregulatory defects, can also be treated with the ABP or other components of the present invention.

[0132] "Allergy": Allergy refers to a disorder in which tissue damage occurs as a result of a humoral or cellular response to antigens of endogenous or exogenous origin, and is classified into four types. Type I allergy (also often referred to as anaphylactic, immediate, atopic, reagenic, IgE-mediated, allergic reaction or allergy) generally results from the release of pharmacologically active substances such as histamine, slow-reacting substance of anaphylaxis (SRS-A) and eosinophil chemotactic factor (ECF) from IgE-sensitized basophils and mast cells after contact with specific exogenous antigens. Type I allergy includes, but is not limited to, allergic extrinsic asthma, seasonal allergic rhinitis and systemic anaphylaxis. Type II allergy (also referred to as cytotoxic, cytolytic, complement-dependent or cell-stimulating allergic reaction) occurs when antibodies react with antigenic components of cells or tissue elements, or antigens or haptens that have become closely bound to cells or tissue. Examples of type II allergy include, but are not limited to, autoimmune hemolytic anemia, erythroblastosis fetalis and Goodpasture's disease. Type III allergy (also referred to as allergic reaction due to toxic complexes, soluble complexes or immune complexes) is caused by the deposition of soluble circulating antigen-antibody complexes in blood vessels or tissues, accompanied by an acute inflammatory reaction at the site of deposition of the immune complexes. Examples of type III allergy include, but are not limited to, the Arthus reaction, serum sickness, systemic lupus erythematosus and certain types of glomerulonephritis. Type IV allergy (also often called cell-mediated hypersensitivity reaction, cellular hypersensitivity reaction, delayed-type hypersensitivity reaction or tuberculin-type hypersensitivity reaction) is caused by sensitized T lymphocytes resulting from contact with specific antigens. Examples of type IV allergy include, but are not limited to, contact dermatitis and allograft rejection (Richard A. et al. Immunology, Fifth Edition, 2003, W.H. FREEMAN AND COMPANY).

[0133] "Diseases associated with excessive stimulation of the host immune system by microorganisms": The invasion of microorganisms can, in severe cases, sometimes cause a systemic inflammatory response in a subject, leading to diseases associated with excessive stimulation of the host immune system by microorganisms. Events occurring during the onset of such diseases, such as in the case of influenza A (H5N1) or bacterial infections, include significantly elevated blood concentrations of TNFa, interleukin-1 (IL-1), IL-6, IL-12, interferon alpha (IFN-a), interferon beta (IFN-β), interferon gamma (IFN-γ), chemokines, interferon-induced protein 10, monocyte chemoattractant protein 1, interleukin-8, interleukin-1β, and monocyte chemoattractant protein 1. Such responses can lead to cytokine-mediated lethal shock, which is partially involved in sepsis, ARDS, and multiple organ failure observed in many patients (The Writing Committee of the World Health Organization (WHO) Consultation on Human Influenza A H5. Avian Influenza A (H5N1) Infection in Humans. N Engl J Med 2005;353:1374-85). A significant increase in the blood concentration of cytokines after microbial infection is called hypercytokinemia or cytokine storm. Studies have suggested that patients exposed to avian influenza or SARS may require drugs that suppress the immune response in addition to antiviral drugs. Therefore, the ABP of the present invention may be useful for the treatment of viral diseases associated with an overactive immune response, such as COVID-19 caused by SARS-CoV2 infection.

[0134] The compounds of the present invention can be used for treating and / or preventing diseases associated with stimulation of the host immune system by microorganisms in a subject. Microorganisms that cause these diseases include, but are not limited to, viruses, bacteria, fungi, parasites, and the causative agents of spongiform encephalopathies.

[0135] Viruses that cause diseases associated with overstimulation of the host immune system by microorganisms include SARS CoV, especially SARS-CoV2, influenza virus, avian influenza virus, HIV-1, poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, echovirus, equine encephalitis virus, rubella virus, dengue virus, encephalitis virus, yellow fever virus, coronavirus, vesicular stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, influenza virus, hantavirus, bunga virus, phlebovirus, nairovirus, hemorrhagic fever virus, reovirus, orbivirus and rotavirus, hepatitis B virus, parvovirus, papillomavirus, polyomavirus, adenovirus, herpes simplex virus (HSV) 1 and HSV-2, varicella-zoster virus, cytomegalovirus (CMV), herpesvirus, smallpox virus, vaccinia virus, poxvirus, African swine fever virus, causative agent of spongiform encephalopathy, hepatitis D virus, hepatitis C virus, foot-and-mouth disease virus and avian influenza virus.Diseases associated with excessive stimulation of the host immune system by microorganisms, such as sepsis, can be caused by bacteria including Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacterium species (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Group A streptococcus, Group B streptococcus, Streptococcus spp., Streptococcus faecalis, Streptococcus bovis, Streptococcus spp. (anaerobic species), Streptococcus pneumoniae, pathogenic Campylobacter spp., Enterococcus spp., Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium spp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogeytes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides spp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira spp. and Actinomyces israelii. Fungi that can cause diseases associated with excessive stimulation of the host immune system by microorganisms include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.Parasites that can cause diseases associated with overstimulation of the host immune system by microorganisms include Plasmodium falciparum and Toxoplasma gondii.

[0136] "Graft rejection": Graft rejection (graft-versus-host disease) is an immune-mediated disorder caused by organ transplantation or tissue transplantation. Transplantation means the transfer of transplanted tissue (graft) from a donor to a recipient. A graft is a living cell, tissue or organ transplanted from a donor to a recipient. An autograft is a graft of one's own tissue moved from one location to another, an isograft is a graft between identical twins, an allograft (homograft) is a graft between genetically dissimilar members of the same species, and a xenograft (heterograft) is a transplanted tissue between members of different species. When a subject is a recipient of an allograft or xenograft, their body can mount an immune response against the donor tissue. In this situation, there is a clear need to suppress that immune response to avoid rejection of the graft (Richard A. et al. Immunology, Fifth Edition, 2003, W.H. FREEMAN AND COMPANY). Examples of grafts include the heart, kidney, liver, bone marrow, skin, cornea, lung, pancreas, small intestine (intestinum tenue), limb, muscle, nerve, duodenum, small bowel, pancreatic islet cells, stem cells, etc.

[0137] The subject treated with the above fusion protein can be a human or a non-human animal. Such animals are preferably mammals, such as humans, pigs, cows, rabbits, mice, rats, primates, goats, sheep, chickens or horses, and most preferably humans.

[0138] The ABP of the present invention can also be used for the diagnosis of diseases such as diseases as described herein. The ABP can be labeled with a suitable detectable signal label for this purpose. Such labeled ABP can enable the detection or quantification of TIRC7 levels, or cancer such as leukemia or any of the cancers described above, or a subject. When designed for in vivo use, the detectable signal label is preferably a label detectable in vivo.

[0139] The labeled ABP can be used in immunoimaging methods. And its detectable signal label can be selected, for example, based on the immunoimaging method used for diagnosis. For example, in the case of gamma camera imaging method / SPECT, it can be a radionuclide (or gamma emitter) that emits gamma rays, and in the case of MRI or PET imaging method, it can be a metal or a positron emitter, respectively. In this regard, one or more detectable signal labels of the present disclosure include agents that can be imaged with a gamma camera, agents that can be imaged with PET, and agents that can be imaged with MRI, such as radionuclides, fluorophores, fluorogens, chromophores, chromogens, phosphors, chemiluminescent agents, and bioluminescent agents.

[0140] A suitable detectable signal label can be a radionuclide. The radionuclide can be selected from the group consisting of 3H, 14C, 35S, 99Tc, 123I, 125I, 131I, IIIIn, 97Ru, 67Ga, 68Ga, 72As, 89Zr, and 201T1.

[0141] A suitable detectable signal label may also be a fluorophore or a fluorogen. The fluorophore or fluorogen can be selected from the group consisting of fluorescein, rhodamine, dansyl, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, fluorescein derivatives, Oregon Green, Rhodamine Green, Rhodol Green, or Texas Red.

[0142] The ABP to be labeled can be directly or indirectly bound to a detectable signal label. For example, the ABP can be directly (e.g., via a tyrosine residue of the ABP) or indirectly (e.g., via a linker as a metal chelating agent) bound to the detectable signal label. In some other embodiments, the ABP can be bound to a molecule that can bind to a detectable signal label (in vitro or in vivo) at the time of use and at the place of use.

[0143] The detectable signal label can be bound to the ABP via one or more diethylenetriaminepentaacetic acid (DTPA) residues bound to the ABP.

[0144] An in vitro method for detecting or diagnosing a disease as defined herein is also contemplated by the present invention. Such a method can include contacting a sample obtained from a subject with a preferably labeled ABP of the present invention. The sample can be a blood, urine or cerebrospinal fluid sample, but preferably can be a liquid sample or a biopsy sample. The disease to be detected or diagnosed is preferably leukemia such as ALL or AML.

[0145] In a ninth aspect, the present invention relates to a method of modulating a cellular immune response in a human cell expressing human TIRC7, the method comprising contacting said cell with the components described in the seventh aspect in the presence of immune cells such as T cells, thereby modulating, preferably inhibiting, the cellular immune response.

[0146] In a tenth aspect, the present invention relates to a method for preventing and / or treating a disorder associated with a pathological immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of the components described in the seventh aspect, wherein the disorder associated with the pathological immune response is characterized by the expression of TIRC7 in cells associated with the disorder.

[0147] Furthermore, the present invention relates to the following series of item-by-item embodiments. Item 1: (i) comprising CDRH1 (GYTFTTYV) of SEQ ID NO: 01, CDRH2 (INPYNDGT) of SEQ ID NO: 02 and CDRH3 (AEFITKTVGGSNWYLDV) of SEQ ID NO: 03, or, independently in each case, where CDRH1, CDRH2 and / or CDRH3 each comprise a sequence having 3 or fewer, preferably 1 or fewer, amino acid substitutions, deletions or insertions compared to SEQ ID NO: 01, SEQ ID NO: 02 or SEQ ID NO: 03, one, preferably two, heavy chain variable regions, and (ii) an antigen-binding protein (ABP) capable of binding to T cell immune response cDNA 7 (TIRC7), comprising CDRL1 (SSISY) of SEQ ID NO: 05, CDRL2 (DTS) of SEQ ID NO: 06 and CDRL3 (HQRSSYTWT) of SEQ ID NO: 07, or, independently in each case, where CDRL1, CDRL2 and / or CDRL3 each comprise a sequence having 3 or fewer, preferably 1 or fewer, amino acid substitutions, deletions or insertions compared to SEQ ID NO: 05, SEQ ID NO: 06 or SEQ ID NO: 07, one, preferably two, light chain variable regions, wherein one, preferably two, of its heavy chain variable domains and one, preferably two, of its light chain variable regions each comprise an antibody framework region having at least a portion of a human antibody consensus framework sequence, Antigen-binding protein (ABP). Item 2: The ABP according to Item 1, wherein the human antibody consensus framework sequence of the heavy chain variable region is derived from IGHV1-2 (accession number X07448), IGHV7-4-1*02 or IGHV1-46*01, and / or the human antibody consensus framework sequence of the light chain variable region is derived from IGKV1-9 (accession number Z00013), IGKV3-11 (accession number X01668), IGKV6-21*01 or IGKV1-17*03. Item 3: The ABP according to Item 1 or 2, wherein the ABP comprises one, preferably two, antibody heavy chain variable sequences, and in one, preferably two, of its antibody heavy chain variable sequences, one of positions 78, 80, 82 and / or 85 is deleted or mutated, and the numbering follows the IMGT nomenclature. Item 4: The ABP comprises one, preferably two, antibody heavy chain variable sequences, one of which, preferably two, antibody heavy chain variable sequences comprises 78L, 80S, 82K and / or 85S, the numbering of which follows the IMGT nomenclature, the ABP according to any one of Items 1 to 3. Item 5: The ABP comprises one, preferably two, antibody heavy chain variable sequences, one of which, preferably two, antibody heavy chain variable sequences comprises 78L, the numbering of which follows the IMGT nomenclature, the ABP according to any one of Items 1 to 3. Item 6: The heavy chain variable region comprises an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 or 85, or independently in each case, optionally, contains 10, 9, 8, 7, 6, 5, 4 or fewer, preferably 3, 2 or 1 or fewer amino acid substitutions, insertions or deletions compared to these sequences, and / or the light chain variable region comprises an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 or 86, or independently in each case, optionally, contains 10, 9, 8, 7, 6, 5, 4 or fewer, preferably 3, 2 or 1 or fewer amino acid substitutions, insertions or deletions compared to these sequences, the ABP according to any one of Items 1 to 5. Item 7: The ABP is an antibody or an antigen-binding fragment thereof, composed of at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences, the heavy chain sequence and light chain sequence of which each contain variable region sequences in one of the following combinations, the ABP according to any one of Items 1 to 6. JPEG0007691977000002.jpg Item 8: The ABP according to any one of Items 1 to 7, comprising an effector group and / or being labeled. Item 9: The ABP according to any one of Items 1 to 8, which is isolated and / or substantially pure. Item 10: The ABP according to any one of Items 1 to 9, which is an antibody such as a monoclonal antibody or an antibody fragment such as a fragment of a monoclonal antibody. Item 11: The ABP according to Item 10, wherein the antibody is a chimeric antibody such as a human chimeric antibody. Item 12: The ABP according to Item 10 or 11, wherein the antibody is an IgG, IgE, IgD, IgA or IgM immunoglobulin, preferably an IgG immunoglobulin. Item 13: The ABP according to any one of Items 10 to 12, which is an antibody fragment selected from the list consisting of Fab, Fab’-SH, Fv, scFv and F(ab’)2. Item 14: The ABP according to any one of Items 1 to 13, wherein the ABP is modified or engineered to enhance antibody-dependent cell cytotoxicity (ADCC), preferably the ABP is defucosylated. Item 15: The ABP according to any one of Items 1 to 14, which is a chimeric antigen receptor (CAR). Item 16: The ABP according to any one of Items 1 to 15, comprising one or more additional antigen-binding domains that bind to an antigen other than TIRC7, such as an antigen present on mammalian T cells, most preferably human CD3. Item 17: The ABP according to Item 16, which is bispecific, preferably comprising one or two binding sites that bind to TIRC7 and one or two binding sites that bind to an antigen other than TIRC7, such as an antigen present on mammalian T cells, most preferably human CD3. Item 18: An antigen-binding protein (ABP) or an antigen-binding fragment thereof that can bind to human TIRC7 and can compete with the binding of the ABP according to any one of Items 1 to 17 to TIRC7. Item 19: An isolated nucleic acid comprising an ABP or an antigen-binding fragment or monomer of an ABP as described in any one of Items 1 to 18, for example, a sequence encoding a heavy chain or a light chain. Item 20: A nucleic acid construct (NAC) comprising the nucleic acid as described in Item 19 and one or more further sequence features enabling the expression of the encoded ABP or a component of the ABP (for example, a heavy chain or a light chain of an antibody) in a (host) cell. Item 21: A recombinant host cell comprising the nucleic acid as described in Item 19 or the NAC as described in Item 20. Item 22: A pharmaceutical composition comprising (i) an ABP as described in any one of Items 1 to 18, or (ii) the nucleic acid as described in Item 19 or the NAC as described in Item 20, or (iii) the recombinant host cell as described in Item 21, and a pharmaceutically acceptable carrier, stabilizer and / or excipient. Item 23: A component for use in a medicament, the component being selected from the list consisting of an ABP as described in any one of Items 1 to 18, the isolated nucleic acid as described in Item 19, the NAC as described in Item 20, the recombinant host cell as described in Item 21, and the pharmaceutical composition as described in Item 22. Item 24: The component for use as described in Item 23, wherein the component is for use in regulating the cellular immune response of a subject. Item 25: The component for use as described in Item 24, wherein the regulation of the immune response is an inhibition of the cellular immune response. Item 26: The component for use as described in Item 25, wherein the inhibition is an inhibition of the proliferation of immune cells such as lymphocytes and / or an inhibition of cytokine expression in immune cells such as lymphocytes. Item 27: The component for use as described in Item 24 or 25, wherein the inhibition of the cellular immune response is a reduction in the proliferation / activity of effector memory T cells and / or an increase in the proliferation / activity of regulatory T cells (TREG). Item 28: The component for use as described in any one of Items 23 to 27, wherein the use is for treating a pathological immune response, preferably for use in treating an inflammatory disease, an autoimmune disease, allograft rejection (graft-versus-host disease). Item 29: A method for regulating the cellular immune response in human cells expressing human TIRC7, the method comprising contacting the cells with the component according to item 23 in the presence of immune cells such as T cells, thereby regulating, preferably inhibiting, the cellular immune response. Item 30: A method for preventing and / or treating a disorder associated with a pathological immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of the component according to item 23, wherein the disorder associated with the pathological immune response is characterized by the expression / activity of TIRC7 in the cells associated with the disorder.

[0148] As used herein, the terms "[the] invention", "according to the invention", "relating to the invention", etc. are intended to refer to all aspects and embodiments of the invention described and / or claimed herein.

[0149] As used herein, the term "comprising" should be interpreted to include both "including" and "consisting of", and both of these meanings are clearly intended according to the present invention, and thus are embodiments that are separately disclosed. "And / or", as used herein, should be interpreted as a clear disclosure of each of two specific features or components, with or without the other. For example, "A and / or B" should be interpreted as a clear disclosure of (i) A, (ii) B, and (iii) A and B, as if each were separately shown herein. In the context of the present invention, the terms "about" and "approximately" refer to the range of accuracy that a person skilled in the art understands to still ensure the technical effect of the feature in question. This term typically indicates a deviation of ±20%, ±15%, ±10%, and for example ±5% from the stated numerical value. As will be recognized by those skilled in the art, such a specific deviation of a given numerical value for a given technical effect depends on the nature of that technical effect. For example, a natural or biological technical effect may generally have a greater such deviation than an artificial or engineering technical effect. As will be recognized by those skilled in the art, such a specific deviation of a given numerical value for a given technical effect depends on the nature of that technical effect. For example, a natural or biological technical effect may generally have a greater such deviation than an artificial or engineering technical effect. When using an indefinite or definite article, such as "a", "an", or "the", when referring to a singular noun, this includes a plurality of that noun, unless something else is clearly stated.

[0150] It should be understood that applying the teachings of the present invention to a particular problem or environment, and including variations of the present invention or its further features (e.g., further aspects and embodiments), would be within the capabilities of those skilled in the art in light of the teachings included herein.

[0151] Unless the context indicates otherwise, the descriptions and definitions of the features set forth above are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0152] All references, patents, and publications cited in this specification are hereby incorporated by reference in their entirety.

[0153] In view of the above, it will be recognized that the present invention also relates to the following itemized embodiments.

Brief Description of the Drawings

[0154] Brief Description of the Drawings and Arrangements The drawings show the following.

[0155]

Figure 1

[0156]

Figure 2

[0157]

Figure 3

[0158]

Figure 4

[0159]

Figure 5

[0160]

Figure 6

[0161]

Figure 7

[0162]

Figure 8

[0163]

Figure 9

[0164]

Figure 10

[0165]

Figure 11

[0166]

Figure 12

[0167] All ABPs of the present invention are described in the sequence listing and Table 1 below. [Table 1] JPEG0007691977000004.jpg201140JPEG0007691977000005.jpg194140JPEG0007691977000006.jpg190140JPEG0007691977000007.jpg194140JPEG0007691977000008.jpg190140JPEG0007691977000009.jpg194140JPEG0007691977000010.jpg190140JPEG0007691977000011.jpg194140JPEG0007691977000012.jpg190140JPEG0007691977000013.jpg194140JPEG0007691977000014.jpg190140JPEG0007691977000015.jpg60140

[0168] Examples Hereinafter, certain aspects and embodiments of the present invention will be described by way of example with reference to the descriptions, drawings, and tables shown in this specification. Such examples of the methods, uses, and other aspects of the present invention are merely representative examples and should not be construed as limiting the scope of the present invention to only such representative examples.

[0169] The examples are as follows.

[0170] Example 1: Preparation of Variant Anti-TIRC7 Antibody and Humanized Anti-TIRC7 Antibody

[0171] For the purpose of identifying complementarity-determining regions (CDRs) and analyzing the most matching germline sequences, the IMGT Domain Gap Align tool was used. http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi.

[0172] Based on the homology to the crystal structures of previously published antibodies, molecular models of the VH and VL domains were constructed using in-house software. PDB files can be provided upon request for viewing with any molecular visualization software. Images were generated using PyMol.

[0173] To enable cloning into Absolute Antibody® cloning and expression vectors, variable heavy and light chain domains with appropriate restriction enzyme recognition sites at the 5' and 3' termini were designed. The codons of the variable domain sequences were optimized for expression in human cells. After gene synthesis, the variable domains were cloned into the appropriate species and type of Absolute Antibody® vectors. The correct sequence was confirmed by Sanger sequencing using DNASTAR® Lasergene software to analyze the raw data. Once confirmed, plasmid DNA of the appropriate size was prepared to generate a sufficient amount of high-quality DNA for transfection.

[0174] HEK293 (human embryonic kidney 293) mammalian cells were passaged to a stage optimal for transient transfection. The heavy chain expression vector and the light chain expression vector were transiently transfected into the cells, and the cells were cultured for an additional 6 days. The culture was collected by centrifugation at 4000 rpm and filtered through a 0.22 μM filter. The first step of purification was performed by protein A affinity chromatography, eluting with citrate buffer pH 3.0 and then neutralizing with 0.5 M Tris, pH 9.0. Subsequently, the buffer of the eluted protein was exchanged to PBS using a desalting column. The antibody concentration was measured by UV spectroscopy and the antibody was concentrated if necessary.

[0175] The purity of the antibody was measured by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and HPLC (high performance liquid chromatography). SEC-HPLC was performed on an Agilent 1100 series instrument using an appropriate size exclusion column (SEC). The expression titer of the antibody was measured by protein A HPLC.

[0176] The VH and VL sequences of 17 - 20 were subjected to the IGMT Gap Align tool and analyzed against all known antibody germline sequences (Figure 1). CDR regions were assigned using the IMGT definition. As expected, the sequences aligned most closely to those of the mouse, specifically IGHV1-2, IGHV1-46*01 and IGHV7-4-1*02 for the heavy chain, and IGKV1-9, IGKV3-11, IGKV6-21*01 and IGKV1-17*03 for the light chain.

[0177] To enable humanization based on structural information, models were constructed for the 17 - 20 mouse VH and VL sequences as shown in Figure 2.

[0178] Those VH and VL sequences were aligned with the Absolute Antibody® database of human germline sequences. Table 1 shows the germline sequences selected as frameworks for humanization.

[0179] Tables 2 and 3: Germline sequences of the heavy and light chains selected as humanization frameworks, and their percent identity to the original mouse VH and VL sequences

Table 2

Table 3

[0180] The VH and VL sequences described above were subjected to a CDR grafting algorithm, and their CDRs were transferred from the mouse antibody 17-20 to selected human germline sequences. CDRs are defined as being mainly involved in binding to the antigen, but amino acids within the regions known as framework regions, which are outside these regions, can be directly involved in binding or play a role in correctly orienting the CDRs. Which framework amino acids among the original mouse amino acids to retain to maintain binding integrity was determined using a structure-based approach (Figures 3 and 6). Table 1 contains the sequences produced.

[0181] Example 2: Combinations of germlines and non-intuitive back mutations were required to obtain a functional humanized anti-TIRC7 antibody.

[0182] As described in Example 1, humanized heavy and light chains were designed. Each of these was synthesized separately and cloned into a human IgG1 heavy chain expression vector and a human kappa light chain expression vector, respectively. At the time of transfection, all possible combinations of their humanized sequences were made to produce a total of 12 different humanized antibodies. In addition to these, the original mouse antibody as well as a chimeric human IgG1 were produced as controls.

[0183] Most of the humanized chains produced either did not express, expressed at low monomer content, or lost activity. A total of five humanization attempts were required to identify some valid humanized sequences, and in addition to normal grafting procedures and back mutations near the CDR regions, further non-intuitive back mutations in the heavy chain sequence were required. This was surprising.

[0184] Alignments of some of the antibodies shown in Table 1 above are provided in Figures 4 and 5.

[0185] In one attempt to humanize the heavy chain, the inventors generated a humanized heavy chain using the IGHV7-4-1 human germline sequence. Unfortunately, none of the sequences generated were able to express at all (see Figure 7. Antibodies with heavy chain sequences cAB2714-VH or cAB2712-VH).

[0186] In a further attempt, the human germline sequence IGHV1-46 was used for transplantation. The chains obtained were cAB2288-VH and cAB2287-VH, of which cAB2287-VH was unable to express at all, while cAB2288-VH showed some expression. However, the antibody with the cAB2288-VH chain did not show a monomer content when expressed (Figure 7).

[0187] When IGHV1-2, another attempt, was used for transplantation, the cAB1901VH chain was obtained. The antibody with this chain was also unable to express at all.

[0188] In two further humanizations, chains based on all three of the germline sequences described above were generated by introducing various mutations at selected positions in order to combine human framework regions from different germline sequences (which is a normal approach) (in particular, the chains cAB1458-VH, cAB1462-VH, cAB1466-VH, cAB2021-VH and cAB2023-VH). The heavy chain sequences generated in at least some of the antibodies were able to express, at least to a low degree. However, the antibodies containing the cAB2021-VH or cAB2023-VH chains had a low or undetectable monomer content and were excluded from the final selection.

[0189] The remaining chains were compared by the biological activity retained by the antibodies containing them. The results are shown in Figure 8. Most surprisingly, antibodies containing a heavy chain in which three non-intuitive revertant mutations were introduced into framework 3 (FR3) showed surprising biological activity while having favorable protein expression and monomer content (chain cAB1466-VH). In summary, the antibodies designated cAB1467-10.0, cAB1468-10.0 and cAB1459-10.0 are the best candidates, and cAB1467-10.0, cAB1468-10.0 (both containing the cAB1466-VH chain) are the best.

[0190] Conclusion:

[0191] After standard humanization, analysis was performed to determine which human germline genes were closest to the original mouse hybridoma sequences and could therefore be used as donor frameworks for CDR grafting. In many variants of the humanization process, combinations of framework donors from two different human VH genes were also tried. Extensive experiments revealed donor combinations that gave better results. Sequence comparisons highlighting the positions of interest are shown in Figures 9 (heavy chain) and 10 (light chain). In these figures, the numbering and CDR positioning follow Chothia et al. (1992) J. Mol. Biol., 227, 776-798, Tomlinson et al. (1995) EMBO J., 14, 4628-4638 and Williams et al. (1996) J. Mol. Biol., 264, 220-232). An example is shown at VBase. https: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#JHEX. · IGHV1-2 was used as the donor for FR1 and FR2 (indicated by the highlights) · IGHV1-46*01 was used as the donor for FR3 (indicated by the highlights)

[0192] To achieve the high affinity and high stability of the humanized antibody, irregular back mutations to the original mouse sequences were required in Framework 3. · Back mutation of human donor R (Arg, basic) to mouse L (Leu, hydrophobic) at position 71 · Back mutation of human donor T (Thr, nucleophilic) to mouse K (Lys, basic) at position 73 · Back mutation of human donor M (Met, hydrophobic) to mouse L (Leu, hydrophobic) at position 69. Although a minor change, it seems to make cAb1466-VH better than cAb2021.

[0193] Irregular mutations were also created in Framework 2. · In the original VH17-20, all proposed human donor VHs, and most of the humanized candidates, position 43 was Q (Gln, amide). However, in the best humanized VH (cAb1466-VH), the mutation at position 43 from Q (Gln, amide) to K (Lys, basic) is considered important for better affinity / stability than the second-best humanized VH (cAb2021-VH).

[0194] Therefore, it is demonstrated that only combinations of multiple human germline sequences and the non-intuitive introduction of mouse back mutations can result in a few final candidate antibodies with improved expression and biological activity.

[0195] The wide diversity of humanized light chains that were able to function well in both the affinity and stability of the intact mAb against human lymphocytes suggests that most of the success of that mAb is due to the heavy chain, and that the heavy chain is rather disordered in light chain selection.

[0196] Example 3: Effect of the improved anti-TIRC7 antibody of the present invention on human lymphocytes

[0197] The antibodies of the present invention were tested in an immune assay for mixed lymphocyte proliferation and in an immune assay by PHA stimulation of lymphocytes. The results show the effect of the improved antibodies of the present invention, which is surprisingly stronger than that of the parental chimeric antibodies. The results are shown in Figures 11 and 12, respectively.

[0198] Example 4: Surface Plasmon Resonance Test for Antibody Affinity

[0199] To compare the target binding of the produced antibodies with the parental molecule, the binding constants of the parental antibody and the selected produced humanized version antibodies were obtained from a BiaCore® surface plasmon resonance test. The largest extracellular domain of TIRC7, which is a known binding substance for the parental antibody, was immobilized on the carboxymethylated dextran surface of a CM5 sensor chip (CM5 chip, research grade). A Biacore T200 instrument was used at an analysis temperature of 25 °C and a flow rate of 50 μl / min for the quantitative analysis of kinetic interactions. The analysis buffer was 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20.

[0200] To measure the rate constant or absorption effect, multiple Biacore SPR analysis columns were performed for each antibody at various concentrations. The specific constant was a very reproducible constant for the conditions of this buffer. The parental antibody showed an average K of 300 + / - 100 pM. D Measurements were performed for three humanized antibodies in the same setting. The results are provided in Table 4. Surprisingly, those results showed that the AB affinity of the selected humanized AB variants for TIRC7 was at least one order of magnitude higher than that of the parental molecule.

[0201] [Table 4]

[0202] The following references are cited. 1. Utku N, Heinemann T, Tullius SG, Bulwin GC, Beinke S, Blumberg RS, Beato F, Randall J, Kojima R, Busconi L, Robertson ES et al: Prevention of acute allograft rejection by antibody targeting of TIRC7, a novel T cell membrane protein. Immunity (1998) 9(4):509 - 518. 2. Liang P, Pardee AB: Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science (1992) 257(5072):967 - 971. 3. Dangond F, Hafler DA, Tong JK, Randall J, Kojima R, Utku N, Gullans SR: Differential display cloning of a novel human histone deacetylase (HDAC3) cDNA from PHA - activated immune cells. Biochem Biphys Res Commun (1998) 242(3):648 - 652. 4. Utku N, Bulwin GC, Beinke S, Heinemann T, Beato F, Randall J, Schnieders B, Sandhoff K, Volk H - D, Milford E, Gullans SR: The human homologue of Drosophila cornichon protein is differentially expressed in alloactivated T cells. Biochim Biophys Acta (1999) 1449(3):203 - 210. 5. Heinemann T, Bulwin GC, Randall J, Schnieders B, Sandhoff K, Volk HD, Milford E, Gullans SR, Uktu N: Genomic organization of the gene coding for TIRC7, a novel membrane protein essential for T cell activation. Genomics (1999) 57(3):398-406. 6. Schneider HS, da Rocha Dias S, Hu H, Rudd CE: A regulatory role for cytoplasmic YVKM motif in CTLA-4 inhibition of TCR signaling. Eur J Immunol (2001) 31(7):2042-2050. 7. Klabunde T, Hessler G: Drug design strategies for targeting G-protein-coupled receptors. ChemBioChem (2002) 3(10):928-944. 8. Sexton PM, Christopoulos A: G protein-coupled receptor drug targets. Curr Pharm Des (2006) 12(14):1681-1682. 9. Li YP, Chen W, Stashenko P: Molecular cloning and characterization of a putative novel human osteoclast-specific 116-kDa vacuolar proton pump subunit. Biochem Biophys Res Commun (1996) 218(3):813-321. · First demonstration that cDNA OC116 was isolated from a human osteosarcoma cell line and is expressed restrictedly in cells obtained from human giant cell tumors of bone and pancreatic cancer cells. 10. Smirnova AS, Morgun A, Shulzhenko N, Silva ID, Gerbase-DeLima M: Identification of new alternative splice events in the TCIRG1 gene in different human tissues. Biochem Biophys Res Commun (2005) 330(3):943-949. 11. Li YP, Chen W, Liang Y, Li W, Stashenko P: Atp6i-deficient mice exhibit severe osteopetrosis due to loss of osteoclast-mediated extracellular acidification. Nat Genet (1999) 23(4):447-451 12. Lee C, Ghoshal K, Beaman KD: Cloning of a cDNA or a T cell produced molecule with a putative immune regulatory role. Mol Immunol (1990) 27(11):1137-1144. 13. Manolson MF, Proteau D, Jones EW: Evidence for a conserved 95-120 kDa subunit associated with and essential for activity of V-ATPases. J Exp Biol (1992) 172:105-112. 14. Utku N, Heinemann T, Winter M, Bulwin GC, Schlawinsky M, Fraser P, Nieuwenhuis EE, Volk HD, Blumberg RS: Antibody targeting of TIRC7 results in significant therapeutic effects on collagen-induced arthritis in mice. Clin Exp Immunol (2006) 144(1):142-151. 15. Bulwin GC, Heinemann T, Bugge V, Winter M, Lohan A, Schlawinsky M, Schulze A, Walter S, Sabat R, Schulein R, Wiesner B et al: TIRC7 inhibits T cell proliferation by modulation of CTLA-4 expression. J Immunol (2006) 177(10): 6833-6841. 16. Bromley SK, Burack WR, Johnson KG, Somersalo K, Sims TN, Sumen C, Davis MM, Shaw AS, Allen PM, Dustin ML: The immunological synapse. Annu Rev Immunol (2001) 19: 375-396. 17. Grakoui A, Bromley SK, Sumen C, Davis MM, Shaw AS, Allen PM, Dustin ML: The immunological synapse: A molecular machine controlling T cell activation. Science (1999) 285(5425): 221-227. 18. Monks CR, Freiberg BA, Kupfer H, Sciaky N, Kupfer A: Three-dimensional segregation of supramolecular activation clusters in T cells. Nature (1998) 395(6697): 82-86. 19. Egen JG, Allison JP: Cytotoxic T lymphocyte antigen-4 accumulation in the immunological synapse is regulated by TCR signal strength. Immunity (2002) 16(1): 23-35. 20. Linsley PS, Bradshaw J, Greene J, Peach R, Bennett KL, Mittler RS: Intracellular trafficking of CTLA-4 and focal localization towards sites of TCR engagement. Immunity (1996) 4(6):535-543. 21. Kumamoto Y, Tomschegg A, Bennai-Sanfourche F, Boerner A, Kaser A, Schmidt-Knosalla I, Heinemann T, Schlawinsky M, Blumberg RS, Volk HD, Utku N: Monoclonal antibody specific for TIRC7 induces donor-specific anergy and prevents rejection of cardiac allografts in mice. Am J Transplant (2004) 4(4):505-514. 22. Kumamoto Y, Tamura A, Volk HD, Reinke P, Lohler J, Tullius SG, Utku N: TIRC7 is induced in rejected human kidneys and anti-TIRC7 mAb with FK506 prolongs survival of kidney allografts in rats. Transpl Immunol (2006) 16(3-4):238-244. 23. Utku N, Boerner A, Tomschegg A, Bennai-Sanfourche F, Bulwin GC, Heinemann T, Loehler J, Blumberg RS, Volk HD: TIRC7 deficiency causes in vitro and in vivo augmentation of T and B cell activation and cytokine response. J Immunol (2004) 173(4):2342-2352. 24. Frischer J, Schmidt S, Milford EL, Lassmann H, Utku N Role of TIRC7 and HLA-DR axis in lymphocytes involved in multiple sclerosis. Multiple Sclerosis Journal (2014), 20.9: 1171 - 1181, IF: 4.671 25. Cantrell D: T cell antigen receptor signal transduction pathways. Annu Rev Immunol (1996) 14: 259 - 274.

Claims

**Claim 1** A humanized anti-TIRC7 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein (i) the heavy chain variable region comprising complementarity determining regions (CDRs) is such that CDRH1 comprises the amino acid sequence of SEQ ID NO: 01, CDRH2 comprises the amino acid sequence of SEQ ID NO: 02, CDRH3 comprises the amino acid sequence of SEQ ID NO: 03, and (ii) the light chain variable region comprising complementarity determining regions (CDRs) is such that CDRL1 comprises the amino acid sequence of SEQ ID NO: 05, CDRL2 comprises the amino acid sequence of SEQ ID NO: 06, CDRL3 comprises the amino acid sequence of SEQ ID NO: 07, and (iii) the heavy chain variable region and the light chain variable region are of the following amino acid sequences; SEQ ID NO: 09 and SEQ ID NO: 10; SEQ ID NO: 09 and SEQ ID NO: 12; SEQ ID NO: 17 and SEQ ID NO: 12; SEQ ID NO: 17 and SEQ ID NO: 14; SEQ ID NO: 17 and SEQ ID NO: 16; SEQ ID NO: 25 and SEQ ID NO: 10; SEQ ID NO: 25 and SEQ ID NO: 12; SEQ ID NO: 25 and SEQ ID NO: 14; SEQ ID NO: 25 and SEQ ID NO: 16; SEQ ID NO: 35 and SEQ ID NO: 36; SEQ ID NO: 35 and SEQ ID NO: 38; SEQ ID NO: 39 and SEQ ID NO: 38; or SEQ ID NO: 45 and SEQ ID NO: 44; and a humanized anti-TIRC7 antibody or an antigen-binding fragment thereof. **Claim 2** The humanized anti-TIRC7 antibody or an antigen-binding fragment thereof according to claim 1, which suppresses activation and / or proliferation of immune cells when contacting the immune cells. **Claim 3** The humanized anti-TIRC7 antibody or an antigen-binding fragment thereof according to claim 1 or 2, which comprises an effector group and / or is labeled. **Claim 4** The humanized anti-TIRC7 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the binding to Fc receptor is attenuated. **Claim 5** The humanized anti-TIRC7 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4, which is isolated and / or substantially pure. **Claim 6** The humanized anti-TIRC7 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5, which is an antigen-binding fragment selected from the list consisting of Fab, Fab'-SH, Fv, scFv and F(ab')2. **Claim 7** The humanized anti-TIRC7 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which binds to the extracellular domain of TIRC7 with a KD of less than 100 pM, preferably less than 50 pM, when measured by surface plasmon resonance.

8. An isolated nucleic acid comprising a sequence encoding the humanized anti-TIRC7 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. A recombinant host cell comprising the nucleic acid according to claim 8.

10. The recombinant host cell according to claim 9, which is a Chinese hamster ovary (CHO) cell or a human immune cell.

11. A pharmaceutical composition comprising (i) the humanized anti-TIRC7 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or (ii) the nucleic acid according to claim 8, or (iii) the recombinant host cell according to claim 9 or 10, and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

12. A component for use in medicine, the component being selected from the list consisting of the humanized anti-TIRC7 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the isolated nucleic acid according to claim 8, the recombinant host cell according to claim 9 or 10, and the pharmaceutical composition according to claim 11.

13. The component for use according to claim 12, wherein the component is for use in regulating the cellular immune response of a subject.

14. The component for use according to claim 13, wherein the regulation of the immune response is an inhibition of the cellular immune response.

15. The component for use according to claim 14, wherein the inhibition of the cellular immune response is an inhibition of the proliferation of immune cells such as lymphocytes and / or an inhibition of cytokine expression in immune cells such as lymphocytes.

16. The component for use according to claim 14 or 15, wherein the inhibition of the cellular immune response is a reduction in the proliferation / activity of effector memory T cells and / or an increase in the proliferation / activity of regulatory T cells (TREG).

17. The component for use according to any one of claims 12 to 16, wherein the use is for treating a pathological immune response, preferably for use in treating inflammatory diseases, autoimmune diseases, allograft rejection (graft-versus-host disease).

18. The component for use according to claim 17, wherein the disease is an autoimmune disease such as an autoimmune disease selected from psoriatic arthritis, graft-versus-host disease, autoimmune hepatitis, primary sclerosing cholangitis, primary biliary cirrhosis, IgG4-related autoimmune disease, fibrotic disease such as pulmonary fibrosis, Sjögren's syndrome, systemic lupus erythematosus, Graves' disease, uveitis or uveitis associated with tubulointerstitial nephritis, vasculitis, chronic fatigue syndrome, and systemic scleroderma.

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