Anti-human CD45RC binding domains and uses thereof

Abstract

The present invention relates to antigen-binding domains that specifically bind to CD45RC, as well as antibodies and chimeric antigen receptors comprising same. The present invention further relates to their use in various methods for therapeutic purposes, including for preventing and / or alleviating transplant rejection, in particular graft-versus-host disease (GvHD).

Description

[Technical field]

[0001] The present invention relates to antigen-binding domains that specifically bind to CD45RC, as well as antibodies and chimeric antigen receptors comprising same. The present invention further relates to their use in various methods for therapeutic purposes, including preventing and / or reducing transplant rejection, in particular graft-versus-host disease (GvHD). [Background technology]

[0002] CD45 (leukocyte common antigen (LCA), EC3.1.3.48, also known as T200, Ly5, and PTPRC) constitutes the first prototypic receptor-like protein tyrosine phosphatase (RPTP). Its expression is restricted to leukocytes, where it is one of the most abundant cell surface glycoproteins, constituting nearly 10% of the cell surface (Trowbridge & Thomas, 1994. Annu Rev Immunol. 12:85-116; Hermiston et al., 2003. Annu Rev Immunol. 21:107-37; Holmes, 2006. Immunology. 117(2):145-55).

[0003] CD45 is composed of an extracellular domain, a single transmembrane domain, and a large cytoplasmic domain. The transmembrane and cytoplasmic domains are highly conserved among species. In particular, the cytoplasmic domain of CD45 contains two tandemly duplicated phosphatase domains, of which only the membrane-proximal domain has enzymatic activity (Desai et al., 1994. EMBO J. 13(17):4002-10). The function of the more C-terminal second phosphatase domain in CD45 remains unclear, although it has been suggested that it may indirectly contribute to CD45 activity by stabilizing the first domain. Through this cytoplasmic domain, CD45 functions as a central regulator of phosphotyrosine levels, at least in lymphocytes, by modulating the activity of the Src family of tyrosine protein kinases, such as Lck in T cells, or Lyn, Fyn, and Lck in B cells (Palacios & Weiss, 2004. Oncogene. 23(48):7990-8000; Lowell, 2004. Mol Immunol. 41(6-7):631-43).

[0004] In contrast to the transmembrane and cytoplasmic domains, the N-terminal extracellular domain of CD45 shows higher polymorphism among different leukocyte lineages. Indeed, this extracellular domain is highly glycosylated and contains three alternatively spliced ​​exons (4, 5, and 6 - which code for the A, B, and C determinants, respectively), both of which are O-linked glycosylated and sialylated (Hermistone et al., 2003. Annu Rev Immunol. 21:107-37; Holmes, 2006. Immunology. 117(2):145-55). CD45 isoforms that differ in size, shape, and charge may therefore be generated by dynamically regulated alternative splicing during both leukocyte differentiation and cell activation, resulting in changes in the extracellular domain of the molecule (Hall et al., 1988. J Immunol. 141(8):2781-7; Lynch, 2004. Nat Rev Immunol. 4(12):931-40).

[0005] The largest CD45 isoform containing all three alternatively spliced ​​exons, CD45RABC, is approximately 235 kDa, while the smallest isoform not containing all three exons, CD45RO, is approximately 180 kDa. In between are possible isoforms that contain only two (e.g., CD45RAB, CD45RBC) or only one (e.g., CD45RB) of the three exons.

[0006] Although the function of the different CD45 isoforms is unclear, differential expression of these isoforms correlates with the level of T cell activation and allows the dissociation of naive and memory T cells (Birkeland et al., 1989. Proc Natl Acad Sci USA. 86(17):6734-8). For example, CD45RA is expressed by peripheral naive mature CD4 + CD45RO is present on activated memory CD4 + CD45RABC is expressed on T cells. CD45RABC is expressed on B cells and their precursors, on a subgroup of dendritic cells and other antigen-presenting cells. A subtype of terminally differentiated memory T cells, effector memory RA T cells (TEMRA), also re-express the naive T cell marker CD45RA (Koch et al., 2008. Immun Ageing. 5:6). Importantly, this pattern of isoform expression is highly conserved across species, highlighting its functional role and importance (Hermiston et al., 2003. Annu Rev Immunol. 21:107-37).

[0007] CD4 + and CD8 + The specific expression patterns of CD45RC isoforms on T cells allow us to distinguish functionally distinct alloreactive T cell subsets that behave differently with respect to proliferation and cytokine secretion. For example, in rodents, CD4 + and CD8 + CD45RC highBoth precursor and / or potent T cells can promote transplant rejection and organ inflammation. h 1 effector cells (Spickett et al., 1983. J Exp Med. 158(3):795-810; Xystrakis et al., 2004. Eur J Immunol. 34(2):408-17), while expressing undetectable or low levels of CD45RC low / - T cells expressing T h CD45RC2 and regulatory T cells, which inhibit allograft rejection, graft-versus-host disease (GvHD) and cellular autoimmune diseases (Xystrakis et al., 2004. Blood. 104(10):3294-30; Guillonneau et al., 2007. J Clin Invest. 117(4):1096-106; Powrie & Mason, 1990. J Exp Med. 172(6):1701-8). In humans, CD45RC1 is expressed prior to transplantation. + CD8 + High percentages of T cells correlate with poor graft survival in kidney transplant patients (Ordonez et al., 2013. PLoS One. 8(7):e69791).

[0008] CD45RC high Depletion of T cell populations is therefore a promising approach for inducing immune tolerance in humans and thus for preventing, alleviating and / or treating transplant rejection (especially GvHD) and autoimmune diseases.

[0009] GvHD (graft-versus-host disease) is a significant cause of morbidity and mortality in stem cell transplant patients. It is a T-cell-mediated immune response process in which donor cells react against recipient cells. Currently, immunosuppression with immunomodulatory drugs such as corticosteroids is the mainstay of GvHD prevention. Although there has been progress in improving survival outcomes over time, corticosteroids do not prevent GvHD in a high percentage of patients (less than 50% of patients with acute GvHD and 40-50% of patients with chronic GvHD, depending on the severity of the initial disease - Garnett et al., 2013. Ther Adv Hematol. 4(6):366-378), are associated with significant toxicity, and many of the currently available salvage therapies are accompanied by increased immunosuppression and infectious complications. Thus, there remains an unmet need for the development of new therapeutic strategies for GvHD to improve long-term post-transplant outcomes.

[0010] The present inventors have high We previously described that T cell depletion is a novel therapeutic strategy to prevent or attenuate transplant rejection by decreasing aggressive effector T cells while increasing tolerogenic regulatory T cells. Indeed, transient anti-CD45RC mAb treatment induced rapid CD45RC mAb upregulation while preserving memory immunity. high Furthermore, the inventors have shown that short-term anti-CD45RC antibody treatment results in durable allograft survival without signs of chronic rejection (International Patent Application 2016 / 016442; Picarda et al., 2017. JCI Insight. 2(3):e90088). The inventors have also surprisingly shown that CD45RC high They demonstrated that treatment with anti-CD45RC antibodies, which specifically deplete T cells, improved muscle strength (Ouisse et al., 2019. Front Immunol. 10:2131) and prevented and treated monogenic diseases such as Duchenne muscular dystrophy (DMD) or autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) (International Patent Application 2019 / 115791).

[0011] Recently, the inventors generated a de novo humanized anti-human CD45RC antibody that showed higher efficiency in inducing targeted T cell apoptosis and improved therapeutic efficacy compared to the commercially available anti-CD45RC antibody MT2, particularly in an immune-humanized NSG mouse model of xenogeneic GvHD (International Patent Application 2020 / 058495).

[0012] Here, the inventors have gone a step further and developed an improved version of the already improved anti-human CD45RC antibody of WO 2020 / 058495, which has even better therapeutic efficacy. Summary of the Invention

[0013] The present invention relates to an antigen-binding domain that specifically binds to CD45RC, said antigen-binding domain comprising: (a) a heavy chain variable region (HCVR) comprising the following three CDRs: (i) V of the sequence of SEQ ID NO: 10 H -CDR1; (ii) V of the sequence of SEQ ID NO: 11 H - CDR2; and (iii) V of the sequence of SEQ ID NO: 12 H -CDR3; and (b) a light chain variable region (LCVR) comprising the following three CDRs: (i) V of the sequence of SEQ ID NO: 13 L -CDR1; (ii) V of the sequence of SEQ ID NO: 14 L - CDR2; and (iii) V of the sequence of SEQ ID NO: 15 L -CDR3, Including, X in SEQ ID NO:13 is absent or selected from the group consisting of Asn (N), Ser (S), and Gly (G); preferably, X in SEQ ID NO:13 is absent.

[0014] In one embodiment, the antigen binding domain comprises: 1) the HCVR of sequence SEQ ID NO: 24 and the LCVR of sequence SEQ ID NO: 25; or 2) an HCVR comprising three CDRs having SEQ ID NOs: 10, 11, and 12 and a framework region sharing at least 70% sequence identity with the framework region of SEQ ID NO: 24; and an LCVR comprising three CDRs having SEQ ID NOs: 13, 14, and 15 and a framework region sharing at least 70% sequence identity with the framework region of SEQ ID NO: 25; wherein the antigen-binding domain retains its binding specificity for CD45RC; wherein X1 in SEQ ID NO:25 is absent or selected from the group consisting of Asn (N), Ser (S), and Gly (G); preferably, X in SEQ ID NO:13 is absent; X2 in SEQ ID NO:25 is selected from the group consisting of Tyr (Y) and Phe (F), preferably, X2 in SEQ ID NO:25 is Tyr (Y).

[0015] In one embodiment, the antigen binding domain is selected from the group consisting of a single chain variable fragment (scFv), tandem-di-scFv, tandem-tri-scFv, scFv-Fc, minibody, maxibody, diabody, triabody, Fv, Fab, Fab', Fab'-SH, and F(ab')2.

[0016] The present invention further relates to an antibody or antigen-binding fragment thereof that specifically binds to CD45RC, wherein said antibody comprises an antigen-binding domain according to the invention.

[0017] In one embodiment, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region (HCCR) and a light chain constant region (LCCR), Preferably, the HCCR comprises an amino acid sequence of any one of SEQ ID NOs: 26 to 28, more preferably an amino acid sequence that shares at least 70% sequence identity with SEQ ID NO: 26, and the LCCR comprises an amino acid sequence that shares at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 29; The antibody or antigen-binding fragment thereof retains binding specificity for CD45RC.

[0018] The present invention further comprises: i) at least one extracellular antigen-binding domain according to the invention; ii) optionally, an extracellular hinge domain; iii) at least one transmembrane domain, Preferably, it is selected from the group consisting of the CD8 transmembrane domain and the CD28 transmembrane domain, More preferably, a transmembrane domain selected from the group consisting of a CD8 transmembrane domain having an amino acid sequence that shares at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 49, and a CD28 transmembrane domain having an amino acid sequence that shares at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 50; iv) at least one intracellular signaling domain comprising at least one primary signaling domain and, optionally, one or more costimulatory signaling domains; Preferably, at least one primary signaling domain is the signaling domain of CD3ζ; Preferably, at least one primary signaling intracellular signaling domain is a signaling domain of CD3ε having an amino acid sequence that shares at least 70% sequence identity with the amino acid sequence of SEQ ID NO:51. The present invention relates to a chimeric antigen receptor (CAR) that specifically binds to CD45RC, comprising:

[0019] The present invention further relates to an immune cell population expressing a chimeric antigen receptor according to the invention on its cell surface.

[0020] In one embodiment, the immune cells of the population are CD4 + T cells, CD8 + The antibody is selected from the group comprising or consisting of a T cell, a double positive T cell, a double negative T cell, a γδ T cell, a NK cell, a NKT cell, a B cell, a macrophage, or a dendritic cell.

[0021] The present invention further relates to a nucleic acid encoding an antigen-binding domain according to the invention, an antibody or antigen-binding fragment thereof according to the invention, or a CAR according to the invention, which is preferably under the control of a regulatory element.

[0022] The present invention relates to an antigen-binding domain according to the invention, an antibody or antigen-binding fragment thereof according to the invention, an immune cell population according to the invention, or a nucleic acid according to the invention, for use as a medicament.

[0023] The present invention further relates to an antigen-binding domain according to the invention, an antibody or antigen-binding fragment thereof according to the invention, an immune cell population according to the invention, or a nucleic acid according to the invention, for use in inducing immune tolerance in a subject in need thereof.

[0024] The present invention further relates to the use of CD45RC in a subject in need thereof. high The present invention relates to an antigen-binding domain according to the invention, an antibody or antigen-binding fragment thereof according to the invention, an immune cell population according to the invention, or a nucleic acid according to the invention for use in depleting cells.

[0025] The present invention further relates to an antigen-binding domain according to the invention, an antibody or antigen-binding fragment thereof according to the invention, an immune cell population according to the invention, or a nucleic acid according to the invention for use in preventing and / or reducing transplant rejection in a subject in need thereof.

[0026] In one embodiment, preventing and / or reducing transplant rejection comprises or consists of preventing and / or reducing graft-versus-host disease (GvHD).

[0027] The present invention further relates to the use of CD45RC in a subject in need thereof. highThe present invention relates to an antigen-binding domain according to the invention, an antibody or antigen-binding fragment thereof according to the invention, an immune cell population according to the invention, or a nucleic acid according to the invention, preferably a CD45RC associated disease, disorder or condition, for use in preventing, reducing and / or treating the disease, disorder or condition. high The associated disease, disorder or condition is selected from the group consisting of an autoimmune disease, an unwanted immune response, a monogenic disease, lymphoma, and cancer.

[0028] definition "Antibodies" or "Immunoglobulins" As used herein, the term "immunoglobulin" refers to a protein having a combination of two heavy chains and two light chains, regardless of whether it has any associated specific immunoreactivity. "Antibody" refers to an aggregate having significant known specific immunoreactive activity against an antigen of interest (e.g., human CD45RC). The term "anti-hCD45RC antibody" is used herein to refer to an antibody exhibiting immunological specificity for the human CD45RC protein. As explained elsewhere herein, "specificity" for human CD45RC does not exclude cross-reactivity with species in which CD45RC is expressed. Antibodies and immunoglobulins comprise light and heavy chains, with or without interchain covalent linkage between them. The basic immunoglobulin structure in vertebrate systems is relatively well understood. The collective term "immunoglobulin" includes five different classes of antibodies that are biochemically distinguishable. Although the following discussion is generally directed to the IgG class of immunoglobulin molecules, all five classes of antibodies are within the scope of the present invention. With respect to IgG, immunoglobulins comprise two identical light polypeptide chains of approximately 23 kDa molecular weight, and two identical heavy chains of approximately 53-70 kDa molecular weight. The four chains are joined by disulfide bonds in a "Y" configuration, with the light chains enveloping the heavy chains that begin at the mouth of the "Y" and continue through the variable region. The light chains of antibodies are classified as either kappa (κ) or lambda (λ). Each heavy chain class may be associated with either κ or λ light chains. Generally, the light and heavy chains are covalently linked to each other, and the "tail" regions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when the immunoglobulin is produced by either a hybridoma, a B cell, or a genetically engineered host cell. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain. Those skilled in the art will appreciate that heavy chains are classified as gamma (γ), mu (μ), alpha (α), delta (Δ) or epsilon (ε), with several subclasses within them (e.g., γ1-γ4). It is the nature of the chain that determines the "class" of the antibody as IgG, IgM, IgA IgD, or IgE, respectively. Immunoglobulin subclasses or "isotypes" (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) are well characterized and are known to provide functional specialization. Modified versions of each of these classes and isotypes are readily discernible to one of skill in the art in light of the instant disclosure, and are therefore within the scope of the instant invention. As discussed above, the variable region of an antibody enables the antibody to select, recognize and specifically bind to an epitope on an antigen. That is, the light chain variable domain (V L domain) and heavy chain variable domain (V HThe four dimensional antibody structure forms an antigen binding site at the end of each arm of the "Y". More specifically, the antigen binding site is composed of V H Chain and V L Each of the chains is defined by three complementarity determining regions (CDRs).

[0029] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which the Fc region of an antibody bound to an antigen-bearing target cell binds Fcγ receptors (FcγR) of the CD16a type, which are present primarily on NK cells (DiLillo & Ravetch, 2015. Cancer Immunol Res. 3(7):704-13). Binding of certain IgG isotypes to other FcγRs, such as CD32 or CD64, leads to activation of cells such as monocytes, macrophages, and PMNs, and secretion of various substances, such as lyases, perforins, granzymes, and TFNs, that mediate the destruction of inflammatory target cells. Engagement of CD16a on NK cells also leads to the production of cytokines (Anegon et al., 1988. J Exp Med. 167(2):452-72). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Pat. No. 5,500,362 or 5,821,337 may be performed.

[0030] "Antibody-Dependent Cell-Mediated Phagocytosis" or "ADCP" As used herein, the term "antibody-dependent cell-mediated phagocytosis" or "ADCP" refers to a form of phagocytosis in which the Fc region of an antibody bound to an antigen-bearing target cell binds Fcγ receptors (FcγR) present on certain phagocytes (e.g., neutrophils, monocytes, macrophages, and dendritic cells). This binding triggers the initiation of the phagocytic process to mediate the destruction of the target cell.

[0031] "Antigen-binding fragment" or "antigen-binding domain" As used herein, the term "antigen-binding fragment" or "antigen-binding domain" refers to a portion or region of an antibody or immunoglobulin that contains fewer amino acid residues than a whole antibody but is capable of binding antigen and / or competing with the whole antibody for antigen binding (e.g., specific binding to human CD45RC). Antigen-binding fragments include, but are not limited to, single chain antibodies, Fv, Fab, Fab', Fab'-SH, and F(ab')2.

[0032] "Complement-dependent cytotoxicity (CDC)" or "CDC" The term "complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system to antibodies that are bound to their cognate antigen. To assess complement activation, a CDC assay (e.g., as described in Gazzano-Santoro et al., 1997. J Immunol Methods. 202(2):163-71) may be performed.

[0033] "CD45" As used herein, the term "CD45" (also known as CD45R or PTPRC) refers to a transmembrane glycoprotein that exists in different isoforms. These different isoforms of CD45 differ in their extracellular domain structure, resulting from alternative splicing of three variable exons (exons 4, 5, and 6) that code for the A, B, and C determinants of the CD45 extracellular region, respectively. Antibodies that react with restricted epitopes are clustered as "CD45R". Thus, anti-CD45RA, anti-CD45RB, and anti-CD45RC antibodies recognize CD45 isoforms that contain expression of A, B, and C determinants, respectively. When all three A, B, and C determinants are absent, the isoform is referred to as CD45RO. The various isoforms of CD45 have different extracellular domains but share the same extracellular sequence proximal to the membrane, as well as a large cytoplasmic tail segment that contains a transmembrane domain and two tandemly homologous highly conserved phosphatase domains of approximately 300 residues. CD45 and its isoforms are non-covalently associated with lymphocyte phosphatase-associated phosphoprotein (LPAP) on T and B lymphocytes. CD45 has been reported to associate with several other cell surface antigens, including CD1, CD2, CD3, and CD4. CD45 is involved in signal transduction of lymphocyte activation. When preceded by the letter "h" (e.g., hCD45), it means that CD45 is of human origin.

[0034] "CD45RC" As used herein, the term "CD45RC" refers to a 200-220 kDa single-chain type I membrane glycoprotein well known to those skilled in the art. CD45RC is an alternatively spliced ​​isoform of CD45 that contains exon 6, which encodes the C determinant (hence the term CD45RC, i.e., C determinant-restricted ( R estricted to the CThe amino acid sequence of human CD45RC is shown in SEQ ID NO: 1 (corresponding to UniProt accession number P08575-10). This CD45RC isoform is expressed in B cells and NK cells, as well as in CD8 + T cells and CD4 + Expressed on a subset of T cells, but not CD8 + or CD4 + Tregs, CD14 + It is not expressed on monocytes or PMNs (Picarda et al., 2017. JCI Insight. 2(3):e90088). Some monoclonal antibodies are able to recognize epitopes in the CD45 portion that are common to all the different isoforms (these are called anti-CD45 antibodies), whereas others have restricted specificity to a given isoform, depending on the determinant (A, B or C) they recognize. If preceded by the letter "h" (e.g., hCD45RC), it means that CD45RC is of human origin.

[0035] "CDR" or "Complementarity Determining Region" As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen combining sites found in the variable regions of both heavy and light chain polypeptides. The CDRs are identified according to the rules in Table 1, as deduced from Kabat et al., (1991. Sequences of proteins of immunological interest (5th ed.). Bethesda, MD: USDep. of Health and Human Services; and Chothia & Lesk, 1987. J Mol Biol. 196(4):901-17). [Table 1]

[0036] "Manipulated" As used herein, the terms "engineered" or "modified" refer to cells that have been transfected, transformed, or transduced.

[0037] "Epitope" As used herein, the term "epitope" refers to a specific arrangement of amino acids located on a protein(s) to which an antibody or binding fragment thereof binds. Epitopes are often composed of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics and specific charge characteristics. Epitopes may be linear (or contiguous) or conformational, i.e., involving two or more sequences of amino acids in different regions of the antigen, which may not necessarily be contiguous.

[0038] "Framework Region" or "FR" or "Non-CDR Region" As used herein, the term "framework region" or "FR" or "non-CD region" includes amino acid residues that are part of the variable region but are not part of the CDRs (e.g., using the Kabat / Chothia definition of CDRs). Thus, a variable region framework is about 100-120 amino acids long, but includes only amino acids outside the CDRs. Examples of HCVRs and CDRs as defined by Kabat / Chothia: - FR1 may correspond to the domain of the variable region including amino acids 1-25 according to the Chothia / AbM definition, or 5 residues later according to the Kabat definition; - FR2 may correspond to the domain of the variable region comprising amino acids 36 to 49; - FR3 may correspond to the domain of the variable region comprising amino acids 67 to 98; -FR4 may correspond to the domain of the variable region from amino acids 104-110 to the end of the variable region. The framework regions of the light chain are similarly separated by each of the CDRs of the LCVR. In naturally occurring antibodies, the six CDRs present in each monomeric antibody are short non-contiguous sequences of amino acids that are specifically positioned to form an antigen-binding site when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining parts of the heavy and light chain variable domains have less inter-molecular variation in amino acid sequence and are called framework regions. The framework regions mainly adopt a β-sheet conformation, and the CDRs form loops that connect and in some cases form part of the β-sheet structure. Thus, these framework regions act to form a scaffold that provides positioning of the six CDRs in the correct orientation by non-covalent interactions between the chains. The antigen-binding site formed by the positioned CDRs defines a surface that is complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to the immunoreactive antigen epitope. The position of the CDRs can be easily identified by those skilled in the art.

[0039] "Heavy Chain Region" As used herein, the term "heavy chain region" includes amino acid sequences derived from the constant domain of an immunoglobulin heavy chain. A protein that includes a heavy chain region is a C H 1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, C H 2 domain, C H In one embodiment, the antibody or binding fragment thereof according to the invention comprises at least one of the Fc region of an immunoglobulin heavy chain (e.g., hinge portion, C H 2 domain, and C H In another embodiment, the antibody or binding fragment thereof according to the invention may comprise at least a region of a constant domain (e.g., C HIn certain embodiments, at least one, and preferably all, of the constant domains are derived from a human immunoglobulin heavy chain. For example, in one preferred embodiment, the heavy chain region comprises a fully human hinge domain. In other preferred embodiments, the heavy chain region comprises a fully human Fc region (e.g., a hinge, C domain, or a portion of a C domain from a human immunoglobulin). H 2 and C H In one embodiment, the constituent constant domains of the heavy chain region are derived from different immunoglobulin molecules. For example, the heavy chain region of the protein comprises a C 3 domain sequence derived from an IgG1 molecule. H In another embodiment, the constant domain is a chimeric domain comprising regions of different immunoglobulin molecules. For example, the hinge may comprise a first region from an IgG1 molecule and a second region from an IgG3 or IgG4 molecule. As mentioned above, the skilled artisan will appreciate that the constant domains of the heavy chain region may be modified such that they differ in amino acid sequence from a naturally occurring (wild type) immunoglobulin molecule. That is, the antibody or binding fragment thereof according to the invention comprises a heavy chain constant domain (C H 1, hinge, C H 2 or C H 3) and / or the light chain constant domain (C L ) in the sequence or sequence of the polypeptide. Exemplary modifications include the addition, deletion, or substitution of one or more amino acids in one or more domains.

[0040] "Hinge Region" or "Hinge Domain" As used herein, the terms "hinge region" or "hinge domain" are used interchangeably and refer to a C H 1 domain C HThe hinge region comprises the region of an immunoglobulin heavy chain that connects the two N-terminal antigen-binding domains. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding domains to move independently. The hinge region can be subdivided into three different domains: the upper, middle, and lower hinge domains (Roux et al., 1998. J Immunol. 161(8):4083-90). In the context of chimeric antigen receptors (CARs), the term "hinge region" or "hinge domain" refers to the region that connects the extracellular binding domain with the transmembrane domain.

[0041] "Sameness" or "Identical" As used herein, the term "identity" or "identical," when used in the context of two or more sequences of amino acid sequences or two or more sequences of nucleic acid sequences, refers to the degree of sequence relatedness between the amino acid or nucleic acid sequences, as determined by the number of matches between strings of two or more amino acid or nucleic acid residues. "Identity" measures the percentage of identical matches between the smaller of two or more sequences, with gap alignment (if any) as specified by a particular mathematical model or computer program (i.e., "algorithm"). The identity of related amino acid or nucleic acid sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in: Lesk, 1988. Computational molecular biology: Sources and methods for sequence analysis. New York, NY: Oxford University Press; Smith, 1993. Biocomputing: Informatics and Genomics Projects. San Diego, CA: Academic Press; Griffin & Griffin, 1994. Computer analysis of sequence data, Part 1. Totowa, NJ: Humana Press; von Heijne, 1987. Sequence analysis in molecular biology: treasure trove or trivial pursuit. San Diego, CA: Academic press; Gribskov & Devereux, 1991. Sequence analysis primer. New York, NY: Stockton Press; Carillo et al., 1988. SIAM J Appl Math. 48(5):1073-82. Preferred methods for determining identity are designed to obtain the maximum match between the sequences tested. Methods for determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, such as GAP (Genetics Computer Group, University of Wisconsin, Madison, WI; Devereux et al., 1984. Nucleic Acids Res. 12(1 Pt 1):387-95), BLASTP, BLASTN, and FASTA (Altschul et al., 1990. J Mol Biol. 215(3):403-10). The BLASTX program is available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894). The well-known SmithWaterman algorithm may also be used to determine identity.

[0042] "Immunospecific," "specific for," or "specifically binds" As used herein, an antibody or binding fragment thereof is said to be "immunospecific," "specific," or "specifically binds" to an antigen (e.g., hCD45RC) if it reacts with the antigen at a detectable level, preferably within about 10 6 M -1 More than 10, preferably about 10 7 M -1 , 10 8 M -1 , 5x10 8 M -1 , 10 9 M -1 , 5x10 9 M -1 or greater. The affinity of an antibody or its binding fragment for its cognate antigen is also generally expressed as the equilibrium dissociation constant (K DThe antibody or binding fragment thereof is preferably expressed as a 10-fold increase in its detectable affinity to the antigen (e.g., hCD45RC). -6 M or more, preferably 10 -7 M or less, 5x10 -8 M, 10 -8 M, 5x10 -9 M, 10 -9 K below M D When an antigen reacts with an antigen, it is said to be "immunospecific," "specific for," or "specifically binds" to that antigen. The affinity of an antibody or binding fragment thereof can be readily determined using conventional techniques, such as those described in Scatchard, 1949. Ann NY Acad Sci. 51:660-672. The binding characteristics of an antibody or binding fragment thereof to an antigen, cell or tissue can generally be determined and assessed using immunodetection methods, such as ELISA, immunofluorescence-based assays, such as immunohistochemistry (IHC) and / or fluorescence activated cell sorting (FACS), or surface plasmon resonance (SPR, e.g., using BIAcore®), and the like.

[0043] "Monoclonal antibodies" As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies in the population are identical except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies or binding fragments thereof according to the invention may be prepared by the hybridoma methodology first described by Kohler et al., 1975. Nature. 256(5517):495-7, or may be produced using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al., 1991. Nature. 352(6336):624-8 and Marks et al., 1991. J Mol Biol. 222(3):581-97, for example.

[0044] "Prevent" or "preventing" or "prevention" As used herein, the terms "prevent," "preventing," and "prevention" refer to prophylactic and preventative measures, the purpose of which is to reduce the likelihood that a subject will develop a pathological condition or disorder over a given period of time. Such a reduction may be reflected, for example, in a delay in the onset of at least one symptom of the pathological condition or disorder in the subject.

[0045] "Single-chain variable fragment" As used herein, the term "single-chain variable fragment", also abbreviated as "sFv" or "scFv", refers to VFs connected into a single amino acid chain. H and V L The scFv amino acid sequence preferably has a V domain that enables the scFv to form the desired structure for antigen binding. H Domain and V L (Pluckthun, 1994. Antibodies from Escherichia coli. In Rosenberg & Moore (Eds.), The pharmacology of monoclonal antibodies. Handbook of Experimental Pharmacology, 113:269-315. Springer: Berlin, Heidelberg). Bivalent, trivalent and higher valent scFvs, called tandem-di-scFv, tandem-tri-scFv, etc., can be engineered by linking two, three or more scFvs together. Alternatively, two scFvs can be linked together using a V H Domain and V L Using too short a linker peptide (typically about 5-10 amino acids) between the domains can force dimerization into diabodies. Diabodies are described in more detail in European Patent EP 0 404 097, International Publication WO 1993 / 011161, and Holliger et al., 1993. Proc Natl Acad Sci USA. 90(14):6444-8. Alternatively, even shorter linker peptides (typically about 1-2 amino acids) result in the formation of scFv trimers, also called tribodies or tribodies.

[0046] "subject" As used herein, the term "subject" refers to a mammal, preferably a human. In one embodiment, the subject may be a "patient", i.e., a warm-blooded animal, more preferably a human, awaiting medical treatment or receiving medical treatment, or has been / is / will be the subject of medical treatment, or is being monitored for the development of a disease. The term "mammal" herein refers to any mammal, including humans, farm animals, and zoo, sport, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, and the like. Preferably, the mammal is a primate, more preferably a human.

[0047] "Treatment" or "Treatment" or "Amelioration" As used herein, the terms "treating" or "treatment" or "alleviating" refer to therapeutic treatment, excluding prophylactic or preventative measures, where the objective is to slow down (reduce) the targeted pathological condition or disorder. Those in need of treatment include those already with the disorder as well as those suspected of having the disorder. A subject is successfully "treated" for a targeted condition or disorder if, after receiving a therapeutic amount of an isolated antibody or binding fragment thereof, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the invention, said subject exhibits an observable and / or measurable reduction or absence of one or more of the following: CD45RC high Decreased number of cells;CD45RC high A reduction in the percentage of total cells that are cells; abatement, to some extent, of one or more of the symptoms associated with a particular disease or condition; a reduction in morbidity and mortality; and / or an improvement in quality of life issues. The above parameters for determining successful treatment and disease improvement are readily measurable by routine procedures familiar to physicians.

[0048] "Variable Region" or "Variable Domain" As used herein, the term "variable" refers to a variable domain V H and V LIt refers to the fact that certain regions of the variable domains of antibodies differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its target antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is the V domains that form part of the antigen-binding site that are the most important loci in the antibody. L Domains and V H In each domain, it is concentrated into three segments called "hypervariable loops." The first, second, and third hypervariable loops of a Vλ light chain domain are referred to herein as L1(λ), ​​L2(λ) and L3(λ), and may be defined as comprising residues 24-33 (L1(λ), ​​consisting of 9, 10 or 11 amino acid residues), 49-53 (L2(λ), consisting of 3 residues) and 90-96 (L3(λ), consisting of 6 residues) in the VL domain (Morea et al., 2000. Methods. 20(3):267-79). The first, second, and third hypervariable loops of the Vκ light chain domain are referred to herein as L1(κ), L2(κ) and L3(κ), and V L Domains can be defined as those containing residues 25-33 (L1(κ), consisting of 6, 7, 8, 11, 12 or 13 residues), 49-53 (L2(κ), consisting of 3 residues) and 90-97 (L3(κ), consisting of 6 residues) (Morea et al., 2000. Methods. 20(3):267-79). V H The first, second, and third hypervariable loops of a domain are referred to herein as H1, H2, and H3, and V H Domains can be defined as those containing residues 25-33 (H1, consisting of 7, 8 or 9 residues), 52-56 (H2, consisting of 3 or 4 residues) and 91-105 (H3, which varies greatly in length) (Morea et al, 2000. Methods. 20(3):267-79). Unless otherwise specified, the terms L1, L2, and L3 refer to the first, second, and third hypervariable loops, respectively, of the VL domain, and include hypervariable loops from both Vκ and Vλ isotypes. HIt refers to the first, second, and third hypervariable loops of a domain, including hypervariable loops from any of the known heavy chain isotypes, including gamma (γ), mu (μ), alpha (α), delta () or epsilon (ε). Hypervariable loops L1, L2, L3, H1, H2, and H3 may each comprise a portion of a "complementarity determining region" or "CDR," as defined above.

[0049] "vector" As used herein, the term "vector" refers to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. The nucleic acid sequence can be "exogenous", meaning that it is foreign to the cell into which the vector is introduced, or that the sequence is homologous to a sequence in the cell, but is at a location within the nucleic acid of the host cell in which it is not normally found. Vectors include DNA vectors, RNA vectors, plasmids, phagemids, phage derivatives, viruses (e.g., bacteriophages, animal viruses, and plant viruses), cosmids, and artificial chromosomes (e.g., YACs). A person skilled in the art will be fully equipped to construct vectors through standard recombinant techniques known in the art. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] In a first aspect, the present invention relates to an antigen-binding domain that specifically binds to CD45RC, preferably human CD45RC (hCD45RC). Thus, the antigen-binding domain of the present invention is a "CD45RC-binding domain", preferably an "hCD45RC-binding domain".

[0051] In one embodiment, the CD45RC binding domain specifically binds to hCD45RC having SEQ ID NO:1. [ka]

[0052] In one embodiment, the CD45RC binding domain specifically binds to the extracellular domain of hCD45RC. In one embodiment, the CD45RC binding domain specifically binds to at least one epitope present on the extracellular domain of hCD45RC.

[0053] In one embodiment, the CD45RC binding domain specifically binds to a C determinant encoded by exon 6 of hCD45. In one embodiment, the CD45RC binding domain specifically binds to at least one epitope on a C determinant encoded by exon 6 of hCD45.

[0054] In one embodiment, the amino acid sequence of the C determinant encoded by exon 6 of hCD45 comprises or consists of SEQ ID NO:2. [ka]

[0055] In one embodiment, the CD45RC binding domain specifically binds to at least one epitope comprising or consisting of a sequence sharing at least about 70%, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO:2 or a fragment thereof.

[0056] In one embodiment, the CD45RC binding domain specifically binds to at least one epitope comprising or consisting of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47 amino acids of SEQ ID NO:2; or a sequence sharing at least about 70%, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:2.

[0057] In one embodiment, the CD45RC binding domain specifically binds to at least one epitope comprising or consisting of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47 contiguous amino acids of SEQ ID NO:2; or a sequence sharing at least about 70%, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:2.

[0058] In one embodiment, at least one epitopic fragment comprising or consisting of SEQ ID NO:2 comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47 amino acid residues, preferably consecutive amino acid residues.

[0059] In one embodiment, at least one epitopic fragment comprising or consisting of SEQ ID NO:2 is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 100 0, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 73, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100 or more contiguous sequences of a sequence comprising or consisting of SEQ ID NO:2 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47 amino acid residues spanning the range of amino acid residues.

[0060] In one embodiment, the sequence comprising SEQ ID NO:2 is the sequence of hCD45RC set forth in SEQ ID NO:1.

[0061] In one embodiment, the sequence comprising SEQ ID NO:2 is the sequence of hCD45 (i.e., including the A, B, and C determinants), also known as hCD45RABC, set forth in SEQ ID NO:3, which corresponds to UniProt accession number P08575-3. [ka]

[0062] In one embodiment, the CD45RC binding domain does not bind to the A determinant encoded by exon 4 of hCD45. In one embodiment, the CD45RC binding domain does not bind to at least one epitope on the A determinant encoded by exon 4 of hCD45.

[0063] In one embodiment, the amino acid sequence of the C determinant encoded by exon 4 of hCD45 comprises or consists of SEQ ID NO:4. [ka]

[0064] In one embodiment, the CD45RC binding domain does not bind to the B determinant encoded by exon 5 of hCD45. In one embodiment, the CD45RC binding domain does not bind to at least one epitope on the B determinant encoded by exon 5 of hCD45.

[0065] In one embodiment, the amino acid sequence of the B determinant encoded by exon 4 of hCD45 comprises or consists of SEQ ID NO:5. [ka]

[0066] In one embodiment, the CD45RC binding domain does not bind to hCD45RA.In one embodiment, the CD45RC binding domain does not bind to at least one epitope of hCD45RA.

[0067] In one embodiment, the amino acid sequence of hCD45RA comprises or consists of SEQ ID NO: 6, which corresponds to UniProt Accession No. P08575-8. [ka]

[0068] In one embodiment, the CD45RC binding domain does not bind to hCD45RB.In one embodiment, the CD45RC binding domain does not bind to at least one epitope of hCD45RB.

[0069] In one embodiment, the amino acid sequence of hCD45RB comprises or consists of SEQ ID NO: 7, which corresponds to UniProt Accession No. P08575-9. [ka]

[0070] In one embodiment, the CD45RC binding domain does not bind to hCD45RAB.In one embodiment, the CD45RC binding domain does not bind to at least one epitope of hCD45RAB.

[0071] In one embodiment, the amino acid sequence of hCD45RAB comprises or consists of SEQ ID NO: 8, which corresponds to UniProt Accession No. P08575-5. [ka]

[0072] In one embodiment, the CD45RC binding domain does not bind to hCD45RO.In one embodiment, the CD45RC binding domain does not bind to at least one epitope of hCD45RO.

[0073] In one embodiment, the amino acid sequence of hCD45RB comprises or consists of SEQ ID NO: 9, which corresponds to UniProt Accession No. P08575-4. [ka]

[0074] In one embodiment, at least one epitope is a conformational epitope. In another embodiment, at least one epitope is a continuous epitope.

[0075] In one embodiment, the CD45RC binding domain is approximately 5×10, as can be measured, for example, by Biacore on immobilized hCD45RC. -7 M or less, preferably about 2.5×10 -7 M or less, approximately 1×10 -7 M or less, approximately 7.5 x 10 -8 M or less, about 5 x 10 -8 M or less, approximately 2.5 x 10 -8 M or less, approximately 1×10 -8 M or less, approximately 7.5 x 10 -9 M or less, about 5 x 10 -9 M or less, approximately 2.5 x 10 -9 M or less, approximately 1×10 -9 The equilibrium dissociation constant (K d ) binds to hCD45RC.

[0076] In one embodiment, the CD45RC binding domain has a K of less than 10 nM, such as 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, or less, as can be determined by Biacore on immobilized hCD45RC. d In one embodiment, the CD45RC binding domain binds to hCD45RC with a K of about 2.24 nM as measured by Biacore on immobilized hCD45RC. d Binds to hCD45RC.

[0077] In one embodiment, the CD45RC binding domain is about 1×10 4 M -1 seconds -1 or more, preferably about 5×10 4 M -1 seconds -1 That's about 1 x 10 5 M -1 seconds -1 That's about 2.5 x 10 5 M -1 seconds -1 That's about 5 x 10 5 M -1 seconds -1 Association rate (K on ) and binds to hCD45RC.

[0078] In one embodiment, the CD45RC binding domain is about 5×10 -2 seconds -1 Less than or equal to about 4 x 10 -2 seconds -1 Below, approximately 3×10 -2 seconds -1 Below, approximately 2×10 -2 seconds -1 Below, approximately 1.5×10 -2 seconds -1 The following dissociation rate (K off ) binds to hCD45RC.

[0079] In one embodiment, the CD45RC binding domain comprises: -About 5×10 -7 M or less, preferably about 2.5×10 -7 M or less, approximately 1×10 -7 M or less, approximately 7.5 x 10 -8 M or less, about 5 x 10 -8 M or less, approximately 1×10 -8 The equilibrium dissociation constant (K d ); -About 1×10 4 M -1 seconds -1 or more, preferably about 5×10 4 M -1 seconds -1 That's about 1 x 10 5 M -1 seconds -1 That's about 2.5 x 10 5 M -1 seconds -1 The above is about 5×105M. - 1 second -1 Association rate (K on ); and -About 5×10 -2 seconds -1 Less than or equal to about 4 x 10 -2 seconds -1 Below, approximately 3×10 -2 seconds -1 Below, approximately 2×10 -2 seconds -1 Below, approximately 1.5×10 -2 seconds -1 The following dissociation rate (K off) and binds to hCD45RC.

[0080] Methods for determining the affinity of an antigen-binding domain or an antibody for its ligand (e.g., K d , k off and k on Methods for determining the affinity of a protein (including determining the affinity of a protein) are well known in the art and include, but are not limited to, surface plasmon resonance (SPR), fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), AlphaLISA, and KinExA.

[0081] A preferred method is BIAcore®, which relies on SPR using immobilized CD45RC to determine the affinity of an antigen-binding domain or of an antibody.

[0082] In one embodiment, the CD45RC binding domain is an isolated CD45RC binding domain.

[0083] "Isolated" means that the CD45RC binding domain is substantially free of other antigen binding domains having different antigen specificity; in other words, the CD45RC binding domain is substantially free of antigen binding domains that specifically bind to antigens other than CD45RC. However, an isolated CD45RC binding domain may have cross-reactivity to other antigens, particularly CD45RC proteins from other species. Furthermore, an isolated CD45RC binding domain will be substantially free of other cellular material and / or chemicals, particularly including but not limited to enzymes, hormones, and other proteinaceous or non-proteinaceous components, that would interfere with the diagnostic or therapeutic use of the CD45RC binding domain.

[0084] In one embodiment, the isolated CD45RC binding domain is thus purified.

[0085] In one embodiment, the isolated CD45RC binding domain comprises: (1) to greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% or more by weight of the CD45RC binding domain, as can be determined by the Lowry method, and most preferably to greater than 96%, 97%, 98% or 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer; or (3) to homogeneity, as can be shown by SDS-PAGE under reducing or non-reducing conditions and using Coomassie blue or, preferably, silver staining; It is refined.

[0086] In one embodiment, the antigen binding domain is a molecule selected from the group comprising or consisting of a single chain antibody, Fv, Fab, Fab', Fab'-SH, and F(ab')2.

[0087] In one embodiment, the antigen-binding domain is a single chain antibody. In one embodiment, the single chain antibody is a single chain variable fragment (scFv), ascFv-Fc, tandem-di-scFv, tandem-tri-scFv, scFv-Fc, (scFv-C H 3) 2 (also called minibody), (scFv-C H 2-C H 3) selected from the group including or consisting of bibodies (also called maxibodies), diabodies, and triabodies.

[0088] In one embodiment, the antigen binding domain is an scFv.

[0089] In one embodiment, the scFv has a V H Domain and V L It includes a linker between the domains, in one embodiment, the linker is a short polypeptide, preferably having a length in the range of 2 to 25 amino acids.

[0090] For example, glycine-serine linkers provide particularly suitable linkers. Examples of glycine-serine linkers include, but are not limited to, (GS) n Linker, (G2S) n Linker, (G3S) n Linker, (G4S) n linker, etc., where n is a positive integer. In one embodiment, n is equal to 1, 2, 3, 4, 5 or more.

[0091] Specific examples of glycine-serine linkers include, but are not limited to, SEQ ID NOs: 30-39. [ka]

[0092] In what follows, unless otherwise stated, the numbering and definitions of the CDRs are according to the Kabat / Chothia definitions.

[0093] In one embodiment, a CD45RC binding domain comprises a heavy chain variable region (HCVR) that comprises at least one, preferably at least two, and more preferably the following three CDRs: V H - CDR1: NYYIG (SEQ ID NO: 10); V H - CDR2: DIFPGGDYANYNEKFQG (SEQ ID NO: 11); and V H - CDR3: RNFDY (SEQ ID NO: 12).

[0094] In one embodiment, a CD45RC binding domain comprises an HCVR that comprises the following three CDRs: V H - CDR1: NYYIG (SEQ ID NO: 10); V H - CDR2: DIFPGGDYANYNEKFQG (SEQ ID NO: 11); and V H - CDR3: RNFDY (SEQ ID NO: 12).

[0095] In one embodiment, a CD45RC binding domain comprises at least one, preferably at least two, and more preferably the following three CDRs: V L - CDR1: RASSSVS-X-YMH (SEQ ID NO: 13); V L - CDR2: NTANLPS (SEQ ID NO: 14); and V L -CDR3: QQRSSYPLTF (SEQ ID NO: 15) and a light chain variable region (LCVR) comprising: X is absent or is selected from the group comprising or consisting of Asn (N), Ser (S) and Gly (G), preferably X is absent.

[0096] In one embodiment, a CD45RC binding domain has the following three CDRs: V L - CDR1: RASSSVS-X-YMH (SEQ ID NO: 13); V L - CDR2: NTANLPS (SEQ ID NO: 14); and V L -CDR3: QQRSSYPLTF (SEQ ID NO: 15) LCVR, X is absent or is selected from the group comprising or consisting of Asn (N), Ser (S) and Gly (G), preferably X is absent.

[0097] In one embodiment, the CD45RC binding domain comprises: - an HCVR comprising at least one, preferably at least two, more preferably the following three CDRs: V H - CDR1: NYYIG (SEQ ID NO: 10); V H - CDR2: DIFPGGDYANYNEKFQG (SEQ ID NO: 11); and V H - CDR3: RNFDY (SEQ ID NO: 12); and - an LCVR comprising at least one, preferably at least two, more preferably the following three CDRs: V L - CDR1: RASSSVS-X-YMH (SEQ ID NO: 13); V L - CDR2: NTANLPS (SEQ ID NO: 14); and V L -CDR3: QQRSSYPLTF (SEQ ID NO: 15) Including, X is absent or is selected from the group comprising or consisting of Asn (N), Ser (S) and Gly (G), preferably X is absent.

[0098] In one embodiment, the CD45RC binding domain comprises: -HCVR containing three of the following CDRs: V H - CDR1: NYYIG (SEQ ID NO: 10); V H - CDR2: DIFPGGDYANYNEKFQG (SEQ ID NO: 11); and V H - CDR3: RNFDY (SEQ ID NO: 12); and -LCVR containing the following three CDRs: V L - CDR1: RASSSVS-X-YMH (SEQ ID NO: 13); V L - CDR2: NTANLPS (SEQ ID NO: 14); and V L -CDR3: QQRSSYPLTF (SEQ ID NO: 15) Including, X is absent or is selected from the group comprising or consisting of Asn (N), Ser (S) and Gly (G), preferably X is absent.

[0099] In one embodiment, V as defined above H -CDR1, V H -CDR2, V H -CDR3, V L -CDR1, VL - CDR2 and / or V L - Any of the CDR3 may be characterized as having an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with a particular CDR or set of CDRs defined above, while the CD45RC binding domain retains its overall functional properties, in particular its binding specificity for CD45RC, preferably for hCD45RC.

[0100] In one embodiment, a CD45RC binding domain comprises an HCVR that comprises at least one, preferably at least two, more preferably at least three, and even more preferably four of the following framework regions (FRs): V H - FR1: QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 16); V H - FR2: WVRQAPGQGLEWIG (SEQ ID NO: 17); V H - FR3: RVTLTADTSISTAYMELSRLRSDDTVVYYCVR (SEQ ID NO: 18); V H - FR4:WGQGTLVTVSS (sequence number: 19).

[0101] In one embodiment, a CD45RC binding domain comprises an HCVR that includes the four following FRs: V H - FR1: QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 16); V H - FR2: WVRQAPGQGLEWIG (SEQ ID NO: 17); V H - FR3: RVTLTADTSISTAYMELSRLRSDDTVVYYCVR (SEQ ID NO: 18); V H - FR4:WGQGTLVTVSS (sequence number: 19).

[0102] In one embodiment, a CD45RC binding domain comprises at least one, preferably at least two, more preferably at least three, and even more preferably at least four of the following FRs: V L - FR1:DIQLTQSPSFLSASVGDRVTITC (SEQ ID NO: 20); V L - FR2: WYQQKPGKAPKLWIY (SEQ ID NO: 21); V L - FR3: GVPSRFSGSGSGTE-X-TLTISSLQPEDFATYYC (SEQ ID NO: 22); V L -FR4: GGGTKVEIK (SEQ ID NO: 23) LCVR, X is selected from the group comprising or consisting of Tyr (Y) and Phe (F), preferably X is Tyr (Y).

[0103] In one embodiment, a CD45RC binding domain comprises the four following FRs: V L - FR1:DIQLTQSPSFLSASVGDRVTITC (SEQ ID NO: 20); V L - FR2: WYQQKPGKAPKLWIY (SEQ ID NO: 21); V L - FR3: GVPSRFSGSGSGTE-X-TLTISSLQPEDFATYYC (SEQ ID NO: 22); V L -FR4: GGGTKVEIK (SEQ ID NO: 23) LCVR, X is selected from the group comprising or consisting of Tyr (Y) and Phe (F), preferably X is Tyr (Y).

[0104] In one embodiment, the CD45RC binding domain comprises a HCVR comprising or consisting of the amino acid sequence of SEQ ID NO:24.

[0105] In one embodiment, the CD45RC binding domain comprises three CDRs having SEQ ID NOs: 10, 11, and 12, and a HCVR comprising framework regions sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the framework regions of SEQ ID NO:24. [ka]

[0106] In one embodiment, a CD45RC binding domain comprises a LCVR comprising or consisting of the amino acid sequence of SEQ ID NO:25.

[0107] In one embodiment, a CD45RC binding domain comprises three CDRs having SEQ ID NOs: 13, 14, and 15, and an LCVR comprising framework regions that share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the framework regions of SEQ ID NO: 25; - X1 is absent or is selected from the group comprising or consisting of Asn (N), Ser (S) and Gly (G), preferably X1 is absent; and -X2 is selected from the group comprising or consisting of Tyr (Y) and Phe (F), preferably X2 is Tyr (Y). [ka]

[0108] In one embodiment, X1 is absent and X2 is Tyr (Y).

[0109] In one embodiment, the CD45RC binding domain comprises: - a HCVR comprising or consisting of the amino acid sequence of SEQ ID NO: 24; and - LCVR comprising or consisting of the amino acid sequence of SEQ ID NO: 25 Includes.

[0110] In one embodiment, the CD45RC binding domain comprises: - a HCVR comprising three CDRs having SEQ ID NOs: 10, 11, and 12 and framework regions sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the framework regions of SEQ ID NO: 24; and - an LCVR comprising three CDRs having SEQ ID NOs: 13, 14, and 15 and framework regions that share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the framework regions of SEQ ID NO: 25; Includes.

[0111] In one embodiment, the HCVR and / or LCVR defined above may be characterized as having an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with a particular HCVR and / or LCVR defined above, whilst the CD45RC binding domain retains its overall functional properties, in particular its binding specificity for CD45RC, preferably for hCD45RC.

[0112] In one embodiment, the sequences of the framework regions of the HCVRs and / or LCVRs defined above may be characterized as having an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences of the framework regions of a particular HCVR and / or LCVR defined above, whilst the CD45RC binding domain retains its overall functional properties, in particular its binding specificity for CD45RC, preferably for hCD45RC.

[0113] In one embodiment, the CD45RC binding domain is conjugated to the payload, optionally via a linker.

[0114] Examples of suitable payloads include, but are not limited to, peptides, polypeptides, proteins, polymers, nucleic acid molecules, small molecules, mimetics, synthetic drugs, inorganic molecules, organic molecules, and radioisotopes.

[0115] Alternatively or additionally, examples of suitable payloads include, but are not limited to, chemotherapeutic agents, targeted therapeutic agents, cytotoxic agents (or cytotoxins), cell cycle synchronizers, ligands for cell receptor(s), immunomodulatory agents, pro-apoptotic agents, lytic peptides, anti-angiogenic agents, cytokines, growth factors, hormones, coding or non-coding oligonucleotides, optically detectable labels, imaging agents, radiolabels, and the like.

[0116] It is clear that some payloads can fall into multiple categories.

[0117] In one embodiment, the payload is a chemotherapeutic agent. As used herein, the term "chemotherapeutic agent" refers to any molecule that is effective in inhibiting tumor growth.

[0118] Suitable examples of chemotherapeutic agents include those listed in subgroup L01 of the Anatomical Therapeutic Chemical Classification System.

[0119] Suitable examples of chemotherapeutic agents include, but are not limited to, the following: - alkylating agents, such as: Nitrogen mustards, such as chloromethine, cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, melphalan, prednimustine, bendamustine, uramustine, chlornaphazine, chlorophosphamide, estramustine, mechlorethamine, mechlorethamine oxide hydrochloride, novembichin, phenesterine, uracil mustard, etc.; Nitrosoureas, such as carmustine, lomustine, semustine, fotemustine, nimustine, ranimustine, streptozocin, chlorozotocin, etc.; Alkyl sulfonates, such as busulfan, mannosulfan, treosulfan, etc.; Aziridines, such as carboquone, thiotepa, triaziquone, triethylenemelamine, benzodopa, meturedopa, uredopa, etc.; hydrazines, such as procarbazine, etc.; Triazenes, such as dacarbazine, temozolomide, etc.; ethylenimines and methylameramines, such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolmelamine, etc.; · and others, such as mitobronitol, pipobroman, actinomycin, bleomycin, mitomycin (such as mitomycin C), plicamycin, etc.; -Acetogenins, such as bullatacin, bullatacinone, etc.; - benzodiazepines, for example 2-oxoquazepam, 3-hydroxyphenazepam, bromazepam, camazepam, carbrazepam, chlordiazepoxide, cinazepam, cinolazepam, clonazepam, cloniprazepam, clorazepate, ciprazepam, delorazepam, demoxepam, desmethylflunitrazepam, debuazepide, diazepam, diclazepam, difludiazepam, doxefazepam, elfazepam, ethylcarfluzepate, ethyldirazepate, loflazepate Ethyl azepamate, flubromazepam, flurazepam, fludiazepam, flunitrazepam, flurazepam, flutemazepam, flutoprazepam, fosazepam, gidazepam, halazepam, iclazepam, irazepine, kenazepine, ketazolam, lorazepam, lormetazepam, rufurad, meclonazepam, medazepam, menitrazepam, metaclazepam, motrazepam, N-desalkylflurazepam, nifoxapam , nimetazepam, nitemazepam, nitrazepam, nitrazepate, nordazepam, nortetrazepam, oxazepam, phenazepam, pinazepam, pivoxazepam, prazepam, proflazepam, quazepam, QH-II-66, leclazepam, RO4491533, Ro5-4864, SH-I-048A, surazepam, temazepam, tetrazepam, thifluadom, torfazepam, triflunordazepam, tuclazepam, uldazepam, alfendazam, Clobazam, CP-1414S, lofendazam, triflubazam, gilisopam, GYKI-52466, GYKI-52895, nerisopam, talampanel, tofisopam, adinazolam, alprazolam, bromazolam, clonazolam, estazolam, fluralprazolam, flubromazolam, flunitrazolam, nitrazolam, pyrazolam, triazolam, bretazenil, climazolam, EVT-201, FG-8205, flumazenil, GL-II-73, imidazenil, 123I-iomazenil, L-655,708, Loprazolam, Midazolam, PWZ-029, Remimazolam, Ro15-4513, Ro48-6791, Ro48-8684, Ro4938581, Salmazenil, SH-053-R-CH3-2'F, Cloxazolam, Flutazolam, Haloxazolam, Mexazolam, Oxazolam, Bentazepam, Clotiazepam, Brotizolam, Cyclo Tizolam, deschloretizolam, etizolam, fluclotizolam, israpafant, JQ1, metizolam, olanzapine, telenzepine, lopirazepam, zapizolam, lazobazam, ripazepam, zolazepam, zomebazam, zometapine, premazepam, clazolam, anthramycin, avizafone, rilmazafone, etc; -Antimetabolites, e.g.: Folate antagonists, including aminopterin, methotrexate, pemetrexed, pralatrexate, pteropterin, raltitrexed, denopterin, trimetrexate, pemetrexed, etc.; Purine analogues, including pentostatin, cladribine, clofarabine, fludarabine, nelarabine, thioguanine, mercaptopurine, etc.; Pyrimidine analogues, including fluorouracil, capecitabine, doxifluridine, tegafur, tegafur / gimeracil / oteracil, carmofur, floxuridine, cytarabine, gemcitabine, azacitidine, decitabine, and others; and Hydroxycarbamide; -Androgens, such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, etc.; -Antiadrenal drugs, such as aminoglutethimide, mitotane, trilostane, etc.; -Folic acid supplements, such as folic acid; -Maytansinoids, e.g. maytansine, ansamitocins, etc.; - Platinum analogues, for example, platinum, carboplatin, cisplatin, dicycloplatin, nedaplatin, oxaliplatin, satraplatin, etc.; -Anti-hormonal drugs, such as: ·Antiestrogens, including tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxyphene, keoxyphene, LY117018, onapristone, toremifene, etc.; Antiandrogens, including flutamide, nilutamide, bicalutamide, leuprolide, goserelin, etc.; - Trichothecenes, such as T-2 toxin, verlacrin A, roridin A, anguidine, etc.; - Toxoids, such as cabazitaxel, docetaxel, larotaxel, ortataxel, paclitaxel, tesetaxel, etc.; -Topoisomerase I inhibitors, such as belotecan, camptothecin, cositecan, etirinotecan pegol, exatecan, gimatecan, irinotecan, lurtotecan, rubitecan, ciratecan, topotecan, etc.; -Topoisomerase II inhibitors, such as etoposide, teniposide, etc.; - others, for example, camptothecins (such as synthetic analogues topotecan); bryostatins; kallistatins; CC-1065 (such as adozelesin), carzelesin and bizelesin synthetic analogues; cryptophycins (such as cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (such as their synthetic analogues KW-2189 and CBI-TMI); eleutherobin; pancratistatins; sarcodictyins; spongistatins; aclacinomycins; autramycins ( authramycin;azaserine;bleomycin;cactinomycin;carabicin;carminomycin;carzinophilin;chromomycin;dactinomycin;daunorubicin;detorubicin;6-diazo-5-oxo-L-norleucine;doxorubicin (morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, deoxydoxorubicin, etc.);epirubicin;esorubicin;idanrbicin;marcelomycin;mycophenolic acid;nogalamycin (noga larnycin;olivomycin;peplomycin;potfiromycin;puromycin;curamycin;rodorubicin;streptomgrin;streptozocin;tubercidin;ubenimex;zinostatin;zorubicin;aceglatone;aldophosphanide glycosides;aminolevulinic acid;amsacrine;bestrabucil;bisantrene;edatraxate;defofamine;demecolcine;diaziquone;eflornithine ;Elliptinium acetate;Epothilone;Etoglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidamine;Mitoguazone;Mitoxantrone;Mopidamol;Nitracrine;Phenamet;Pirarubicin;Podophyllic acid;2-Ethylhydrazide;PSK(registered trademark);Razoxane;Rhizoxin;Sizofiran;Spirogennanium;Tenuazonic acid;2,2',2''-Trichlorotriethylamine;Urethane;Vindesine;Dacarbazine;Mannomustine;Mitobromtol; Mitolactol; Pipobroman; Gacytosine; Arabinoside; 6-thioguanine; Vinblastine; Etoposide; Vincristine; Vinorelbine; Navelbine; Novantrone; Teniposide; Daunomycin; Xeloda; Ibandronate; CPT-11; Topoisomerase inhibitor RFS2000; Topoisomerase I inhibitor SN38; Difluoromethylornithine; Retinoic acid, etc.;

[0120] In one embodiment, the payload is a targeted therapeutic agent.

[0121] As used herein, the term "targeted therapeutic agent" refers to any molecule that is directed to one or more specific target molecules (e.g., proteins, etc.) involved in tumor origin, tumor progression, tumor metastasis, tumor cell proliferation, cell repair, and the like.

[0122] Suitable examples of targeted therapeutic agents include, but are not limited to, tyrosine kinase inhibitors, serine / threonine kinase inhibitors, monoclonal antibodies, and the like.

[0123] Suitable examples of targeted therapeutic agents include, but are not limited to, HER1 / EGFR inhibitors (e.g., brigatinib, erlotinib, gefitinib, olmutinib, osimertinib, rociletinib, vandetanib, etc.); HER2 / neu inhibitors (e.g., afatinib, lapatinib, neratinib, etc.); C-kit and PDGFR inhibitors (e.g., axitinib, masitinib, pazopanib, sunitinib, sorafenib, toceranib, etc.); FLT3 inhibitors (e.g., lestaurtinib, etc.); VEGFR inhibitors (e.g., axitinib, nib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, etc.; RET inhibitors (e.g., vandetanib, elektinib, etc.); c-MET inhibitors (e.g., cabozantinib, etc.); bcr-abl inhibitors (e.g., imatinib, dasatinib, nilotinib, ponatinib, radotinib, etc.); Src inhibitors (e.g., bosutinib, dasatinib, etc.); Janus kinase inhibitors (e.g., lestaurtinib, momelotinib, ruxolitinib, etc.) nib, pacritinib, etc.); MAP2K inhibitors (e.g., cobimetinib, selumetinib, trametinib, binimetinib, etc.); EML4-ALK inhibitors (e.g., alectinib, brigatinib, ceritinib, crizotinib, etc.); Bruton's inhibitors (e.g., ibrutinib, etc.); mTOR inhibitors (e.g., everolimus, temsirolimus, etc.); Hedgehog inhibitors (e.g., sonidegib, vismodegib, etc.); CDK inhibitors (e.g., palbociclib, ribociclib, etc.); anti-HER1 / EGFR monoclonal antibodies (e.g., cetuximab, necitumumab, panitumumab, etc.); anti-HER2 / neu monoclonal antibodies (e.g., ado-trastuzumab emtansine, pertuzumab, trastuzumab, trastuzumab-dkst, etc.); anti-EpCAM monoclonal antibodies (e.g., catumaxomab, edrecolomab, etc.); anti-VEGF monoclonal antibodies (e.g., bevacizumab, bevacizumab-awwb, etc.); anti-CD20 monoclonal antibodies (e.g., ibritumomab, obinutuzumab, ocrelizumab, ofatumumab, rituximab, tositumomab, etc.);Anti-CD30 monoclonal antibodies (e.g., brentuximab, etc.); anti-CD33 monoclonal antibodies (e.g., gemtuzumab, etc.); and anti-CD52 monoclonal antibodies (e.g., alemtuzumab, etc.);

[0124] In one embodiment, the payload is a cytotoxic agent.

[0125] As used herein, the term "cytotoxic agent" or "cytotoxin" refers to any molecule that brings about cell death by any mechanism.

[0126] Suitable examples of cytotoxic agents include, but are not limited to, taxanes, anthracyclines, alkylating agents, vinca alkaloids, antimetabolites, platinum agents, steroids, chemotherapeutic agents and auristatins.

[0127] Suitable examples of taxanes include, but are not limited to, cabazitaxel, docetaxel, larotaxel, ortataxel, paclitaxel, and tesetaxel.

[0128] Suitable examples of anthracyclines include, but are not limited to, aclarubicin, amrubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, pirarubicin, valrubicin, and zorubicin.

[0129] Suitable examples of alkylating agents include, but are not limited to, nitrogen mustards (e.g., chlormethine, cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, melphalan, prednimustine, bendamustine, uramustine, etc.), nitrosoureas (e.g., carmustine, lomustine, semustine, fotemustine, nimustine, ranimustine, streptozocin, etc.), alkylsulfonates (e.g., busulfan, mannosulfan, treosulfan, etc.), etc.), aziridines (e.g., carboquone, thiotepa, triaziquone, triethylenemelamine, benzodopa, meturedopa, uredopa, etc.), hydrazines (e.g., procarbazine, etc.), triazenes (e.g., dacarbazine, temozolomide, etc.), altretamine, mitobronitol, pipobroman, actinomycin, bleomycin, mitomycin, and plicamycin.

[0130] Suitable examples of vinca alkaloids include, but are not limited to, vinblastine, vincristine, vinflunine, vindesine, and vinorelbine.

[0131] Suitable examples of antimetabolites include, but are not limited to, folate antagonists (e.g., aminopterin, methotrexate, pemetrexed, pralatrexate, raltitrexed, pemetrexed, etc.), purine analogs (e.g., pentostatin, cladribine, clofarabine, fludarabine, nelarabine, thioguanine, mercaptopurine, etc.), pyrimidine analogs (e.g., fluorouracil, capecitabine, doxifluridine, tegafur, tegafur / gimeracil / oteracil, carmofur, floxuridine, cytarabine, gemcitabine, azacitidine, decitabine, etc.), and hydroxycarbamide.

[0132] Suitable examples of platinum agents include, but are not limited to, carboplatin, cisplatin, dicycloplatin, nedaplatin, oxaliplatin, and satraplatin.

[0133] Suitable examples of steroids include, but are not limited to, estrogen receptor modulators, androgen receptor modulators, and progesterone receptor modulators.

[0134] Suitable examples of chemotherapeutic agents are described above.

[0135] Suitable examples of auristatins include, but are not limited to, auristatin E, auristatin F, monomethylauristatin E, and monomethylauristatin F.

[0136] In one embodiment, the payload is a cell cycle synchronizing agent.

[0137] As used herein, the term "cell cycle synchronizing agent" refers to any molecule that, upon administration, is capable of synchronizing the cell cycle of a population of cells into the same phase.

[0138] Suitable examples of cell cycle synchronizers include, but are not limited to, aphidicolin, butyrolactone I, colchicine, cycloheximide, demecolcine, dimethylsulfoxide, 5-fluorodeoxyuridine, Hoechst 33342, mimosine, nocodazole, roscovitine, and thymidine.

[0139] In one embodiment, the payload is a ligand for a cellular receptor.

[0140] As used herein, the term "ligand for a cellular receptor" refers to any molecule that binds to a cellular receptor (e.g., a cell surface receptor, an intracellular receptor or a co-receptor, such as a transcription factor), and includes agonists and antagonists, partial agonists, inverse agonists, and allosteric modulators.

[0141] Suitable examples of ligands for cell receptors include, but are not limited to, AATYK receptors, acetylcholine receptors, ADGRG receptors, adiponectin receptors, adrenergic alpha 1 receptors, adrenergic alpha 2 receptors, adrenergic beta 1 receptors, adrenergic beta 2 receptors, adrenergic beta 3 receptors, adrenomedullin receptors, AMPA receptors, anaphylatoxin receptors, angiopoietin receptors, angiotensin receptors, anti-Mullerian hormone receptors, apelin receptors, asialoglycoprotein receptors, AXL receptors, and the like. Receptor, benzodiazepine receptor, bile acid receptor, bombesin receptor, bone morphogenetic protein receptor, bradykinin receptor, brain-specific angiogenesis inhibitor, cadherin receptor, calcitonin receptor, calcitonin receptor-like receptor, calcium-sensing receptor, cannabinoid receptor, CD97 receptor, chemokine receptor, cholecystokinin receptor, complement receptor, corticotropin-releasing hormone receptor, CysLT receptor, cytokine receptor, DDR receptor, dopamine receptor, EBI2 receptor, ectodysplasin A receptor, EGFR F module-containing mucin-like hormone receptor, EGF receptor, endothelin receptor, EPH receptor, estrogen receptor, FGF receptor, free fatty acid receptor, frizzled receptor, FSH receptor, GABAB receptor, galanin receptor, GHB receptor, ghrelin receptor, glucagon receptor, glucagon-like peptide receptor, glutamate receptor, glycine receptor, gonadotropin receptor, gonadotropin-releasing hormone receptor, GPRC6A receptor, growth factor receptor, growth hormone receptor, growth hormone-releasing hormone receptor, guanylate cyclase-coupled receptor body, HGF receptor, histamine receptor, hydroxycarboxylic acid receptor, immunoglobulin immune receptor, insulin receptor, kainate receptor, KiSS1-inducing peptide receptor, latrophin receptor, leptin receptor, leukotriene B4 receptor, lipoprotein receptor-related protein receptor, LTK receptor, luteinizing hormone / chorionic gonadotropin receptor, lysophosphatidic acid receptor, lysophospholipid receptor, mannose receptor, MAS receptor, melanin-concentrating hormone receptor, melanocortin receptor, melatonin receptor, methuselah-like protein receptor,Motilin receptor, MuSK receptor, N-acetylglucosamine receptor, Neuromedin receptor, Neuropeptide B / W receptor, Neuropeptide FF receptor, Neuropeptide S receptor, Neuropeptide Y receptor, Neuropilin receptor, Neurotensin receptor, N-formyl peptide receptor, Nicotinic acetylcholine receptor, NMDA receptor, Nuclear receptor, Olfactory receptor, Opioid receptor, Opsin receptor, Orexin receptor, Oxoeicosanoid receptor, Oxoglutarate receptor, Oxytocin receptor, Parathyroid hormone receptor, PDGF receptor, Pituitary adenylate cyclase-activating polypeptide type I receptor, Platelet-activating factor receptor, Progestin and adipoQ receptor, Prokineticin receptor, Prolactin receptor, Prolactin-releasing peptide receptor, Prostacyclin receptor, Prostaglandin receptor, Protease-activated receptor, PTK7 receptor, Purinergic receptor adenosine receptors, purinergic P2X receptors, purinergic P2Y receptors, relaxin receptors, RET receptors, retinoic acid-inducible orphan G protein-coupled receptors, ROR receptors, ROS receptors, RYK receptors, scavenger receptors, secretin receptors, serine / threonine-specific receptor protein kinase receptors, serotonin receptors, smoothened receptors, somatostatin receptors, sphingosine-1-phosphate receptors, SREB receptors, stimulator of interferon genes (STING) receptors, succinate receptors, tachykinin receptors, thromboxane receptors, thyrotropin receptors, thyrotropin-releasing hormone receptors, toll-like receptors, trace amine-related receptors, transferrin receptors, Trk receptors, tumor necrosis factor receptors, tyrosine phosphatase receptors, urotensin II receptors, vasoactive intestinal peptide receptors These include ligands that bind to intestinal peptide receptors, vasoactive intestine peptide receptors, vasopressin receptors, VEGF receptors, vomeronasal receptors, and zinc-activated ion channel receptors.

[0142] In one embodiment, the payload is an immunomodulatory agent.

[0143] Suitable examples of immunomodulatory agents include, but are not limited to, immunostimulants and immunosuppressants.

[0144] Suitable examples of immune stimulants include those described in subgroup L03 of the Anatomical Therapeutic Chemical Classification System.

[0145] Suitable examples of immune stimulants include, but are not limited to, cytokines (e.g., filgrastim, pegfilgrastim, lenograstim, molgramostim, sargramostim, ansestim, albin interferon, interferon alpha natural, interferon alpha 2a, pegylated interferon alpha 2a, interferon alpha 2b, pegylated interferon alpha-2b, interferon alpha n1, interferon alfacon-1, interferon α-n3, interferon beta natural, interferon beta 1a, interferon beta 1b, interferon gamma, aldesleukin, oprelvekin, etc.); immune checkpoint inhibitors (e.g., inhibitors of CTLA4, PD-1, PD-L1, LAG-3, B7-H3, B7-H4, TIM3, A2AR, and / or ID, e.g., nivolumab, pembrolizumab, pidilizumab, AMP-224, MPDL3280A, MDX -1105, MEDI-4736, allerumab, ipilimumab, tremelimumab, pidilizumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870, 893, mogamulizumab, varlilumab, avelumab, galiximab, AMP-514, AUNP12, indoximod, NLG-919, INCB024360, etc.);Toll-like receptor agonists (e.g., buprenorphine, carbamazepine, ethanol, fentanyl, GS-9620, imiquimod, lefitolimod, levorphanol, methadone, morphine, (+)-morphine, morphine-3-glucuronide, oxcarbazepine, oxycodone, pethidine, etc.), resiquimod, SD-101, tapentadol, tilsotolimod, VTX-2337, glucuronoxylomannan from Cryptococcus, MALP-2 from Mycoplasma, MALP-404 from Mycoplasma, OspA from Borrelia, porin from Neisseria or Haemophilus, hsp60, hemagglutinin, e.g., erythropoietin ... LcrV from Lucinia, bacterial flagellin, lipopolysaccharide, lipoteichoic acid, lipomannan from Mycobacterium, glycosylphosphatidylinositol, lysophosphatidylserine, lipophosphoglycan from Leishmania, zymosan from Saccharomyces, Pam2CGDPKHPKSF, Pam3CSK4, CpG oligodeoxynucleotides, poly(I:C) nucleic acid sequences, poly(A:U) nucleic acid sequences, double-stranded viral RNA, etc.); STING receptor agonists (e.g., those described in WO2017100305, vadimezan, CL656, ADU-S100, 3'3'-cGAMP, 2'3'-cGAMP, ML RR-S2 CDG, ML RR-S2 cGAMP, cyclic di-GMP, DMXAA, DiABZI, etc.);CD1 ligand;growth hormone;immunocyanin;pegademase;prolactin;tasonermin;female sex steroids;histamine dihydrochloride;poly ICLC;vitamin D;lentinan;plerixafor;roquinimex;mifamurtide;glatiramer acetate;thymopentin;thymosin alpha 1;thymulin;polyinosinic acid:polycytidylic acid;pidotimod;bacillus Calmette-Guerin;melanoma vaccine;sipuleucel-T;etc.;

[0146] Suitable examples of immunosuppressants include those described in subgroup L04 of the Anatomical Therapeutic Chemical Classification System.

[0147] Suitable examples of immunosuppressants include, but are not limited to, the following: -Antimetabolites, e.g.: Folate antagonists, including aminopterin, methotrexate, pemetrexed, pralatrexate, pteropterin, raltitrexed, denopterin, trimetrexate, pemetrexed, etc.; Purine analogues, including pentostatin, cladribine, clofarabine, fludarabine, nelarabine, thioguanine, mercaptopurine, etc.; Pyrimidine analogues, including fluorouracil, capecitabine, doxifluridine, tegafur, tegafur / gimeracil / oteracil, carmofur, floxuridine, cytarabine, gemcitabine, azacitidine, decitabine, and others; and Hydroxycarbamide; - Macrolides, e.g. tacrolimus, cyclosporine, pimecrolimus, abetimus, gusperimus, etc.; - Immunomodulatory imide drugs, such as lenalidomide, pomalidomide, thalidomide, apremilast, etc.; -IL-1 receptor antagonists, such as anakinra; mTOR inhibitors, such as sirolimus, everolimus, ridaforolimus, temsirolimus, umirolimus, zotarolimus, etc.; Serum-targeted antibodies, such as eculizumab, adalimumab, afelimomab, certolizumab pegol, golimumab, infliximab, nerelimomab, mepolizumab, omalizumab, faralimomab, elsilimomab, lebrikizumab, ustekinumab, secukinumab, etc.; - cell-targeting antibodies, such as muromonab-CD3, otelixizumab, teplizumab, vicilizumab, clenoliximab, keliximab, zanolimumab, efalizumab, erlizumab, obinutuzumab, rituximab, ocrelizumab, pascolizumab, gomiliximab, lumiliximab, teneliximab, toralizumab, acelizumab, galixima , gavilimomab, ruplizumab, belimumab, brisibimod, ipilimumab, tremelimumab, bertilimumab, lerdelimumab, metelimumab, natalizumab, tocilizumab, odulimomab, basiliximab, daclizumab, inolimomab, zolimomab Zolimomab aritox, atorolimumab, cedelizumab, fontolizumab, maslimomab, morolimumab, pexelizumab, reslizumab, lovelizumab, siplizumab, talizumab, telimomab aritox, vapaliximab, vepalimomab, etc; - Fusion antibodies, such as abatacept, belatacept, etanercept, pegsunercept, aflibercept, alefacept, rilonacept, etc.

[0148] In one embodiment, the payload is a pro-apoptotic agent.

[0149] As used herein, the term "pro-apoptotic agent" refers to any molecule that is capable of inducing apoptosis, or programmed cell death, in a cell upon administration.

[0150] Suitable examples of pro-apoptotic agents include, but are not limited to, histone deacetylase inhibitors (e.g., sodium butyrate, depsipeptide, etc.), bortezomib, deguelin, flavopiridol, phenylene nitrile, fludarabine, kaempferol, miltefosine, narciclasine, obatoclax, oblimersen, and oncrasin.

[0151] In one embodiment, the payload is a lytic peptide.

[0152] As used herein, the term "lytic peptide" refers to a peptide that has the ability to lyse or penetrate a membrane.

[0153] Examples of lytic peptides include, but are not limited to, LL-37, α-defensin, These include β-defensins, θ-defensins, dermcidins, histatins, perforins, cecropins, dermaseptins, magainon, melittin, nisin, pneumolysin, and toxins.

[0154] Examples of toxins include, but are not limited to, bacterial toxins (e.g., tetanus tetanus toxin, Clostridium difficile tetanolysin, Clostridium perfringens alpha toxin, Clostridium perfringens enterotoxin, Clostridium difficile toxin A, Clostridium difficile toxin B, Clostridium botulinum toxin, Clostridium botulinum C3 exoenzyme, anthrax toxin, listeriolysin O, Staphylococcus aureus alpha toxin, Staphylococcus aureus beta toxin, Staphylococcus aureus delta toxin, Staphylococcus aureus clumping factor A, Staphylococcus aureus fibronectin-binding protein A, exfoliatin, toxic shock syndrome toxin, enterotoxin type B, cord factor, diphtheria toxin, streptolysin, leucocidin, shiga toxin, shiga-like toxin, heat-stable enterotoxin, cholera toxin, pertussis toxin, Pseudomonas exotoxin, extracellular adenylate cyclase, Bacillus subtilis, thuringiensis delta endotoxin), mycotoxins (e.g., aflatoxins, alpha-amanitin, beta-amanitin, gamma-amanitin, epsilon-amanitin, β-nitropropionic acid, citrinin, cytochalasins, ergotamine, fumonisin B1, fumonisin B2, fumonisin B3, fumonisin B4, gliotoxin, ibotenic acid, lolitrem B, muscimol, ochratoxin, patulin, phalloy gin, sterigmatocystin, trichothecenes, vomitoxin, zeranol, zearalenone), plant toxins (e.g. amygdalin, anisatin, anthialine, brucine, chaconine, cyctoxin, daphnine, delphinin, divicin, djenkolic acid, falcarinol, gossypol, helenalin, ledol, linamarin, lotaustralin, mimosine, enanthotoxin, oleandrin, persin, protoanemonin, etc.), pseudoconitine, retronecin, resiniferatoxin;scopolamine, solamargine, solanidine, solanine, solasodamine, solasodine, solasonine, solauricidine, solauricine, strychnine, swainsonine, tagetitoxin, tiniatoxin, tomatine, abrin, ricin, tutin), invertebrate toxins (e.g., Androctonus australis Hector insect venom, charybdotoxin , maurotoxin, agitoxin, margatoxin, slotoxin, scyllatoxin, hefutoxin, HgeTx1, HsTx1, Lq2, biltoxin, bestoxin, BmKAEP, phaiodotoxin, imperatoxin, Pi3, latrotoxin, CSTX, cupiennins, PhTx 3, stromatoxin, vanillotoxin, huentoxin, conotoxin, eledoisin, onquidar, saxitoxin, tetrodotoxin), and vertebrate toxins (e.g., ciguatera, tetrodotoxin, (+)-alloprimiliotoxin 267A, batrachotoxin, arenobufagin, bufotalin, bufotenin, cinobufagin, marinobufagin, epibatidine, histrionicotoxin, pumiliotoxin 251D, samandarin, samandaridin, talicatoxin, zetekitoxin AB, α-bungarotoxin, β-bungarotoxin, calciceptin, taicatoxin, calcicludin, cardiotoxin III);

[0155] In one embodiment, the payload is an anti-angiogenic agent.

[0156] As used herein, the term "anti-angiogenic agent" refers to a molecule that reduces or prevents angiogenesis, which is responsible for the growth and development of blood vessels.

[0157] Suitable examples of antiangiogenic agents include, but are not limited to, inhibitors of any of the vascular endothelial growth factors VEGF-A, VEGF-B, VEGF-C, or VEGF-D, which are major inducers of angiogenesis under normal and pathological conditions and are essential for fetal vascular formation.

[0158] Additionally or alternatively, anti-angiogenic agents can also inhibit other angiogenic factors, such as, but not limited to, members of the fibroblast growth factor (FGF) family, e.g., FGF-1 (acidic), FGF-2 (basic), FGF-4 or FGF-5; or angiopoietin-1, a factor that signals through the endothelial cell-specific Tie2 receptor tyrosine kinase; or receptors for any of these angiogenic factors.

[0159] In one embodiment, the payload is a cytokine.

[0160] Suitable examples of cytokines include, but are not limited to, chemokines, tumor necrosis factors, interleukins, and colony stimulating factors.

[0161] Suitable examples of chemokines include, but are not limited to, chemokine CC motif ligand (CCL) 1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, , CCL27, CCL28, chemokine C-X-C motif ligand (CXCL) 1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, fractalkine, chemokine C motif ligand (XCL) 1, and XCL2.

[0162] Suitable examples of tumor necrosis factors include, but are not limited to, tumor necrosis factor (TNF) alpha, lymphotoxin, OX40L, CD40LG, Fas ligand, CD70, CD153, 4-1BB ligand, TNF-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor kappa-B ligand (RANKL), proliferation-inducing ligand (APRIL), B-cell activating factor (BAFF), and ectodysplasin A (EDA).

[0163] Suitable examples of interleukins include, but are not limited to, interleukin-(IL-) 1α, IL-1β, IL-1Ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, 1, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL- 35, IL-36α, IL-36β, IL-36γ, IL-36Ra, IL-37, IL-38, interferon (IFN)α, IFNβ, IFNκ, and IFNω.

[0164] Suitable examples of colony-stimulating factors include, but are not limited to, granulocyte-macrophage colony-stimulating factor (GM-CSF) (such as granulocyte-colony-stimulating factor (G-CSF) and macrophage-colony-stimulating factor (M-CSF)), hematopoietin, and thrombopoietin.

[0165] In one embodiment, the payload is a growth factor.

[0166] Suitable examples of growth factors include, but are not limited to, fibroblast growth factor (FGF) 1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF23, transforming growth factor (TGF) alpha, epidermal growth factor (EGF), heparin-binding EGF-like growth factor (HB-EGF), These include transforming growth factor (TGF) beta, insulin-like growth factor (IGF) 1, IGF2, platelet-derived growth factor (PDGF) subunit A (PDGFA), PDGF subunit B (PDGFB), PDGF subunit C (PDGFC), PDGF subunit D (PDGFD), vascular endothelial growth factor (VEGF)-A, VEGF-B, VEGF-C, VEGF-D, placenta growth factor (PGF), nerve growth factor (NGF), and hepatocyte growth factor (HGF).

[0167] In one embodiment, the payload is a hormone.

[0168] Suitable examples of hormones include, but are not limited to, GnRH, TRH, dopamine, CRH, GHRH, somatostatin, MCH, oxytocin, vasopressin, FSH, LH, TSH, prolactin, POMC, CLIP, ACTH, MSH, endorphins, lipotropin, GH, aldosterone, cortisol, cortisone, DHEA, DHEA-S, androstenedione, epinephrine, noradrenaline, thyroid hormone T3, thyroid hormone T4, calcitonin, PTH, testosterone, AMH, inhibin, estradiol, progesterone, activin, and rifapril. These include lacin, GnSAF, hCG, HPL, estrogen, glucagon, insulin, amylin, pancreatic polypeptide, melatonin, N,N-dimethyltryptamine, 5-methoxy-N,N-dimethyltryptamine, thymosin alpha 1, beta thymosin, thymopoietin, thymulin, gastrin, ghrelin, CCK, GIP, GLP-1, secretin, motilin, VIP, enteroglucagon, peptide YY, IGF-1, IGF-2, leptin, adiponectin, resistin, osteocalcin, renin, EPO, calcitriol, prostaglandins, ANP, and BNP.

[0169] In one embodiment, the payload is a coding or non-coding oligonucleotide.

[0170] Suitable examples of coding or non-coding oligonucleotides include, but are not limited to, messenger RNA (mRNA), antisense RNA (asRNA), small interfering RNA (siRNA), microRNA (miRNA), long non-coding RNA (lncRNA) (e.g., transfer RNA [tRNA], ribosomal RNA [rRNA], etc.), small temporal RNA (stRNA), trans-acting siRNA, short hairpin RNA (shRNA), cis-natural antisense transcripts (NAT), crisp RNA, long non-coding RNA, piwi-interacting RNA (piRNA), repeat-associated siRNA (rasiRNA), RNA aptamers, ribozymes, and the like.

[0171] Further suitable examples of coding or non-coding oligonucleotides include, but are not limited to, recapuldencel-T, TriMix, BI-1361849, nusinersen, voranesorsen sodium, eteplirsen, ATL1105, ASM-8, inclisiran, patisiran, RXI-109, fitusiran, semdisiran, QPI-1002, BMS-986263, PF-655, pegaptanib, avasincaptad pegol sodium, olaptesed pegol, emapticap pegol, SPC3649, bevasiranib, AGN-745, QPI-1007, TD101, SYL040012, SYL1001, Excellair, ALN-RSV01, CEQ508, siG12D LODER, TKM-ApoB, TKM-PLK1, ALN-VSP02, ALN-TTR01, Bcr-Abl siRNA, Atu027, I5NP, CALAA-01, FANG vaccine, iPsiRNA, Tat / Rev shRNA, ARC1779, ARC19499, AS1411 (AGRO001), Fovista, NOX-A12, NOX-E36, NOX-H94, NU172, RB006 plus RB007, ARC1905, and those listed in Tables 1 and 2 of Crooke et al., 2018 (Cell Metab. 27(4):714-739), which are incorporated herein by reference.

[0172] In one embodiment, the payload is a light-detectable label.

[0173] As used herein, the term "photodetectable label" or "fluorophore" refers to a moiety that is capable of re-emitting light upon excitation with light.

[0174] Suitable examples of optically detectable labels include, but are not limited to, AlexaFluor® dyes, BODIPY® dyes, fluorescein, 5-carboxyfluorescein, 5-(4,6-dichlorotriazin-2-yl)aminofluorescein, 2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein isothiocyanate (FITC), QFITC, Oregon Green® 488, Oregon Green® 514, rhodamine and its derivatives (e.g., rhodamine green, rhodamine green-X, rhodamine red-X, X-rhodamine, 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), Lissamine rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101 (Texas Red), tetramethylrhodamine, tetramethylrhodamine isothiocyanate (TRITC), eosin, eosin isothiocyanate, erythrosine, erythrosine B, erythrosine isothiocyanate, Texas Red®, Texas Red®-X, naphthofluorescein, malachite green, malachite green isothiocyanate, coumarin derivatives, Pacific Orange, cascade blue, cascade yellow, dansyl chloride, dapoxyl dye, 1-dimethylamine-N(2-azido-ethyl)naphthalene-5-sulfonamide, 6-(6-amino-2-(2-azidoethyl)-1,3-dioxo-1H-benzo(de)-2(3H)isoquinoline, 6-(6-amino-2-(2-propynyl)-1,3-dioxo-1H-benzo(de)-2(3H)isoquinoline, 8-(4-azidoethyloxyphenyl)-2,6-diethyl-1,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene, 8-(4-propynyloxyphenyl)-2,6-diethyl-1,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a,4a-Diaza-s-indacene, 1-(3-azido-propoxy)-7-methylamino-phenoxazin-3-one, 1-(2-propynyl)-7-methylamino-phenoxazin-3-one, N-(5-(3-azidopropylamino))-9H-benzo(a)-phenox-2-yn-9-ylidene)-N-methyl-methanaminium chloride, N-(5-(3-propynyl-amino)-9H-benzo(a)-phenoxazin-9-ylene)-N-methyl-methanaminium chloride , (9-(3-azido-propoxy)-7-piperidin-1-yl-phenoxazin-3-ylidene)-dimethyl-ammonium perchlorate, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, acridine, acridine isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid, 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate, N-(4-anilino-1-naphthyl)maleimide , anthranilamide, brilliant yellow, coumarin, coumarin derivatives, 7-amino-4-methylcoumarin, 7-amino-trifluoromethylcoumarin, cyanosine, 4',6-diminidino-2-phenylindole, 5',5''-dibromopyrogallol-sulfonphthalein, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin-4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid, 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid, Natostilbene-2,2'-disulfonic acid, ethidium, IR144, IR1446, 4-methylumbelliferone, o-cresolphthalein, nitrotyrosine, pararosaniline, phenol red, B-phycoerythrin, o-phthaldialdehyde, pyrene, pyrene butyric acid, 1-pyrene butyric acid succinimidyl, reactive red 4, riboflavin, rosolic acid, lanthanide chelates, quantum dots, cyanines, pyrerium dyes, and squaraines.

[0175] In one embodiment, the payload is a contrast agent.

[0176] As used herein, the term "imaging agent" refers to any molecule used in medical imaging to enhance the contrast of structures or fluids within the body. Imaging agents absorb or alter external electromagnetic or ultrasonic radiation (this differs from radioactive labels, which emit radiation themselves).

[0177] Suitable examples of radiolabels include those described in subgroup V08 of the Anatomical Therapeutic Chemical Classification System.

[0178] Suitable examples of contrast agents include, but are not limited to, diatrizoic acid, metrizoic acid, iodamide, iothalamic acid, ioxithalamic acid, ioglicic acid, acetolyzoic acid, iocarmic acid, methiodal, geodon, metrizamide, iohexol, ioxaglic acid, iopamidol, iopromide, iotrolan, ioversol, iopentol, iodixanol, iomeprol, iobitridol, ioxilan, iodoxamic acid, iotroxic acid, ioglycamic acid, adipiodon, iobenzamic acid, acid), iopanoic acid, iocetamic acid, sodium iopodate, tyroponic acid, calcium iopodate, iopidol, propriodone, iofendylate, lipiodol, barium sulfate, gadobenic acid, gadobutrol, gadodiamide, gadofosbeset, gadolinium, gadopentetic acid, gadoteric acid, gadoteridol, gadoversetamide, gadoxetic acid, ferric ammonium citrate, mangafodipir, ferumoxil, ferristene, perflubron, human albumin microspheres, galactose microparticles, perfrenapent, phospholipid microspheres, sulfur hexafluoride, etc.

[0179] In one embodiment, the payload is a radiolabel.

[0180] As used herein, the term "radiolabel," "radiopharmaceutical," or "radioisotope" refers to any molecule that emits radiation. Radiolabels can be used for therapeutic or diagnostic purposes.

[0181] Suitable examples of radiolabels include those described in subgroups V09 and v10 of the Anatomical Therapeutic Chemical Classification System.

[0182] Suitable examples of radiolabels include, but are not limited to, 99m Tc compounds (e.g., examethazime, medronate, macroaggregated albumin, sestamibi, tetrofosmin, examethazime, sulesomab, tilmanocept, arcitumomab, votumumab, hynic-octreotide, etc.); 123 I, 125 I or 131 I compounds (e.g., iofulpane, iofetamine, iomazenil, sodium iodohippurate, iobenguane, iodocholesterol, minretumomab, tositumomab, etc.); 18 F compounds (e.g., florbetapir, flutemetamol, fluciclovine, fludeoxyglucose, fluoroethyltyrosine, sodium fluoride, etc.); 64Cu compounds (e.g., Cu-ETS2, etc.); 75 Se compounds (e.g., SeHCAT, etc.); 111 In compounds (e.g., imciromab, capromab pendetide, satumomab pendetide, etc.); 82 Rb compounds (e.g. rubidium chloride); 153 Sm compounds (e.g., lexidronam, etc.); 89Sr compounds (e.g., strontium 89 chloride, etc.); 90 Y compounds (e.g., ibritumomab tiuxetan); 223 Ra compounds (e.g. radium 223 chloride, etc.); 177 Lu compound (e.g., oxodotreotide, etc.); and at least one 2 H, 3 H, 11 C.13 N, 14 C. 15 O. 18 F, 22 Na, 24 Na, 32 P, 33 P, 40 K, 47 Ca, 51 Cr, 55 EU, 57 Co, 58 Co, 60 Co, 59 Fe, 64 Cu, 67 Ga, 68 Ga, 75 Se, 81 mKr, 82 Rb, 89 Sr, 90 Sr, 90 Y, 97 Tc, 99 mTc, 103 Pd, 106 Ru, 107 Pd, 111 In, 113 Cd, 121 Sn, 123 I, 124 I, 125 I, 126 Sn, 129 I, 131 I, 133 Xe, 135 Cs, 137 Cs, 153 Sm, 165 Dy, 169Er, 177 Lu, 186 Re, 192 Ir, 198 Au, 201 Tl, 213 Bi, 223 Ra, and / or 225 Any compound containing a Ra atom may be mentioned.

[0183] In one embodiment, the CD45RC binding domain is conjugated to an auristatin, or its analog, derivative, or prodrug. Auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, nucleus and cell division (Woyke et al., 2001. Antimicrob Agents Chemother. 46(12):3802-8), and have anticancer activity (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al., 1998. Antimicrob Agents Chemother. 42(11):2961-5). For example, auristatin E can be reacted with paraacetylbenzoic acid or benzoylvaleric acid to generate AEB and AEVB, respectively. Other typical auristatin derivatives include MMAF (monomethyl auristatin F) and MMAE (monomethyl auristatin E). Suitable auristatins and auristatin analogs, derivatives and prodrugs, as well as suitable linkers for conjugation of auristatins to antibodies, are described, for example, in U.S. Pat. Nos. 5,635,483, 5,780,588 and 6,214,345, and in WO 2002 / 088172, WO 2004 / 010957, WO 2005 / 081711, WO 2005 / 084390, WO 2006 / 132670, WO 2003 / 026577, WO 2007 / 00860, WO 2007 / 011968, and WO 2005 / 082023.

[0184] In one embodiment, the CD45RC binding domain is conjugated to a pyrrolo[2,1-c][1,4]-benzodiazepine (PDB), such as anthramycin, or an analog, derivative, or prodrug thereof. Suitable PDBs and PDB derivatives, and related techniques, are described in the art.

[0185] In one embodiment, the CD45RC binding domain is conjugated to a cytotoxic moiety selected from an anthracycline, a maytansine, a calicheamicin, a duocarmycin, a rachelmycin (CC-1065), dolastatin 10, dolastatin 15, irinotecan, monomethylauristatin E, monomethylauristatin F, PDB, or an analog, derivative, or prodrug thereof.

[0186] In one embodiment, the CD45RC binding domain is conjugated to an anthracycline or an analogue, derivative or prodrug thereof.

[0187] In one embodiment, the CD45RC binding domain is conjugated to maytansine, or an analogue, derivative or prodrug thereof.

[0188] In one embodiment, the CD45RC binding domain is conjugated to a calicheamicin or an analog, derivative or prodrug thereof.

[0189] In one embodiment, the CD45RC binding domain is conjugated to a duocarmycin or an analogue, derivative or prodrug thereof.

[0190] In one embodiment, the CD45RC binding domain is conjugated to rachelmycin (CC-1065) or an analogue, derivative or prodrug thereof.

[0191] In one embodiment, the anti-CD45RC antibody, or antigen-binding fragment thereof, is conjugated to a dolastatin, or an analog, derivative, or prodrug thereof.

[0192] In one embodiment, the CD45RC binding domain is conjugated to monomethyl auristatin E or an analogue, derivative or prodrug thereof.

[0193] In one embodiment, the CD45RC binding domain is conjugated to monomethyl auristatin F or an analog, derivative or prodrug thereof.

[0194] In one embodiment, the CD45RC binding domain is conjugated to irinotecan, or an analog, derivative or prodrug thereof.

[0195] In a second aspect, the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to CD45RC, preferably to hCD45RC. Thus, the antibody or antigen-binding fragment thereof of the present invention is an "anti-CD45RC antibody or antigen-binding fragment thereof", preferably an "anti-hCD45RC antibody or antigen-binding fragment thereof".

[0196] In one embodiment, the anti-CD45RC antibody or antigen-binding fragment thereof comprises at least one CD45RC binding domain, as defined above.

[0197] All features and embodiments described above for the CD45RC binding domain apply mutatis mutandis to anti-CD45RC antibodies or antigen-binding fragments thereof. Additional features are defined hereinafter.

[0198] In one embodiment, the anti-CD45RC antibody or antigen-binding fragment thereof is a polyclonal antibody. Preferably, the anti-CD45RC antibody or antigen-binding fragment thereof is a monoclonal antibody.

[0199] Methods for producing polyclonal and monoclonal antibodies, and fragments thereof, are well known in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988).

[0200] In one embodiment, the anti-CD45RC antibody or antigen-binding fragment thereof has an isotype selected from the group including or consisting of IgG (including IgG1, IgG2, IgG3 and IgG4), IgM, IgA (including IgA1 and IgA2), IgD and IgE.

[0201] The constant region of an antibody is important in the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity and phagocytosis.Therefore, as discussed herein, the isotype of an antibody can be selected based on whether it is desirable for the antibody to mediate cytotoxicity / phagocytosis.The determination or selection of the isotype of an antibody can be performed by methods known in the art.

[0202] In one embodiment, the anti-CD45RC antibody or antigen-binding fragment thereof comprises a heavy chain constant region (HCCR or C H ) and / or the light chain constant region (LCCR or C L (abbreviated as "abbreviated as ").

[0203] In one embodiment, the HCCR and / or light chain constant region LCCR is a murine HCCR and / or LCCR.

[0204] In one embodiment, the HCCR and / or the light chain constant region LCCR, preferably both, are human or humanized.

[0205] In one embodiment, the anti-CD45RC antibody or antigen-binding fragment thereof comprises a human or humanized HCCR comprising or consisting of the amino acid sequence of SEQ ID NO:26; or a human or humanized HCCR comprising or consisting of an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence of SEQ ID NO:26. [ka]

[0206] In one embodiment, the anti-CD45RC antibody or antigen-binding fragment thereof comprises a human or humanized HCCR comprising or consisting of the amino acid sequence of SEQ ID NO:27; or a human or humanized HCCR comprising or consisting of an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence of SEQ ID NO:27. [ka]

[0207] In one embodiment, the anti-CD45RC antibody or antigen-binding fragment thereof comprises a human or humanized HCCR comprising or consisting of the amino acid sequence of SEQ ID NO:28; or a human or humanized HCCR comprising or consisting of an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence of SEQ ID NO:28. [ka]

[0208] In one embodiment, the anti-CD45RC antibody or antigen-binding fragment thereof comprises a human or humanized HCCR comprising or consisting of the amino acid sequence of SEQ ID NO:29; or a human or humanized HCCR comprising or consisting of an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence of SEQ ID NO:29. [ka]

[0209] Thus, in one embodiment, the anti-CD45RC antibody or antigen-binding fragment thereof is - a HCVR comprising or consisting of the amino acid sequence of SEQ ID NO: 24; - a LCVR comprising or consisting of the amino acid sequence of SEQ ID NO: 25; - a HCCR comprising or consisting of the amino acid sequence of SEQ ID NO: 26, 27 or 28, preferably the amino acid sequence of SEQ ID NO: 26; and - LCCR comprising or consisting of the amino acid sequence of SEQ ID NO: 29 Includes.

[0210] In one embodiment, the anti-CD45RC antibody or antigen-binding fragment thereof comprises: - a HCVR comprising three CDRs having SEQ ID NOs: 10, 11, and 12 and framework regions sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the framework regions of SEQ ID NO: 24; - an LCVR comprising three CDRs having SEQ ID NOs: 13, 14, and 15 and framework regions sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the framework regions of SEQ ID NO: 25; - a HCCR comprising or consisting of an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence of any one of SEQ ID NOs: 26, 27 or 28, preferably SEQ ID NO: 26; and - an LCCR comprising or consisting of an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 29; Including, On the other hand, an anti-CD45RC antibody or antigen-binding fragment thereof retains its overall functional properties, in particular its binding specificity for CD45RC, preferably for hCD45RC.

[0211] It will be understood that an anti-CD45RC antibody or antigen-binding fragment thereof can be modified using methods well known in the art, for example, to improve the properties of the isolated antibody or antigen-binding fragment thereof, or to enhance antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). Such modifications are well known in the art.

[0212] For example, to slow down in vivo clearance and obtain a more desirable pharmacokinetic profile, anti-CD45RC antibodies or antigen-binding fragments thereof may be modified with polyethylene glycol (PEG). Methods for site-specific conjugation of PEG to antibodies or antigen-binding fragments thereof are described, for example, in Leong et al., 2001. Cytokine. 16(3): 106-19 or Delgado et al., 1996. Br J Cancer. 73(2): 175-82.

[0213] Another non-limiting example of modification is the modification of the human Fc region of an antibody to increase its affinity for Fcγ receptors. Methods for enhancing Fcγ receptor binding include modification of the primary amino acid sequence of the Fc fragment, particularly the HCCR, and alteration of the carbohydrate structure and content of the Fc fragment.

[0214] Thus, antibodies with reduced fucose content are known to enhance ADCC and / or ADCP responses. Indeed, removal of the core α-1,6-fucose moiety from Fc fragment oligosaccharides has been demonstrated to improve ADCC and / or ADCP activity through improved FcγRIII receptor binding (see, for example, Carter, 2001. Nat Rev Cancer. 1(2): 118-29; Kanda et al., 2007. Glycobiology. 17(1): 104-18; Shields et al., 2002. J Biol Chem. 277(30): 26733-40; Shinkawa et al., 2003. J Biol Chem. 278(5): 3466-73; Niwa et al., 2004. Cancer Res. 64(6): 2127-33; WO 2014 / 140322).

[0215] Thus, an anti-CD45RC antibody or antigen-binding fragment thereof may contain a reduced fucose content.

[0216] As used herein, the term "fucose content" refers to the percentage of fucosylated forms within the N-glycan attached to the N297 residue (corresponding to N180 in SEQ ID NO: 26 or 27) of the Fc fragment of each heavy chain of each antibody.

[0217] As used herein, the term "reduced fucose content" refers to a fucose content of 85% or less. Advantageously, the fucose content is in the range of about 65% to about 85%. Alternatively, the fucose content can be 85% or less, 80%, 75%, 70%, 65%, or even less, such as 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20%. However, the fucose content does not have to be zero, and can be, for example, 5%, 10%, 15%, or 20% or more.

[0218] Levels of another glycoform, the bisecting N-linked carbohydrate, have also been suggested to increase ADCC and / or ADCP (see, e.g., Umana et al., 1999. Nat Biotechnol. 17(2):176-80; Hodoniczky et al., 2005. Biotechnol Prog. 21(6):1644-52).

[0219] In one embodiment, anti-CD45RC antibodies or antigen-binding fragments thereof may further comprise different types of glycosylation (oligomannose or biantennary complex type N-glycans with variable ratios of bisecting N-acetylglucosamine (GlcNAc) residues or galactose residues in the case of biantennary complex type N-glycans), provided that they have a reduced fucose content as defined above (WO 2007 / 048077). For example, antibodies with reduced fucose content can be obtained by the methods described in, for example, European Patent Publication 1 176 195 or WO 2001 / 077181 or WO 2012 / 041768.

[0220] Oligomannose-type N-glycans have a reduced half-life in vivo compared to biantennary complex-type N-glycans. Thus, advantageously, an anti-CD45RC antibody or antigen-binding fragment thereof has a biantennary complex-type glycan structure with reduced fucose content at the N-glycosylation site of the Fc fragment, as defined above.

[0221] Various Fc fragment variants with optimized binding affinity to Fcγ receptors and / or enhanced ADCC and / or ADCP have been described and are known in the art.

[0222] In one embodiment, the anti-CD45RC antibody or antigen-binding fragment thereof is conjugated to a payload, optionally via a linker. Suitable examples of payloads are described above in relation to the CD45RC binding domain, which apply mutatis mutandis here.

[0223] In one embodiment, the payload is a drug. Conjugates between an antibody or antigen-binding fragment thereof and a drug are known in the art as "antibody-drug conjugates" or "ADCs."

[0224] In a third aspect, the present invention relates to a chimeric antigen receptor (CAR) that specifically binds to CD45RC, preferably to hCD45RC. Thus, the CAR of the present invention is an "anti-CD45RC CAR", preferably an "anti-hCD45RC CAR".

[0225] In one embodiment, the anti-CD45RC CAR comprises: (i) at least one extracellular CD45RC binding domain, preferably at least one extracellular hCD45RC binding domain, as described above; (ii) optionally, an extracellular hinge domain; (iii) at least one transmembrane domain; and (iv) at least one intracellular signaling domain, comprising at least one primary signaling domain, and optionally one or more costimulatory signaling domains. Includes.

[0226] In one embodiment, the CAR comprises at least one extracellular CD45RC binding domain, as described above.

[0227] In one embodiment, the extracellular CD45RC binding domain is a CD45RC binding scFv.

[0228] In one embodiment, a CAR comprises two or more extracellular antigen-binding domains, at least a first of which is a CD45RC binding domain as described above, and at least a second of which specifically binds to an antigen other than CD45RC. Such a CAR can thus bind to at least two different antigens, one of which is CD45RC.

[0229] In one embodiment, the extracellular antigen-binding domain, in particular the extracellular CD45RC-binding domain, is connected to the transmembrane domain via a hinge domain.

[0230] In one embodiment, the hinge domain is a short polypeptide linker, preferably having a length in the range of 2 to 25 amino acids. In one embodiment, the hinge domain is a V H Domain and V L A linker similar to the linker between domains.

[0231] In one embodiment, the hinge domain is a glycine-serine linker as described above, e.g., (GS) n Linker, (G2S) n Linker, (G3S) n Linker, (G4S) n linker, etc.; n is a positive integer. In one embodiment, n is equal to 1, 2, 3, 4, 5 or more. In one embodiment, the hinge domain can be a glycine-serine linker having any one of SEQ ID NOs: 30-39.

[0232] Further examples of suitable hinge domains are described in International Patent Publication No. WO 2012 / 138475, which is incorporated herein by reference.

[0233] In one embodiment, the hinge domain may be any one of SEQ ID NOs:40-44. [ka]

[0234] In one embodiment, the hinge domain comprises or consists of an amino acid sequence of a CD8 hinge having SEQ ID NO:45, or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO:45. [ka]

[0235] In another embodiment, the hinge domain comprises or consists of the amino acid sequence of an IgG4 hinge having SEQ ID NO: 46, or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 46. [ka]

[0236] In another embodiment, the hinge domain comprises or consists of an amino acid sequence of an IgD hinge having SEQ ID NO:47, or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO:47. [ka]

[0237] In another embodiment, the hinge domain comprises or consists of an amino acid sequence of a CD28 hinge having SEQ ID NO:48, or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO:48. [ka]

[0238] Examples of transmembrane domains that may be used in a CAR according to the present invention include, but are not limited to, transmembrane domains of the α, β or ζ chains of the T cell receptor, CD28, CD3γ, CD3δ, CD3ε, CD3ζ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1α subunit (CD11a), LFA-1β subunit (CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, C D49a, ITGA4, IA4, CD49d, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, ITGAM, CD11b, PD1, I TGAX, CD11c, ITGB1, CD29, ITGB2, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244), CD84, CD96(Tactile), C These include the transmembrane domains of EACAM1, CRTAM, Ly-9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (Ly-108), SLAMF1 (CD150), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C.

[0239] In one embodiment, the transmembrane domain may comprise the entire transmembrane domain of the molecule from which it is derived, or it may comprise only a functional and / or structural fragment thereof or a variant thereof.

[0240] In one embodiment, the transmembrane domain comprises or consists of the amino acid sequence of a CD8 transmembrane domain having SEQ ID NO:49, or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO:49. [ka]

[0241] In another embodiment, the transmembrane domain comprises or consists of the amino acid sequence of a CD28 transmembrane domain having SEQ ID NO:50, or an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO:50. [ka]

[0242] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes the immune effector function of CAR-containing immune cells. Examples of immune effector functions in CAR immune cells include, but are not limited to, cytolytic activity, suppressive activity, regulatory activity and helper activity, including secretion of cytokines.

[0243] In one embodiment, the intracellular signaling domain comprises at least one primary signaling domain, and optionally, one or more costimulatory signaling domains.

[0244] The primary signaling domain, also called the endodomain, is located in the cytoplasm and transmits the effector function signal upon binding of the extracellular binding domain to its antigen and instructs the CAR-containing immune cell to carry out its specialized function.

[0245] Examples of primary signaling domains include, but are not limited to, the zeta chain of the T cell receptor or any of its homologues, e.g., eta chain, FcεR1 gamma and beta chains, MB1 (Igα) chain, B29 (Igβ) chain, etc.), CD3 polypeptides (e.g., Δ, δ and ε), syk family tyrosine kinases (e.g., Syk, ZAP70, etc.), src family tyrosine kinases (e.g., Lck, Fyn, Lyn, etc.), and other molecules involved in the transduction of T cells, e.g., CD2, CD5 and CD28.

[0246] In one embodiment, the intracellular signaling domain may be the human CD3ε chain, FcγRIII, FcεRI, the cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptor, or a combination thereof. Additional intracellular signaling domains will be apparent to one of skill in the art and may be used in connection with alternative embodiments of the invention.

[0247] In one embodiment, at least one intracellular signaling domain may comprise the entire intracellular portion of the molecule from which it is derived, or the entire naturally occurring intracellular signaling domain, or a functional fragment or derivative thereof.

[0248] In one embodiment, at least one intracellular primary signaling domain comprises or consists of a T cell primary signaling domain (or a sequence derived therefrom).

[0249] In one embodiment, the T cell primary signaling domain comprises or consists of a signaling domain of a protein selected in the group: CD3zeta, CD3gamma, CD3delta, CD3epsilon, common FcRgamma (FCER1G), FcRepsilon (Fc epsilon Rib), CD79a, CD79b, FcgammaRIIa, DAP10, DAP12, and sequences derived therefrom.

[0250] In one embodiment, the T cell primary signaling domain comprises or consists of a functional signaling domain of CD3ζ.

[0251] In one embodiment, the T cell primary signaling domain comprises or consists of a functional signaling domain of CD3zeta having SEQ ID NO:51 or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:51. [ka]

[0252] T cell primary signaling domains that act in a stimulatory manner may additionally or alternatively contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMS).

[0253] Examples of ITAM-containing T cell primary intracellular signaling domains include, but are not limited to, those of (or derived from) CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD5, CD22, CD66b, CD79a, CD79b, DAP10, and DAP12.

[0254] In one embodiment, the T cell primary signaling domain comprises a modified ITAM domain (e.g., a mutated ITAM domain having altered, e.g., increased or decreased, activity compared to the native ITAM domain). In one embodiment, the primary signaling domain comprises a primary intracellular signaling domain containing a modified ITAM, e.g., a primary intracellular signaling domain containing an optimized and / or truncated ITAM. In one embodiment, the primary signaling domain comprises one, two, three, four or more ITAM motifs.

[0255] In one embodiment, the CAR comprises two or more intracellular primary signaling domains, e.g., 2, 3, 4, 5, or more intracellular primary signaling domains.

[0256] In one embodiment, these two or more intracellular primary signaling domains can be linked together in tandem, randomly or in a specific order. Optionally, a linker, such as a flexible peptide linker as described above, can form the link between two different intracellular primary signaling domains. In addition to the glycine-serine linker described above, a single amino acid (such as, for example, an alanine or glycine residue) can also be a suitable linker.

[0257] In one embodiment, the CAR of the invention may optionally comprise one or more costimulatory signaling domains.

[0258] As used herein, the term "costimulatory signaling domain" refers to the intracellular portion of a costimulatory molecule, i.e., its cognate binding partner on a T cell, that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, enhanced cell proliferation, enhanced cell survival and development of memory cells.

[0259] The one or more costimulatory signaling domains may individually be all or a portion of the intracellular domain of a costimulatory molecule, including, but not limited to, 4-1BB (CD137), ICOS (CD278), CD27, CD28, CTLA-4 (CD152), PD-1, MHC class I molecules, BTLA, Toll ligand receptor, OX40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, CDS, ICAM-1, GITR, ARHR, BAFFR, HVE, and the like. M(LIGHTR), SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD160(BY55), CD19, CD19a, CD4, CD8α, CD8β, IL2Rα, IL6Rα, IL2Rβ, IL2 Rγ, IL7Rα, IL-13Rα1, IL-13Rα2, IL-33R, IL-10Rα, IL-10Rβ, IL-4R, IL-5R (CSF2RB), IL-21R, ITGA4, VLA1, CD49a, ITGA4, IA4, CD4 9d, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a / CD18, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD1 8, ITGB7, NKG2D, NKG2C, CD95, TGFbR1 / 2 / 3, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244), CD84, CD96(Tactile), CEACAM1, CRT These include AM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, common gamma chain, a ligand that specifically binds to CD83, NKp44, NKp30, NKp46, NKG2D, and any combination thereof.

[0260] In one embodiment, the CAR comprises at least one costimulatory signaling domain comprising all or a portion of the intracellular domain of 4-1BB, ICOS, OX40, CD28, CTLA4, or PD-1.

[0261] In one embodiment, the intracellular costimulatory signaling domain comprises or consists of all or a portion of the intracellular domain of 4-1BB.

[0262] In one embodiment, the intracellular costimulatory signaling domain comprises or consists of the intracellular domain of 4-1BB having SEQ ID NO:52 or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:52, or a functionally active fragment thereof. [ka]

[0263] In one embodiment, the intracellular costimulatory signaling domain comprises or consists of all or a portion of the intracellular domain of ICOS.

[0264] In one embodiment, the intracellular costimulatory signaling domain comprises or consists of the intracellular domain of ICOS having SEQ ID NO:53 or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:53, or a functionally active fragment thereof. [ka]

[0265] In one embodiment, the intracellular costimulatory signaling domain comprises or consists of all or a portion of the intracellular domain of OX40.

[0266] In one embodiment, the intracellular costimulatory signaling domain comprises or consists of the intracellular domain of OX40 having SEQ ID NO:54 or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:54, or a functionally active fragment thereof. [ka]

[0267] In one embodiment, the intracellular costimulatory signaling domain comprises or consists of all or a portion of the intracellular domain of CD28.

[0268] In one embodiment, the intracellular costimulatory signaling domain comprises or consists of the intracellular domain of CD28 having SEQ ID NO:55 or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:55, or a functionally active fragment thereof. [ka]

[0269] In one embodiment, the intracellular costimulatory signaling domain comprises or consists of all or a portion of the intracellular domain of CTLA4.

[0270] In one embodiment, the intracellular costimulatory signaling domain comprises or consists of the intracellular domain of CTLA4 having SEQ ID NO:56 or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:56, or a functionally active fragment thereof. [ka]

[0271] In one embodiment, the intracellular costimulatory signaling domain comprises or consists of all or a portion of the intracellular domain of PD-1.

[0272] In one embodiment, the intracellular costimulatory signaling domain comprises or consists of the intracellular domain of PD-1 having SEQ ID NO:57 or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:57, or a functionally active fragment thereof. [ka]

[0273] In one embodiment, the CAR comprises two or more intracellular costimulatory signaling domains, e.g., 2, 3, 4, 5, or more intracellular costimulatory signaling domains.

[0274] In one embodiment, these two or more intracellular costimulatory signaling domains can be linked together in tandem, randomly or in a specific order.Optionally, a linker, such as the flexible peptide linker described above, can form the link between two different intracellular costimulatory signaling domains.In addition to the above-mentioned glycine-serine linker, a single amino acid (such as, for example, alanine or glycine residue) can also be a suitable linker.

[0275] In one embodiment, at least one intracellular primary signaling domain and at least one intracellular costimulatory signaling domain can be linked together in tandem, randomly or in a specific order.Optionally, a linker, such as the flexible peptide linker described above, can form the link between the intracellular primary signaling domain and the intracellular costimulatory signaling domain.In addition to the glycine-serine linker described above, a single amino acid (such as, for example, alanine or glycine residue) can also be a suitable linker.

[0276] A CAR according to the invention may be a first, second or third generation CAR.

[0277] The first generation of CARs was developed over 30 years ago (Kuwana et al., 1987. Biochem Biophys Res Commun. 149(3):960-968; Gross et al., 1989. Transplant Proc. 21(1 Pt 1):127-130; Gross et al., 1989. Proc Natl Acad Sci USA. 86(24):10024-10028).

[0278] In one embodiment, the CAR of the invention is a first generation CAR, (i) at least one extracellular CD45RC binding domain, preferably at least one extracellular hCD45RC binding domain, as described above; (ii) optionally, an extracellular hinge domain; (iii) at least one transmembrane domain, and (iv) one or more intracellular primary signaling domain(s). Includes.

[0279] A first generation CAR can be, for example, a CAR in which signaling is provided by CD3ζ, i.e., the primary intracellular signaling domain is CD3ζ.

[0280] Second generation CARs add intracellular costimulatory signaling domains, such as CD28 or 4-1BB, whose engagement improves T cell proliferation, cytokine secretion, resistance to apoptosis, and persistence in vivo.

[0281] In one embodiment, the CAR of the invention is a second generation CAR, (i) at least one extracellular CD45RC binding domain, preferably at least one extracellular hCD45RC binding domain, as described above; (ii) optionally, an extracellular hinge domain; (iii) at least one transmembrane domain; (iv) a costimulatory signaling domain, and (v) one or more intracellular primary signaling domain(s). Includes.

[0282] Third generation CARs combine multiple costimulatory domains, for example CD28-4-1BB or CD28-OX40, to increase T cell activity.

[0283] In one embodiment, the CAR of the invention is a third generation CAR, (i) at least one extracellular CD45RC binding domain, preferably at least one extracellular hCD45RC binding domain, as described above; (ii) optionally, an extracellular hinge domain; (iii) at least one transmembrane domain; (iv) at least two costimulatory signaling domains, and (v) one or more intracellular primary signaling domain(s). Includes.

[0284] In a fourth aspect, the present invention relates to a nucleic acid encoding a CD45RC binding domain, or an anti-CD45RC antibody or antigen-binding fragment thereof, or an anti-CD45RC CAR, as described above.

[0285] The present invention also relates to a vector comprising a nucleic acid encoding a CD45RC binding domain, or an anti-CD45RC antibody or antigen-binding fragment thereof, or an anti-CD45RC CAR, as described above.

[0286] In one embodiment, the vector is an expression vector and further comprises regulatory elements allowing expression of the CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, or anti-CD45RC CAR in the cell.

[0287] "Expression vector" refers to any type of genetic construct that contains a nucleic acid encoding a sequence that can be transcribed. An expression vector can contain a variety of "regulatory elements", which refers to nucleic acid sequences required for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. Examples of such regulatory elements include, but are not limited to, promoters, enhancers, internal ribosome entry sites (IRES), splicing sites, termination signals, and the like.

[0288] Examples of vectors include, but are not limited to, DNA vectors, RNA vectors, plasmids, phagemids, phage derivatives, viruses, and cosmids.

[0289] In one embodiment, the expression vector is a plasmid.

[0290] In one embodiment, the expression vector is a viral vector.Examples of viral vectors include, but are not limited to, adenovirus vector, adeno-associated virus (AAV) vector, retrovirus vector (e.g., lentivirus), vaccinia virus vector, Sindbis virus vector, cytomegalovirus vector, herpes simplex virus vector, etc.

[0291] In one embodiment, the expression vector may be monocistronic. "Monocistronic" means that a single nucleic acid encoding a single protein is expressed in a single expression vector.

[0292] In one embodiment, the expression vector comprises the sequence encoding the HCVR of the CD45RC binding domain, the anti-CD45RC antibody or its antigen-binding fragment, or the anti-CD45RC CAR, preferably operably linked to a regulatory element. In one embodiment, the expression vector comprises the sequence encoding the heavy chain of the CD45RC binding domain, the anti-CD45RC antibody or its antigen-binding fragment, preferably operably linked to a regulatory element, comprising the HCVR and HCCR. In one embodiment, the sequence of the HCVR and HCCR are in the same open reading frame and can therefore be expressed as a single polypeptide chain.

[0293] In one embodiment, the expression vector comprises the sequence encoding the LCVR of the CD45RC binding domain, the anti-CD45RC antibody or its antigen-binding fragment, or the anti-CD45RC CAR, preferably operably linked to a regulatory element. In one embodiment, the expression vector comprises the sequence encoding the light chain, including the LCVR and LCCR, of the CD45RC binding domain, the anti-CD45RC antibody or its antigen-binding fragment, preferably operably linked to a regulatory element. In one embodiment, the sequence of the LCVR and the LCCR are in the same open reading frame, and thus can be expressed as a single polypeptide chain.

[0294] Thus, the present invention also relates to an expression vector system comprising or consisting of at least two expression vectors, one comprising a sequence encoding the HCVR of a CD45RC binding domain, of an anti-CD45RC antibody or antigen-binding fragment thereof, or of an anti-CD45RC CAR, preferably operably linked to a regulatory element, and one comprising a sequence encoding the LCVR of a CD45RC binding domain, of an anti-CD45RC antibody or antigen-binding fragment thereof, or of an anti-CD45RC CAR, preferably operably linked to a regulatory element.

[0295] In one embodiment, an expression vector comprises sequences encoding an extracellular CD45RC-binding domain, optionally an extracellular hinge domain, at least one transmembrane domain, and at least one intracellular signaling domain comprising at least one primary signaling domain and optionally one or more costimulatory signaling domains, preferably operably linked to regulatory elements, in one embodiment, these sequences are in the same open reading frame and thus can be expressed as a single polypeptide chain.

[0296] In one embodiment, the expression vector may be polycistronic. "Polycistronic" means that at least two or more nucleic acids, each encoding a single protein, are expressed in a single expression vector.

[0297] In one embodiment, the expression vector comprises: - a sequence encoding the HCVR of a CD45RC binding domain, of an anti-CD45RC antibody or antigen-binding fragment thereof, or of an anti-CD45RC CAR, preferably operably linked to a regulatory element; - a sequence encoding the LCVR of a CD45RC binding domain, of an anti-CD45RC antibody or antigen-binding fragment thereof, or of an anti-CD45RC CAR, preferably operably linked to a regulatory element Includes.

[0298] It will be readily understood that one of skill in the art can design nucleic acid sequences encoding the HCVRs, LCVRs, HCCRs and / or LCCRs disclosed herein. It is further understood that one of skill in the art is familiar with molecular biology methods aimed at modifying nucleic acid sequences, for example, to improve recombinant production rates, such as by codon optimization. Finally, the present application therefore encompasses any nucleic acid encoding any of the HCVRs, LCVRs, HCCRs and / or LCCRs disclosed herein.

[0299] In one embodiment, the nucleic acid or vector is an isolated nucleic acid or vector.

[0300] "Isolated" means that the nucleic acid or vector is substantially free of genomic DNA sequences and proteins or complexes, such as ribosomes and polymerases, which naturally accompany the native sequence. The term encompasses nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA isolates, as well as chemically synthesized analogs or biologically synthesized analogs from heterologous systems.

[0301] In one embodiment, the isolated nucleic acid or vector is purified.

[0302] In one embodiment, the isolated nucleic acid or vector comprises: (1) Absorbance or fluorescence method (e.g., absorbance at 260 and 280 nm (A 260 / 280 80% by weight, 85% by weight, 90% by weight, 91% by weight, 92% by weight, 93% by weight, 94% by weight, 95% by weight, or more, as determined by, for example, measurement by , and most preferably greater than 96%, 97%, 98%, or 99% by weight nucleic acid; or (2) homogeneity, as demonstrated by agarose gel electrophoresis and using intercalating agents such as ethidium bromide, SYBR Green, and GelGreen; is refined into

[0303] In a fifth aspect, the present invention relates to methods of producing and purifying the above-described CD45RC binding domains, or anti-CD45RC antibodies or antigen-binding fragments thereof.

[0304] In one embodiment, the method comprises: - culturing a host cell containing a nucleic acid, preferably an expression vector as described above, under conditions favorable for expression of the CD45RC binding domain or an anti-CD45RC antibody or antigen-binding fragment thereof; - recovering the expressed CD45RC binding domain or anti-CD45RC antibody or antigen-binding fragment thereof; Includes.

[0305] This recombinant process can be used for large-scale production of antibodies or antigen-binding fragments thereof, including monoclonal antibodies, for in vitro, ex vivo and / or in vivo therapeutic and / or diagnostic uses.

[0306] The nucleic acid or expression vector may be propagated and expressed according to any of a variety of routinely performed procedures for excision, ligation, transformation, and transfection of nucleic acids. In certain embodiments, expression may be performed in prokaryotic host cells (i.e., the host cell containing the nucleic acid or expression vector is a prokaryotic host cell), such as E. coli. In other embodiments, expression may be performed in eukaryotic host cells (i.e., the host cell containing the nucleic acid or expression vector is a eukaryotic host cell), including animal cells (such as mammalian cells), yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Pichia pastoris); and plant cells.

[0307] By methods known to those of skill in the art and based on the present disclosure, a nucleic acid or expression vector may be designed for expressing a foreign sequence in a particular host system, and then a polynucleotide sequence encoding a cellular polypeptide may be inserted. Regulatory elements will vary depending on the particular host.

[0308] All these processes are well known in the art (see, for example, Subramanian (Ed.), 2004. Antibodies (1st ed., Vol. 1: Production and Purification). New York, NY: Springer US).

[0309] In one embodiment, the expressed CD45RC binding domain, or anti-CD45RC antibody or antigen-binding fragment thereof, is further purified.

[0310] Methods for purifying antibodies or antigen-binding fragments thereof are well known in the art (see, e.g., Subramanian (Ed.), 2004. Antibodies (1st ed., Vol. 1: Production and Purification). New York, NY: Springer US).

[0311] In a sixth aspect, the present invention relates to immune cells expressing an anti-CD45RC CAR as described herein, and populations of such immune cells.

[0312] As used herein, the term "immune cells" refers to white blood cells (leukocytes) that are derived from hematopoietic stem cells (HSCs) produced in the bone marrow.

[0313] In one embodiment, the immune cell comprises a nucleic acid or vector, preferably an expression vector, encoding the above-mentioned anti-CD45RC CAR, thereby generating an engineered immune cell that expresses the anti-CD45RC CAR on its cell surface.

[0314] In one embodiment, the immune cell is a mammalian immune cell, e.g., a human immune cell, an immune cell from a farm animal (e.g., a cow, a pig, or a horse), or an immune cell from a pet (e.g., a cat or a dog).

[0315] In one embodiment, the immune cells are autologous, xenogeneic, or allogeneic cells.

[0316] In one embodiment, the immune cells are selected from the group comprising or consisting of lymphocytes, myeloid cells, and any combination thereof.

[0317] In one embodiment, the immune cell is a lymphocyte, e.g., a cell selected from the group including or consisting of a T cell, a B cell, a natural killer (NK) cell, a natural killer T (NKT) cell, and any combination thereof.

[0318] In one embodiment, the immune cell is a T cell, which is a CD4 + T cells, CD8 + The T cells may be selected from the group comprising or consisting of T cells, γδ T cells, double negative (DN) T cells, and any combination thereof.

[0319] In one embodiment, the immune cells are CD4 T cells, such as, for example, helper T cells, regulatory T cells, effector T cells, and any combination thereof. + In one embodiment, the immune cell is, for example, a cytotoxic CD8 + T cells or CD8 + Regulatory T cells, such as CD8 + In one embodiment, the immune cell is a T cell. In one embodiment, the immune cell is an effector γδ T cell. In one embodiment, the immune cell is a T cell engineered to express a defined γδ TCR (TEG cell). In one embodiment, the immune cell is a DN T cell. In one embodiment, the immune cell is a NK cell. In one embodiment, the immune cell is a NKT cell.

[0320] In one embodiment, the immune cell is a regulatory immune cell, such as, for example, any regulatory immune cell suitable for use in cell therapy. In one embodiment, the regulatory immune cell is a regulatory T cell, a CD4 + Regulatory T cells, CD8 + The regulatory cells are selected from the group consisting of regulatory T cells, regulatory γδ T cells, regulatory DN T cells, regulatory B cells, regulatory NK cells, regulatory NK T cells, regulatory macrophages, regulatory dendritic cells, and any combination thereof.

[0321] In one embodiment, the regulatory immune cells are regulatory T cells (Tregs), in particular thymus-derived Tregs or adaptive or inducible Tregs. + In one embodiment, the Tregs are thymus-derived Tregs, or adaptive or inducible Tregs. In one embodiment, the Tregs are CD4 + FoxP3+ Treg or CD4 + In one embodiment, the immune cells are CD4 + FoxP3 + In one embodiment, the immune cells are CD8 + Treg. CD8 + Examples of Tregs include, but are not limited to, CD8 + CD28 - Tregs, CD8 + CD103 + Tregs, CD8 + FoxP3 + Tregs, CD8 + CD122 + Tregs, and any combination thereof.

[0322] In one embodiment, the regulatory immune cells are regulatory γδ T cells. In one embodiment, the regulatory immune cells are regulatory DN T cells. In one embodiment, the regulatory immune cells are regulatory NK cells. In one embodiment, the regulatory immune cells are regulatory NK T cells.

[0323] In one embodiment, the immune cell is an effector immune cell, such as, for example, any effector immune cell suitable for use in cell therapy. In one embodiment, the effector immune cell is an effector T cell, CD4 + Effector T cells, CD8 + The cells are selected from the group comprising or consisting of effector T cells, effector γδ T cells, effector DN T cells, effector NK cells, effector NK T cells, and any combination thereof.

[0324] In one embodiment, the immune cells are effector T cells (Teff). In one embodiment, the effector immune cells are CD4 + Teff. CD4 + Examples of Teff cells include, but are not limited to, T h 1 cell, T h 2 cells, T h9 cells, T h 17 cells, T h 22 cells, CD4 + In one embodiment, the effector immune cells include CD8 + Teff. CD8 + Examples of effector T cells include, but are not limited to, CD8 + CD45RO + CCR7 - CD62L - Teff, CD8 + CD45RA + CCR7 - CD62L - Teff, and any combination thereof.

[0325] In one embodiment, the immune cell is an effector γδ T cell. In one embodiment, the immune cell is an effector DN T cell. In one embodiment, the immune cell is an effector NK cell. In one embodiment, the immune cell is an effector NK T cell.

[0326] The present invention also relates to isolated and / or substantially purified populations of immune cells as described above.

[0327] As used herein, an "isolated population" refers to a population of cells that have been removed from their natural environment (such as peripheral blood) and isolated, purified, or separated, and that are at least about 75% free, 80% free, 85% free, and in some embodiments about 90% free, 95% free, 96% free, 97% free, 98% free, 99% free of other cells that are naturally present with them but lack the cell surface markers based on which they are isolated.

[0328] The present invention also relates to an enriched population of immune cells as described above. In one embodiment, the enriched population comprises at least 30%, preferably at least 40%, 50%, 60%, 70%, 80%, 90% or more of the immune cells of interest.

[0329] In a seventh aspect, the present invention relates to a composition comprising the above-mentioned CD45RC binding domain, or an anti-CD45RC antibody or antigen-binding fragment thereof, or an anti-CD45RC CAR; or a nucleic acid or a vector encoding them, preferably an expression vector, as described above; or an immune cell expressing the above-mentioned anti-CD45RC CAR.

[0330] In one embodiment, the composition is a pharmaceutical composition and further comprises at least one pharma- ceutically acceptable excipient.

[0331] The term "pharmaceutical acceptable excipient" includes any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The excipient does not cause any adverse, allergic or other untoward reactions when administered to animals, preferably humans. For human administration, preparations must meet the sterility, pyrogenicity, and general safety and purity standards required by regulatory authorities, such as the FDA Office or EMA.

[0332] Pharmaceutically acceptable excipients that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances (e.g. sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

[0333] In one embodiment, the pharmaceutical composition comprises a pharma- ceutically acceptable vehicle for the formulation to be injected into a subject. These may be, in particular, isotonic, sterile, saline (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc., or mixtures of such salts), or may be a dry, in particular lyophilized, composition that allows the constitution of an injectable solution upon addition of sterile water or saline, as the case may be.

[0334] In one embodiment, the composition is a pharmaceutical agent.

[0335] In an eighth aspect, the present invention relates to the use of the above-mentioned CD45RC binding domains, anti-CD45RC antibodies or antigen-binding fragments thereof, anti-CD45RC CARs, nucleic acids or vectors encoding them, immune cells expressing anti-CD45RC CARs or populations of such immune cells, and compositions in various methods for therapeutic purposes, as further described below.

[0336] In one embodiment, the present invention relates to a method for inducing immune tolerance in a subject in need thereof by administering to the subject any of the above-mentioned CD45RC binding domains, anti-CD45RC antibodies or antigen-binding fragments thereof, anti-CD45RC CARs, nucleic acids or vectors encoding the same, immune cells expressing anti-CD45RC CARs or a population of such immune cells, or compositions.The present invention also relates to the above-mentioned CD45RC binding domains, anti-CD45RC antibodies or antigen-binding fragments thereof, anti-CD45RC CARs, nucleic acids or vectors encoding the same, immune cells expressing anti-CD45RC CARs or a population of such immune cells, or compositions for use in inducing immune tolerance in a subject in need thereof.The present invention also relates to the use of the above-mentioned CD45RC binding domains, anti-CD45RC antibodies or antigen-binding fragments thereof, anti-CD45RC CARs, nucleic acids or vectors encoding the same, immune cells expressing anti-CD45RC CARs or a population of such immune cells, or compositions in the preparation of a medicament for inducing immune tolerance in a subject in need thereof.

[0337] As used herein, the term "immune tolerance" refers to a state in which the immune system is unresponsive to certain substances or tissues that have the capacity to elicit an immune response while maintaining an immune response against other substances or tissues.

[0338] As used herein, the term "immune response" includes T cell-mediated immune responses and / or B cell-mediated immune responses. Exemplary immune responses include, but are not limited to, T cell responses (e.g., cytokine production and cytotoxicity), and also immune responses indirectly affected by T cell activation (e.g., macrophages). Immune cells involved in an immune response include lymphocytes (e.g., B cells and T cells, e.g., CD4 + , CD8 + , T h 1 and T h2 cells), antigen presenting cells (e.g., professional antigen presenting cells such as dendritic cells), natural killer cells, myeloid cells (e.g., macrophages, eosinophils, mast cells, basophils, and granulocytes).

[0339] In one embodiment, the present invention provides a method for the treatment of CD45RC in a subject in need thereof by administering to the subject any of the above-mentioned CD45RC binding domains, anti-CD45RC antibodies or antigen-binding fragments thereof, anti-CD45RC CARs, nucleic acids or vectors encoding same, immune cells expressing anti-CD45RC CARs or populations of such immune cells, or compositions. high The present invention also relates to a method for depleting CD45RC cells in a subject in need thereof. high The present invention also relates to the above-mentioned CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing anti-CD45RC CAR or population of such immune cells, or composition for use in depleting cells. The present invention also relates to the above-mentioned CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing anti-CD45RC CAR or population of such immune cells, or composition for use in depleting cells. high The present invention relates to the use of the above-mentioned CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing anti-CD45RC CAR or a population of such immune cells, or composition in the preparation of a medicament for depleting cells.

[0340] The relative levels of expression of CD45RC can be measured using flow cytometry. Three types of cells are distinguishable: cells that display high, intermediate, or negative levels of CD45RC expression.

[0341] In one embodiment, the CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing an anti-CD45RC CAR or a population of such immune cells, or composition is high Starve the cells.

[0342] "CD45RC high A "cell" is a cell that expresses high levels of the CD45RC marker, as defined above (eg, a T cell, a NK cell, a NKT cell, a B cell, etc.).

[0343] As used herein, the term "deplete" or "depleting" with respect to cells expressing CD45RC refers to a measurable reduction in the number of cells in a subject. The reduction can be at least about 10%, e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more. In one embodiment, the term refers to the reduction of CD45RC in a subject or sample to below the limit of detection. high Refers to a decrease in the number of cells.

[0344] In particular, the above-mentioned CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell or population of such immune cells expressing anti-CD45RC CAR, or composition inhibits CD45RC by binding to CD45RC and transmitting a pro-apoptotic signal and / or by activating antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). high Starve the cells.

[0345] In one embodiment, the above-mentioned CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing anti-CD45RC CAR or population of such immune cells, or composition mediates complement-dependent cytotoxicity.

[0346] In one embodiment, the above-described CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, or anti-CD45RC CAR may be conjugated to a cytotoxic agent or a growth inhibitory agent.

[0347] In one embodiment, the present invention relates to a method for expanding and / or enhancing regulatory T cells in a subject in need thereof by administering to the subject any of the above-mentioned CD45RC binding domains, anti-CD45RC antibodies or antigen-binding fragments thereof, anti-CD45RC CARs, nucleic acids or vectors encoding the same, immune cells expressing anti-CD45RC CARs or a population of such immune cells, or compositions.The present invention also relates to the above-mentioned CD45RC binding domains, anti-CD45RC antibodies or antigen-binding fragments thereof, anti-CD45RC CARs, nucleic acids or vectors encoding the same, immune cells expressing anti-CD45RC CARs or a population of such immune cells, or compositions for use in expanding and / or enhancing regulatory T cells in a subject in need thereof.The present invention also relates to the use of the above-mentioned CD45RC binding domains, anti-CD45RC antibodies or antigen-binding fragments thereof, anti-CD45RC CARs, nucleic acids or vectors encoding the same, immune cells expressing anti-CD45RC CARs or a population of such immune cells, or compositions in the preparation of a medicament for expanding and / or enhancing regulatory T cells in a subject in need thereof.

[0348] As used herein, the term "expansion" refers to the process of converting and / or amplifying a given cell population (e.g., immune cells such as Tregs). Expansion of a cell population can occur in vivo, in vitro, or ex vivo.

[0349] As used herein, the term "enhancing" refers to the process of increasing the function of a given cell population (e.g., increasing the suppressive capacity of Tregs). Enhancing a cell population can occur in vivo, in vitro, or ex vivo.

[0350] "Regulatory T cells" or "Tregs" are T cells that suppress abnormal or excessive immune responses and are involved in immune tolerance. Tregs typically express the "forkhead box P3 (Foxp3)" gene.+ ) Regulatory T cells" and / or "CD45RC low / - "Cells".

[0351] As used herein, "Forkhead box P3 (Foxp3)" refers to + ) Regulatory T cells" and "CD45RC low / - The term "CD4+ cells" refers to 0.1-10% of CD4+ cells in humans and rodents whose characteristic marker is the transcription factor Foxp3. + and / or CD8 + Refers to T cells.

[0352] In one embodiment, the methods and uses include those directed to Foxp3 + and / or CD45RC low / - To expand and / or enhance Tregs.

[0353] In one embodiment, CD45RC low / - Tregs are expanded by stimulation. In one embodiment, CD45RC low / - Tregs are expanded by stimulation in the presence of IL-2 and / or IL-15. low / - Tregs are expanded by stimulation with anti-CD3 / anti-CD28 antibodies and / or allogeneic antigen-presenting cells (APCs) and / or specific antigens.

[0354] Additionally or alternatively, the present invention provides a method for the detection of CD45RC low / - The present invention relates to in vitro or ex vivo methods for purifying Tregs.

[0355] In one embodiment, CD45RC low / - Tregs are CD8 + / CD4 + In one embodiment, the CD45RC low / - Tregs are CD8 + / CD4 - In one embodiment, the CD45RC low / - Tregs are CD8 - / CD4 +T cells.

[0356] In one embodiment, purified CD45RC low / - Tregs may be further expanded and / or enhanced prior to, concomitantly with, or following administration to a subject in need thereof.

[0357] In one embodiment, the present invention relates to a method for preventing and / or reducing transplant rejection in a subject in need thereof by administering to the subject any of the above-mentioned CD45RC binding domains, anti-CD45RC antibodies or antigen-binding fragments thereof, anti-CD45RC CARs, nucleic acids or vectors encoding same, immune cells expressing anti-CD45RC CARs or populations of such immune cells, or compositions.The present invention also relates to the above-mentioned CD45RC binding domains, anti-CD45RC antibodies or antigen-binding fragments thereof, anti-CD45RC CARs, nucleic acids or vectors encoding same, immune cells expressing anti-CD45RC CARs or populations of such immune cells, or compositions for use in preventing and / or reducing transplant rejection in a subject in need thereof.The present invention also relates to the use of the above-mentioned CD45RC binding domains, anti-CD45RC antibodies or antigen-binding fragments thereof, anti-CD45RC CARs, nucleic acids or vectors encoding same, immune cells expressing anti-CD45RC CARs or populations of such immune cells, or compositions in the preparation of a medicament for preventing and / or reducing transplant rejection in a subject in need thereof.

[0358] The terms "preventing transplant rejection" and "reducing transplant rejection" are meant to include preventing or inhibiting immune transplant rejection, as well as delaying the onset or progression of immune transplant rejection. The terms are also meant to include extending the survival of a graft in a subject, or reversing transplant failure in a subject. Additionally, the terms are meant to include ameliorating symptoms of immune transplant rejection, including ameliorating immunological complications associated with immune rejection, such as, for example, interstitial fibrosis, chronic graft arteriosclerosis, or vasculitis.

[0359] The term "transplantation" and variations thereof refer to the insertion of a transplant (also called a graft) into a recipient, whether the transplant is syngeneic (where donor and recipient are genetically identical), allogeneic (where donor and recipient are of different genetic origins but the same species), or xenogeneic (where donor and recipient are from different species). Thus, in a typical scenario, the host is a human and the graft is a syngeneic graft, derived from a human of the same or different genetic origin. In another scenario, the graft is derived from a phylogenetically widely separated species of animal, a species different from the species into which it is transplanted, including, for example, a baboon heart transplanted into a human host.

[0360] As used herein, the term "transplant rejection" includes both acute and chronic transplant rejection.

[0361] "Acute rejection" refers to the rejection by the immune system of a tissue transplant recipient when the transplanted tissue is immunologically foreign. Acute rejection is characterized by infiltration of the transplanted tissue by the recipient's immune cells, which carry out their effector functions and destroy the transplanted tissue. The onset of acute rejection is rapid and generally occurs within a few weeks after transplant surgery in humans. In general, acute rejection can be inhibited or suppressed by immunosuppressants such as rapamycin, cyclosporine, and anti-CD40L monoclonal antibodies.

[0362] "Chronic rejection" generally occurs in humans within months to years after engraftment, even if immunosuppression of acute rejection is successful. Fibrosis is a common factor in chronic rejection of all types of organ transplants.

[0363] In one embodiment, the transplant rejection is an allogeneic transplant rejection. Thus, in one embodiment, the donor of the transplant is a human. The donor of the transplant may be a living donor or a deceased donor, i.e., a cadaveric donor.

[0364] In one embodiment, the graft is an organ, tissue or cells.

[0365] As used herein, the term "organ" refers to a robust, vascularized organ that performs a specific function or group of functions within an organism. The term organ includes, but is not limited to, the heart, lungs, kidneys, liver, pancreas, skin, uterus, bone, cartilage, small or large intestine, bladder, brain, breasts, blood vessels, esophagus, fallopian tubes, gallbladder, ovaries, pancreas, prostate, placenta, spinal cord, upper and lower limbs, spleen, stomach, testes, thymus, thyroid, trachea, ureters, urethra, and uterus.

[0366] As used herein, the term "tissue" refers to any type of tissue in a human or animal, including, but not limited to, vascular tissue, skin tissue, liver tissue, pancreatic tissue, nervous tissue, urogenital tissue, gastrointestinal tissue, skeletal tissue including bone and cartilage, adipose tissue, connective tissue including tendons and ligaments, amniotic tissue, chorionic tissue, dura mater, pericardium, muscle tissue, glandular tissue, facial tissue, and ocular tissue.

[0367] The term "cells" as used herein refers to compositions enriched with cells of interest, preferably comprising at least 30%, preferably at least 50%, even more preferably at least 65% of cells of interest. In one embodiment, the cells are selected from the group consisting of or comprise pluripotent hematopoietic stem cells derived from bone marrow, peripheral blood, or umbilical cord blood; or pluripotent (i.e., embryonic stem cells [ES] or induced pluripotent stem cells [iPS]), or differentiated cells derived from pluripotent stem cells of different cell lineages, including but not limited to cardiomyocytes, β-pancreatic cells, hepatocytes, neurons.

[0368] In one embodiment, where the transplant is an allogeneic hematopoietic stem cell transplant (HSCT), the cells are selected from the group comprising or consisting of multipotent hematopoietic stem cells, typically derived from bone marrow, peripheral blood, or umbilical cord blood.

[0369] "HSCT" or "hematopoietic stem cell transplantation" is a transplantation therapy that can cure patients suffering from leukemia and lymphoma (including, but not limited to, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), myelodysplastic syndromes (MDS), myeloproliferative disorders, Hodgkin lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL) and multiple myeloma). However, a significant limitation of allogeneic HSCT is the development of graft-versus-host disease (GvHD), which occurs in a severe form in approximately 30-50% of humans who undergo this treatment.

[0370] Thus, in one embodiment, the methods and uses are for preventing and / or alleviating graft-versus-host disease (GvHD).

[0371] In one embodiment, the above-mentioned CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing anti-CD45RC CAR or population of such cells immune cell, or composition is used in combination with pluripotent hematopoietic stem cells to treat malignant diseases such as leukemia and / or lymphoma (including but not limited to acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), myeloproliferative syndrome, Hodgkin's lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), neuroblastoma, Ewing's sarcoma, myelodysplastic syndrome, myeloproliferative syndrome ... In some embodiments, the therapeutic agent may prevent and / or treat diseases such as adrenoleukodystrophy, thalassemia, renal cellular anemia, aplastic anemia, sickle cell anemia, Fanconi anemia, bone marrow failure syndromes, hemoglobinopathies, infantile malignant osteopetrosis, mucopolysaccharidoses, pyruvate kinase deficiency, immunodeficiency syndromes, hemophagocytic lymphohistiocytosis, inborn errors of metabolism, osteosclerosing myeloma (also known as POEMS syndrome), and primary amyloidosis.

[0372] Additionally or alternatively, the above-mentioned CD45RC binding domains, anti-CD45RC antibodies or antigen-binding fragments thereof, anti-CD45RC CARs, nucleic acids or vectors encoding same, immune cells expressing anti-CD45RC CARs or populations of such immune cells, or compositions may be used to engineer the graft.

[0373] In one embodiment, the graft to be transplanted is CD45RC high To deplete the cells, they are treated with the above-mentioned CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cells expressing anti-CD45RC CAR or a population of such immune cells, or composition prior to transplantation.

[0374] In one embodiment, the graft is bone marrow and is CD45RC highTo deplete the cells, prior to transplantation, the bone marrow is treated with the above-mentioned CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing anti-CD45RC CAR or population of such immune cells, or composition. high T cells and CD45RC low / - CD34 containing T cells + Contains cells.

[0375] In one embodiment, the present invention provides a method for the treatment of CD45RC in a subject in need thereof by administering to the subject any of the above-mentioned CD45RC binding domains, anti-CD45RC antibodies or antigen-binding fragments thereof, anti-CD45RC CARs, nucleic acids or vectors encoding same, immune cells expressing anti-CD45RC CARs or populations of such immune cells, or compositions. high The present invention also relates to methods for preventing, alleviating, and / or treating a CD45RC associated disease, disorder, or condition in a subject in need thereof. high The present invention relates to the above-mentioned CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing anti-CD45RC CAR or population of such immune cells, or composition for use in preventing, alleviating, and / or treating an associated disease, disorder, or condition. The present invention also relates to the above-mentioned CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing anti-CD45RC CAR or population of such immune cells, or composition for use in preventing, alleviating, and / or treating an associated disease, disorder, or condition in a subject in need thereof. high The present invention relates to the use of the above-mentioned CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing anti-CD45RC CAR or population of such immune cells, or composition in the preparation of a medicament for preventing, alleviating, and / or treating an associated disease, disorder, or condition.

[0376] As used herein, "CD45RC highThe term "CD45RC associated disease, disorder or condition" refers to a disease, disorder or condition that is caused by, potentially caused by, or characterized by an increased proportion of cells expressing CD45RC in a subject and / or an increased level of expression of CD45RC in the cells of a subject.

[0377] An "increased percentage of cells expressing CD45RC" refers to an increased percentage of cells expressing CD45RC (i.e., CD45RC high cells) in a given subject compared to a reference, such as the number of CD45RC high cells in a substantially healthy subject (i.e., a subject not suffering from the CD45RC high associated disease, disorder, or condition in question). high By this term is meant an increase of about 5%, preferably about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100% or more in the number of cells.

[0378] "Increased levels of expression of CD45RC" refers to a subject that is substantially healthy (i.e., a subject that does not have the CD45RC in question). high By "CD45RC" is meant an increase of about 5%, preferably about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100% or more in the expression level of CD45RC, either at the mRNA level or at the protein level, in the cells of a particular subject, as compared to, for example, the expression level of CD45RC in the cells of a subject not suffering from the relevant disease, disorder or condition.

[0379] In one embodiment, CD45RC high The associated disease, disorder or condition is selected from the group including or consisting of an autoimmune disease, an unwanted immune response, a monogenic disease, and a lymphoma or cancer.

[0380] In one embodiment, CD45RC highThe associated disease, disorder or condition is selected from the group comprising or consisting of an autoimmune disease, an unwanted immune response, and a monogenic disease.

[0381] As used herein, the term "autoimmune disease" refers to a disease in which the immune system mounts an immune response (e.g., a B cell or T cell response) against antigens that are part of the normal host (i.e., self-antigens), resulting in tissue damage. In an autoimmune disease, the host's immune system fails to recognize a particular antigen as "self," and an immune response is mounted against host tissues that express the antigen.

[0382] Exemplary autoimmune diseases contemplated by the present invention include, but are not limited to, rheumatoid arthritis, juvenile oligoarthritis, collagen-induced arthritis, adjuvant-induced arthritis, Sjogren's syndrome, multiple sclerosis, experimental autoimmune encephalomyelitis, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), autoimmune gastric atrophy, pemphigus vulgaris, psoriasis, vitiligo, alopecia areata, type 1 diabetes, non-obese diabetes, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, sclerosing cholangitis, sclerosing sialadenitis, systemic lupus erythematosus, autoimmune thrombotic thrombocytopenic purpura, Goodpasture's syndrome, Addison's disease, systemic sclerosis, polymyositis, dermatomyositis, acquired hemophilia, thrombotic thrombocytopenic purpura, uveitis, IgG4-related autoimmune diseases (e.g., Kleger et al., (2015. Dtsch Arztebl Int. 112(8):128-135), which is incorporated by reference.

[0383] In one embodiment, the autoimmune disease is systemic lupus erythematosus.

[0384] In one embodiment, the autoimmune disease is inflammatory bowel disease, including Crohn's disease, colitis, and ulcerative colitis. In one embodiment, the autoimmune disease is Crohn's disease. In one embodiment, the autoimmune disease is ulcerative colitis.

[0385] As used herein, the term "undesirable immune response" refers to any undesirable immune response, preferably directed to (i) a protein expressed in the course of gene therapy, (ii) a vector (such as a viral vector) used in the course of gene therapy, and / or (iii) a therapeutic protein. Such proteins include, for example, factor VIII (hemophilia A) and other clotting factors, enzyme replacement therapy, monoclonal antibodies (such as natalizumab, rituximab, infliximab), polyclonal antibodies, enzymes and cytokines (such as IFNβ). The term "undesirable immune response" also refers to allergies and allergic reactions.

[0386] In one embodiment, the above-mentioned CD45RC binding domain, anti-CD45RC antibody or its antigen-binding fragment, anti-CD45RC CAR, nucleic acid or vector encoding it, immune cell expressing anti-CD45RC CAR or a group of such cells immune cell, or composition can be administered to a subject to suppress immune response, particularly to prevent immune reaction against a specific protein when the expression of the specific protein is restored by gene therapy in a subject with a corresponding gene defect.Thus, the above-mentioned CD45RC binding domain, anti-CD45RC antibody or its antigen-binding fragment, anti-CD45RC CAR, nucleic acid or vector encoding it, immune cell expressing anti-CD45RC CAR, or a group of such immune cells, or composition can be used to prevent immune reactivity against proteins that are not normally present in a subject due to mutation, while their reconstitution is achieved by gene therapy.In addition, protein therapy is a field of medical innovation that is becoming more popular and involves the application of proteins, such as enzymes, antibodies, cytokines, etc., directly to a subject as a therapeutic product.One of the major obstacles in the delivery of such medicines involves the immune response directed against the therapeutic protein itself. Administration of protein-based therapeutics is often accompanied by administration of immunosuppressants, which are used to promote the longer life span of proteins and thereby increased uptake of proteins into cells and tissues of organisms.General immunosuppressants, however, are disadvantageous due to the non-specific nature of the immunosuppression carried out, and may cause undesirable side effects in patients.Therefore, this approach can be applied to suppress immune responses to therapeutic proteins and peptides, such as therapeutic antibodies, cytokines, enzymes, or any other proteins administered to patients.

[0387] In one embodiment, the above-mentioned CD45RC binding domain, anti-CD45RC antibody or its antigen-binding fragment, anti-CD45RC CAR, nucleic acid or its encoding vector, immune cell expressing anti-CD45RC CAR or the immune cell population of such cells, or composition can be administered to a subject to suppress immune response, particularly to prevent immune reaction against the vector used in gene therapy, particularly the viral vector used in gene therapy.Such viral vectors include, for example, adeno-associated virus (AAV) vector, adenovirus (Ad) vector, lentivirus vector, etc.For a general description, see Nayak&Herzog, 2010.Gene Ther.17(3):295-304.

[0388] As used herein, the term "allergy(s)" refers to an inappropriate response of the immune system. Allergic reactions occur to normally harmless environmental substances known as allergens, and these reactions are acquired, predictable, and rapid. Technically, allergies are one of four forms of hypersensitivity, called Type I (or immediate) hypersensitivity. The disease is characterized by the excessive activation of certain white blood cells called mast cells and basophils by a type of antibody known as IgE, resulting in an extreme inflammatory response. Common allergic reactions include eczema, hives, hay fever, asthma, food allergies, celiac disease, and reactions to the venom of stinging insects such as wasps and bees.

[0389] As used herein, the term "monogenic disease" refers to a disease caused by a mutation in a single gene selected from among the following genes: (i) genes not associated with immune function, but whose deficiencies are associated with inflammation and / or autoimmune responses, such as genes that are defective in the following diseases: Duchenne muscular dystrophy (DMD), cystic fibrosis, lysosomal diseases, α1-antitrypsin deficiency; and (ii) Genes involved in the immune system and whose defects cause inflammatory and / or autoimmune responses, such as genes defective in the following diseases: primary T-cell immunodeficiency, e.g., IPEX (immuno-dysregulation polyendocrinopathy enteropathy X-linked syndrome), autoimmune polyendocrine syndrome type 1 (APS-1) (also known as autoimmune polyendocrine deficiency-candidiasis-ectodermal dystrophy or APECED), autoimmune polyendocrine syndrome type 2 (APS-2) (also known as Schmidt syndrome), autoimmune polyendocrine syndrome type 3 (APS-3) (also known as autoimmune polyendocrinopathy), primary B-cell immunodeficiency, Muckle-Wells syndrome, mixed autoinflammatory and autoimmune syndrome, hereditary NLRP12-associated periodic fever syndrome, tumor necrosis factor receptor 1-associated periodic syndrome.

[0390] Other autoimmune diseases of genetic origin (i.e., monogenic diseases as defined herein) are known in the art and are encompassed herein by the term "monogenic disease" and are described, for example, in Table 1 of Ramos et al., 2015 (J Hum Genet. 60(11):657-64), which is incorporated herein by reference. In particular, autoimmune diseases of genetic origin encompassed herein are those associated with mutations in any one of the following genes: HLA-DR alleles, STAT4, PTPN22, IFIH1, TRAF3IP2, TRAF1-C5, PADI4, TNF, IL-1, IL-6, IL-4, IL-5, OPN, IL12A, and IL12RB2.

[0391] In one embodiment, the monogenic disease is APECED (Autoimmune Polyendocrine Deficiency-Candidosis-Ectodermal Dystrophy).

[0392] In one embodiment, the monogenic disease is Duchenne muscular dystrophy (DMD).

[0393] As used herein, the term "lymphoma or cancer" refers to a lymphoma or cancer that ishigh Includes lymphomas or cancers associated with CD45RC cells. high Exemplary lymphomas or cancers associated with the cells include, but are not limited to, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), myelodysplastic syndromes (MDS) / myeloproliferative disorders, lymphomas (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), chronic lymphocytic leukemia (CLL), and multiple myeloma.

[0394] In one embodiment, CD45RC high The associated disease, disorder or condition is systemic lupus erythematosus, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), APECED (autoimmune polyendocrine deficiency-candidiasis-ectodermal dystrophy), or Duchenne muscular dystrophy (DMD).

[0395] In one embodiment, CD45RC high The associated disease, disorder or condition is systemic lupus erythematosus, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), or APECED (autoimmune polyendocrine deficiency-candidiasis-ectodermal dystrophy).

[0396] In one embodiment, the above-mentioned CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing anti-CD45RC CAR or population of such immune cells, or composition is formulated for administration to a subject.

[0397] In one embodiment, the above-mentioned CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing anti-CD45RC CAR or a population of such immune cells, or composition is administered systemically or locally.

[0398] In one embodiment, the above-mentioned CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing anti-CD45RC CAR or population of immune cells of such cells, or composition is administered by oral, topical, nasal, buccal, rectal, vaginal, intratracheal injection, endoscopic, transmucosal, or transdermal administration.

[0399] In one embodiment, the above-mentioned CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing anti-CD45RC CAR or a population of such immune cells, or composition is injected, preferably systemically injected.

[0400] Examples of formulations adapted for injection include, but are not limited to, liquid solutions (such as, for example, sterile aqueous solutions), gels, dispersions, emulsions, suspensions, solid forms (such as, for example, powders, liposomal forms, etc., suitable for use in preparing solutions or suspensions upon the addition of liquid prior to use).

[0401] Examples of systemic injections include, but are not limited to, intravenous (iv), subcutaneous, intramuscular (im), intradermal (id), intraperitoneal (ip) injection and perfusion.

[0402] It is understood that other suitable routes of administration are also contemplated in the present invention, and the mode of administration will ultimately be determined by the attending physician within the scope of sound medical judgment. Apart from administration by injection (iv, ip, im, etc.), other routes are also available, such as nebulization (Respaud et al., 2014. MAbs. 6(5): 1347-55; Guilleminault et al., 2014. J Control Release. 196: 344-54; Respaud et al., 2015. Expert Opin Drug Deliv. 12(6): 1027-39) or subcutaneous administration (Jackisch et al., 2014. Geburtshilfe Frauenheilkd. 74(4): 343-349; Solal-Celigny, 2015. Expert Rev Hematol. 8(2): 147-53).

[0403] In one embodiment, the above-mentioned CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing anti-CD45RC CAR or population of such immune cells, or composition is administered to a subject in need thereof in a therapeutically effective amount.

[0404] As used herein, the term "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic and / or therapeutic result.

[0405] However, it is understood that the total daily usage of the above-mentioned CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing anti-CD45RC CAR or population of such immune cells, or composition will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will vary depending on a variety of factors, including: the disease being treated and the severity of that disease; the activity of the CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cells expressing anti-CD45RC CAR or population of such immune cells, or composition used; the age, weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the particular CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cells expressing anti-CD45RC CAR or population of such immune cells, or composition used; the duration of treatment; drugs used in combination with or concurrently with the particular CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cells expressing anti-CD45RC CAR or population of such immune cells, or composition used; and similar factors well known in the medical field. For example, it is well within the skill of one in the art to begin administering the compound at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. The total dose required for each treatment may be administered in multiple doses or in a single dose.

[0406] In one embodiment, a therapeutically effective amount of a CD45RC binding domain, or an anti-CD45RC antibody or antigen-binding fragment thereof, is in the range of about 0.1 mg / kg to about 5 mg / kg, about 0.2 mg / kg to about 4 mg / kg, about 0.3 mg / kg to about 3 mg / kg, about 0.4 mg / kg to about 2.5 mg / kg, or about 0.5 mg / kg to about 2 mg / kg.

[0407] In one embodiment, a therapeutically effective amount of a CD45RC binding domain, or an anti-CD45RC antibody or antigen-binding fragment thereof, is in the range of about 10 μg / kg to about 400 μg / kg, about 20 μg / kg to about 300 μg / kg, about 30 μg / kg to about 250 μg / kg, about 35 μg / kg to about 200 μg / kg, or about 40 μg / kg to about 160 μg / kg.

[0408] In one embodiment, a therapeutically effective amount of an immune cell expressing an anti-CD45RC CAR or a population of such immune cells is about 1×10 2 pieces~approx. 1×10 9 cells / kg body weight, preferably about 1 x 10 3 ~Approx. 1×10 8 cells / kg body weight (including all integer values ​​within these ranges).

[0409] In one embodiment, a therapeutically effective amount of an immune cell expressing an anti-CD45RC CAR or a population of such immune cells is at least 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 or 1×10 9 cells / kg body weight.

[0410] In one embodiment, a therapeutically effective amount of a CD45RC binding domain, an anti-CD45RC antibody or antigen-binding fragment thereof, an anti-CD45RC CAR, a nucleic acid or vector encoding same, an immune cell expressing an anti-CD45RC CAR or a population of such immune cells, or a composition is administered once daily, twice daily, three times daily, or more.

[0411] In one embodiment, a therapeutically effective amount of a CD45RC binding domain, an anti-CD45RC antibody or antigen-binding fragment thereof, an anti-CD45RC CAR, a nucleic acid or vector encoding same, an immune cell expressing an anti-CD45RC CAR or a population of such immune cells, or a composition is administered every day, every two days, every three days, every four days, every five days, or every six days.

[0412] In one embodiment, a therapeutically effective amount of a CD45RC binding domain, an anti-CD45RC antibody or antigen-binding fragment thereof, an anti-CD45RC CAR, a nucleic acid or vector encoding same, an immune cell expressing an anti-CD45RC CAR or a population of such immune cells, or a composition is administered every week, every two weeks, or every three weeks.

[0413] In one embodiment, a therapeutically effective amount of the CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing an anti-CD45RC CAR or a population of such immune cells, or composition is administered monthly, every two months, every three months, every four months, every five months, or every six months.

[0414] In preferred embodiments, a therapeutically effective amount of a CD45RC binding domain, an anti-CD45RC antibody or antigen-binding fragment thereof, an anti-CD45RC CAR, a nucleic acid or vector encoding same, an immune cell expressing an anti-CD45RC CAR or a population of such immune cells, or a composition is administered every 12 hours, every 24 hours, every 36 hours, every 48 hours, every 60 hours, every 72 hours, or every 96 hours.

[0415] In a preferred embodiment, a therapeutically effective amount of a CD45RC binding domain, an anti-CD45RC antibody or antigen-binding fragment thereof, an anti-CD45RC CAR, a nucleic acid or vector encoding same, an immune cell expressing an anti-CD45RC CAR or a population of such immune cells, or a composition is administered every 60 hours.

[0416] In one embodiment, the CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing anti-CD45RC CAR or population of such immune cells, or composition is for acute administration.In one embodiment, the CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing anti-CD45RC CAR or population of such immune cells, or composition is for chronic administration.

[0417] In one embodiment, a therapeutically effective amount of a CD45RC binding domain, an anti-CD45RC antibody or antigen-binding fragment thereof, an anti-CD45RC CAR, a nucleic acid or vector encoding same, an immune cell expressing an anti-CD45RC CAR or a population of such immune cells, or a composition is given for about 5 days, 7 days, 10 days, 14 days, 21 days, 28 days, 1 month, 2 months, 3 months, 6 months, 1 year or more.

[0418] In one embodiment, a therapeutically effective amount of a CD45RC binding domain, an anti-CD45RC antibody or antigen-binding fragment thereof, an anti-CD45RC CAR, a nucleic acid or vector encoding same, an immune cell expressing an anti-CD45RC CAR or a population of such immune cells, or a composition is administered for a period ranging from about 1 week to about 8 weeks, from about 2 weeks to about 7 weeks, from about 2 weeks to about 6 weeks, or from about 2 weeks to about 5 weeks.

[0419] In preferred embodiments, a therapeutically effective amount of a CD45RC binding domain, an anti-CD45RC antibody or antigen-binding fragment thereof, an anti-CD45RC CAR, a nucleic acid or vector encoding same, an immune cell expressing an anti-CD45RC CAR or a population of such immune cells, or a composition is administered for a period ranging from about 10 days to about 40 days, from about 15 days to about 35 days, or from about 20 days to about 30 days.

[0420] In one embodiment, the above-mentioned CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing anti-CD45RC CAR or population of such immune cells, or composition is administered prior to, simultaneously with, or after a therapeutic agent.

[0421] Examples of suitable therapeutic agents are described above as suitable payloads to be conjugated to the CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, and include chemotherapeutic agents, targeted therapeutic agents, cytotoxic agents (or cytotoxins), cell cycle synchronizing agents, ligands for cell receptor(s), immunomodulatory agents, pro-apoptotic agents, lytic peptides, anti-angiogenic agents, cytokines, growth factors, and hormones.

[0422] It is therefore readily understood that these therapeutic agents can be administered not only in conjugated form, but also in unconjugated (or free) form in conjunction with the above-mentioned CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing anti-CD45RC CAR or population of such immune cells, or composition.

[0423] It will also be understood by one of ordinary skill in the art that the particular therapeutic agent for co-administration will depend on the disease or condition being prevented and / or treated.

[0424] In an exemplary embodiment, when the above-mentioned CD45RC binding domain, anti-CD45RC antibody or antigen-binding fragment thereof, anti-CD45RC CAR, nucleic acid or vector encoding same, immune cell expressing an anti-CD45RC CAR or population of such immune cells, or composition is intended to induce immune tolerance in a subject in need thereof or to prevent and / or reduce transplant rejection, it may be desirable to co-administer an immunosuppressant. [Brief description of the drawings]

[0425] [Figure 1A-C] A series of three graphs showing the efficacy of three anti-hCD45RC antibodies (ABO-21001, ABO-21007 and ABO-21009) to treat GvHD in NSG immunodeficient mice. Figure 1A: Survival curves. Figure 1B: Weight loss. Figure 1C: Clinical scores.

[0426] Working Example The invention is further illustrated by the following examples. The following nomenclature applies throughout the examples:

[0427] "ABIS-45RC": a mouse anti-hCD45RC antibody corresponding to antibody #1 disclosed in Table 2 of WO2020 / 058495.

[0428] "ABO-21001": one of the best-in-class humanized variants of ABIS-45RC of WO 2020 / 058495, corresponding to antibody #92 disclosed in Table 2 of WO 2020 / 058495.

[0429] "ABO-21009": one of the best-in-class humanized variants of ABIS-45RC of WO 2020 / 058495, corresponding to antibody #103 disclosed in Table 2 of WO 2020 / 058495.

[0430] "ABO-21007": a humanized variant of ABIS-45RC, the subject of the present invention, comprising: - A heavy chain variable region (HCVR) having SEQ ID NO: 24: - a light chain variable region (LCVR) with SEQ ID NO: 25, where X1 is absent and X2 is Tyr (Y); - a heavy chain constant region (HCCR) having SEQ ID NO: 26; and - a light chain constant region (LCCR) with SEQ ID NO: 29.

[0431] Example 1: Treatment of Graft-versus-Host Disease (GvHD) with ABO-21007 material and method Isolation of PBMCs Blood was collected from healthy individuals at the Etablissement Francais du Sang (Nantes, France). Written informed consent was provided in accordance with the institutional guidelines. PBMCs were isolated by Ficoll-Paque density gradient centrifugation (Eurobio, Courtaboeuf, France). Residual red blood cells and platelets were removed by hypotonic solution and centrifugation.

[0432] animal NOD / SCID / IL2R 8-12 weeks old - / - (NSG) mice were bred under SPF conditions in our own animal facility (certificate number C44-278).

[0433] GvHD Model Adult NSG immunodeficient mice were irradiated with sublethal doses of whole body radiation (2 Gy dose on day -1) to induce tissue damage favorable for the development of GvHD. The following day (day 0), 1.5 × 10 7 PBMCs (CD45RC high and CD45RC low / - These mice were intravenously injected with human PBMCs, particularly T cells, which react to and attack mouse tissues inducing lesions. These T cells and the lesions observed in the liver, intestine, lungs, and skin mimic GvHD observed after human bone marrow transplantation, or other GvHD experimental systems using rodents as donors and recipients. In particular, these tissue lesions typically induce weight loss, which begins around day 13 after PBMC injection, depending on the number of PBMCs injected and our experimental system. Weight loss is monitored daily, and animals are sacrificed when weight loss falls to 20% of their original weight to avoid unnecessary suffering.

[0434] treatment NSG mice were treated intraperitoneally with purified anti-CD45RC antibodies ABO-21001, ABO-21007 and ABO-21009, or PBS at 0.8 mg / kg every 2.5 days starting on day 0 for 20 days.

[0435] Global Clinical Scoring An overall clinical score for GvHD (0–12) was determined by summing six individual scores: - weight loss score (0-2); - Posture score (0-2); -skin integrity score (0~2); -activity score (0-2); -Coat score (0-2); and - Paleness score (0-2).

[0436] result Treatment with PBMCs alone (PBS) induced mouse death, starting around day 11, with all mice dying by day 15, as shown in FIG. 1A. Treatment with ABO-21001 and ABO-21009 increased mouse survival compared to PBS-treated mice. At day 35, an average of 40% of treated mice were still alive (FIG. 1A). However, surprisingly, treatment with ABO-21007 significantly increased mouse survival, with an average of 70% of mice still alive at day 35 (FIG. 1A).

[0437] Similarly, when treated with ABO-21001 or ABO-21009, NSG mice lost weight, but significantly less than NSG mice treated with PBS (Figure 1B). Surprisingly, however, treatment with ABO-21007 did not result in any weight loss.

[0438] Finally, GvHD clinical scores were also lowest in ABO-21007-treated mice (Figure 1C).

[0439] Taken together, these results demonstrate improved properties of ABO-21007 compared to the already improved anti-human CD45RC antibody of WO 2020 / 058495.

Claims

1. an antigen-binding domain that specifically binds to CD45RC, (a) a heavy chain variable region (HCVR) comprising the following three CDRs: (i) V of the sequence of SEQ ID NO: 10 Ｈ - CDR1; (ii) V of the sequence of SEQ ID NO: 11 Ｈ - CDR2; and (iii) V of the sequence of SEQ ID NO: 12 Ｈ - CDR3; and (b) a light chain variable region (LCVR) comprising the following three CDRs: (i) V of the sequence of SEQ ID NO: 13 Ｌ - CDR1; (ii) V of the sequence of SEQ ID NO: 14 Ｌ - CDR2; and (iii) V of the sequence of SEQ ID NO: 15 Ｌ - CDR3, Including, X in SEQ ID NO: 13 is either absent or selected from the group consisting of Asn (N), Ser (S), and Gly (G). Antigen-binding domain.

2. 1) the HCVR of sequence SEQ ID NO: 24 and the LCVR of sequence SEQ ID NO: 25; or 2) an HCVR comprising three CDRs having SEQ ID NOs: 10, 11, and 12 and framework regions sharing at least 70% sequence identity with the framework regions of SEQ ID NO: 24, and an LCVR comprising three CDRs having SEQ ID NOs: 13, 14, and 15 and framework regions sharing at least 70% sequence identity with the framework regions of SEQ ID NO: 25, and retains its binding specificity for CD45RC; X in SEQ ID NO: 25 １ is either absent or selected from the group consisting of Asn (N), Ser (S) and Gly (G); and X in SEQ ID NO: 25 ２ is selected from the group consisting of Tyr (Y) and Phe (F); The antigen-binding domain of claim 1.

3. The antigen-binding domain of claim 1 or 2, wherein X in SEQ ID NO: 13 is absent.

4. The antigen-binding domain of claim 2, wherein X 2 in SEQ ID NO: 25 is Tyr (Y).

5. 3. The antigen-binding domain of claim 1, wherein the antigen-binding domain is selected from the group consisting of a single-chain variable fragment (scFv), a tandem-di-scFv, a tandem-tri-scFv, a scFv-Fc, a minibody, a maxibody, a diabody, a triabody, an Fv, a Fab, a Fab', a Fab'-SH, and a F(ab')2.

6. An antibody or antigen-binding fragment thereof that specifically binds to CD45RC, wherein the antibody or antigen-binding fragment thereof comprises the antigen-binding domain of claim 1 or 2.

7. further comprising a heavy chain constant region (HCCR) and a light chain constant region (LCCR); The HCCR comprises an amino acid sequence that shares at least 70% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 26-28, and the LCCR comprises an amino acid sequence that shares at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 29; and retains its binding specificity for CD45RC, The antibody or antigen-binding fragment thereof described in claim 6.

8. A chimeric antigen receptor (CAR) that specifically binds to CD45RC, i) at least one extracellular antigen-binding domain according to claim 1 or 2; ii) at least one transmembrane domain, at least one transmembrane domain, wherein said transmembrane domain is selected from the group consisting of a CD8 transmembrane domain and a CD28 transmembrane domain; iii) at least one intracellular signaling domain comprising at least one primary signaling domain, at least one intracellular signaling domain, wherein the at least one primary signaling domain is the signaling domain of CD3ζ; A chimeric antigen receptor (CAR) comprising:

9. The CAR described in claim 8, wherein the CAR further comprises an extracellular hinge domain and / or the at least one intracellular signaling domain further comprises one or more costimulatory signaling domains.

10. A CAR as described in claim 8, wherein the transmembrane domain is selected from the group consisting of a CD8 transmembrane domain having an amino acid sequence that shares at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 49, and a CD28 transmembrane domain having an amino acid sequence that shares at least 70% sequence identity with the amino acid sequence of SEQ ID NO:

50.

11. A CAR as described in claim 8, wherein at least one primary signaling domain is a signaling domain of CD3ζ having an amino acid sequence that shares at least 70% sequence identity with the amino acid sequence of SEQ ID NO:

51.

12. A population of immune cells expressing the chimeric antigen receptor of claim 8 on their cell surface.

13. The immune cells of the population are CD4 ＋ T cells, CD8 ＋ 13. The immune cell population of claim 12, wherein the immune cell population is selected from the group comprising or consisting of T cells, double positive T cells, double negative T cells, γδ T cells, NK cells, NKT cells, B cells, macrophages, or dendritic cells.

14. A nucleic acid encoding an antigen-binding domain described in claim 1 or 2, an antibody or antigen-binding fragment thereof comprising the antigen-binding domain described in claim 1 or 2, or a CAR comprising at least one extracellular antigen-binding domain described in claim 1 or 2.

15. A pharmaceutical composition for use in the treatment of a disease, comprising an antigen-binding domain according to claim 1 or 2, an antibody or antigen-binding fragment thereof comprising the antigen-binding domain according to claim 1 or 2, an immune cell population expressing a chimeric antigen receptor comprising at least one extracellular antigen-binding domain according to claim 1 or 2, or a nucleic acid encoding an antigen-binding domain according to claim 1 or 2, an antibody or antigen-binding fragment thereof comprising the antigen-binding domain according to claim 1 or 2, or a CAR comprising at least one extracellular antigen-binding domain according to claim 1 or 2.

16. 10. A pharmaceutical composition for use in inducing immune tolerance in a subject in need thereof, comprising: an immune cell population expressing the antigen-binding domain of claim 1 or 2, an antibody or antigen-binding fragment thereof comprising the antigen-binding domain of claim 1 or 2, a chimeric antigen receptor comprising at least one extracellular antigen-binding domain of claim 1 or 2, or a nucleic acid encoding a CAR comprising the antigen-binding domain of claim 1 or 2, an antibody or antigen-binding fragment thereof comprising the antigen-binding domain of claim 1 or 2, or at least one extracellular antigen-binding domain of claim 1 or 2.

17. CD45RC in a subject in need thereof ｈｉｇｈ 10. A pharmaceutical composition for use in depleting a cell, comprising an immune cell population expressing the antigen-binding domain of claim 1 or 2, an antibody or antigen-binding fragment thereof comprising the antigen-binding domain of claim 1 or 2, a chimeric antigen receptor comprising at least one extracellular antigen-binding domain of claim 1 or 2, or a nucleic acid encoding the antigen-binding domain of claim 1 or 2, an antibody or antigen-binding fragment thereof comprising the antigen-binding domain of claim 1 or 2, or a CAR comprising at least one extracellular antigen-binding domain of claim 1 or 2.

18. 10. A pharmaceutical composition for use in preventing and / or alleviating transplant rejection in a subject in need thereof, comprising: an immune cell population expressing the antigen-binding domain of claim 1 or 2, an antibody or antigen-binding fragment thereof comprising the antigen-binding domain of claim 1 or 2, a chimeric antigen receptor comprising at least one extracellular antigen-binding domain of claim 1 or 2, or a nucleic acid encoding a CAR comprising the antigen-binding domain of claim 1 or 2, an antibody or antigen-binding fragment thereof comprising the antigen-binding domain of claim 1 or 2, or at least one extracellular antigen-binding domain of claim 1 or 2.

19. A pharmaceutical composition for use in preventing and / or alleviating graft-versus-host disease (GvHD) in a subject in need thereof, comprising an antigen-binding domain according to claim 1 or 2, an antibody or antigen-binding fragment thereof comprising the antigen-binding domain according to claim 1 or 2, an immune cell population expressing a chimeric antigen receptor comprising at least one extracellular antigen-binding domain according to claim 1 or 2, or a nucleic acid encoding a CAR comprising the antigen-binding domain according to claim 1 or 2, an antibody or antigen-binding fragment thereof comprising the antigen-binding domain according to claim 1 or 2, or at least one extracellular antigen-binding domain according to claim 1 or 2.

20. CD45RC in a subject in need thereof ｈｉｇｈ 10. A pharmaceutical composition for use in preventing, alleviating and / or treating an immune cell population expressing the antigen-binding domain of claim 1 or 2, an antibody or antigen-binding fragment thereof comprising the antigen-binding domain of claim 1 or 2, a chimeric antigen receptor comprising at least one extracellular antigen-binding domain of claim 1 or 2, or a nucleic acid encoding a CAR comprising the antigen-binding domain of claim 1 or 2, an antibody or antigen-binding fragment thereof comprising the antigen-binding domain of claim 1 or 2, or at least one extracellular antigen-binding domain of claim 1 or 2.

21. The pharmaceutical composition of claim 20, wherein the CD45RC high -associated disease, disorder, or condition is selected from the group consisting of an autoimmune disease, an unwanted immune response, a monogenic disease, lymphoma, and cancer.

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