Anti-CD8 alpha depleting antibody

Abstract

The present invention relates to novel anti-CD8α (cluster of differentiation 8 alpha) antibodies and antigen-binding fragments thereof, and therapeutic uses thereof.

Description

[Technical Field]

[0001] The present invention relates to anti-CD8α (cluster of differentiation 8 alpha) antibodies and antigen-binding fragments thereof. [Background technology]

[0002] CD8α (cluster of differentiation 8 alpha) is a glycoprotein that is a member of the immunoglobulin superfamily. CD8α homodimerizes with another CD8α protein or heterodimerizes with CD8β (cluster of differentiation 8 beta) protein to form dimeric CD8 molecules expressed on the surface of CD8+ cytotoxic T cells. In CD8+ T cells, the dimeric CD8 molecule functions as a coreceptor for the T cell receptor (TCR) to recognize antigens presented by antigen-presenting cells in association with class I MHC molecules. Thus, the dimeric CD8 molecule plays a role in enhancing interactions between T cells and antigen-presenting cells.

[0003] Both CD8α and CD8β proteins consist of an immunoglobulin-like extracellular domain and a long glycosylated peptide chain that functions as both a transmembrane and an intracellular domain. Thus, the dimeric CD8 molecule also binds to the tyrosine kinase p56, which is important for the phosphorylation cascade that occurs after antigen recognition. lck It is important for transmitting signals to T cells through its intracellular domain, which recruits IL-1 receptors.

[0004] CD8+ cytotoxic T cells have been shown to mediate the elimination of cancer cells, cells infected with intracellular pathogens (such as viruses or bacteria), and damaged cells. Upon exposure to infected or dysfunctional cells, CD8+ cytotoxic T cells express death-inducing ligands, which also release cytotoxins such as perforin, granzymes, and granulysin, which induce apoptosis or lysis of the target cells. Thus, CD8+ cytotoxic T cells play an important role in the immune response and the fight against intracellular pathogens and tumor cells.

[0005] However, CD8+ cytotoxic T cells are also involved in the development of various diseases and disorders, and therefore, in certain situations, it may be advantageous to be able to deplete CD8+ cytotoxic T cells in a controlled manner.Therefore, there is a need for new therapeutic means that specifically target CD8+ cytotoxic T cells, allowing for the temporary depletion of CD8+ cytotoxic T cells in a controlled manner.

[0006] As a result, the inventors herein aimed to develop novel antibodies that specifically bind to CD8α and allow for the depletion of CD8α-expressing cells, primarily CD8+ cytotoxic T cells. Advantageously, anti-CD8α antibodies as described herein can induce rapid and almost complete depletion of CD8α-expressing cells, followed by controlled repopulation of CD8α-expressing cells. Thus, anti-CD8α antibodies as described herein would create a valuable therapeutic tool for transiently depleting CD8α-expressing cells. Summary of the Invention

[0007] The present invention provides an isolated anti-CD8α antibody, or antigen-binding fragment thereof, comprising: a) The variable region (VH) of the heavy chain of the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises the following three CDRs: -V H -CDR1:NX1X2MN, in the formula, X1 is N or Y, X2 is D, A, or Y; -V H - CDR2: X3ISGSSX4YIX5YADFVKG (SEQ ID NO: 1), wherein X3 is D or S, X4 is S or R, X5 is D, G, or Y; -V H -CDR3:SSX6X7X8X9YX 10 X 11 X 12 X 13 MDV (SEQ ID NO: 2), wherein: X6 is N or does not contain an amino acid; X7 is Y or does not contain an amino acid; X8 is Y or G or does not contain an amino acid; X9 is D or S or does not contain an amino acid; X 10 is S, N or F, X 11 is A or G, X 12 is S or D or N, X 13 is A or G; b) the variable region of the light chain (VL) of the isolated anti-CD8α antibody or an antigen-binding fragment thereof comprises the following three CDRs: -V L -CDR1:AGTSSDVGGX 14 X 15 X 16 VS (SEQ ID NO: 3), wherein: X 14 is G or N or Y, X 15 is S or Y, X 16 is S or Y, -V L -CDR2:X 17 DSX 18 RPS (SEQ ID NO: 4), wherein: X 17 is Q or S or Y, X 18 is Y or S, -V L -CDR3:SSX 19 TX 20 YSTRV (SEQ ID NO: 5), wherein: X 19 is Y or D, X 20 is Y or Q or S, or an antigen-binding fragment thereof.

[0008] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is a) A VH comprising the following three CDRs: -V H CDR1: NNAMN (SEQ ID NO: 6), NYDMN (SEQ ID NO: 12), or NYMN (SEQ ID NO: 18); -V H - CDR2: DISGSSRYIGYADFVKG (SEQ ID NO: 7), DISGSSSYIDYADFVKG (SEQ ID NO: 13), or SISGSSRYIYYADFVKG (SEQ ID NO: 19); -V H - CDR3: SSNYYDYNADAMDV (SEQ ID NO: 8), SSYYSGMDV (SEQ ID NO: 14), or SSGSYFGNAMDV (SEQ ID NO: 20); b) a VL comprising the following three CDRs: -V L - CDR1: AGTSSDVGGNSYVS (SEQ ID NO: 9), AGTSSDVGGGSSVS (SEQ ID NO: 15), or AGTSSDVGGYYSVS (SEQ ID NO: 21); -V L CDR2: SDSSRPS (SEQ ID NO: 10), QDSYRPS (SEQ ID NO: 16), or YDSSRPS (SEQ ID NO: 22) -V L - CDR3: comprising SSYTQYSTRV (SEQ ID NO: 11), SSYTYYSTRV (SEQ ID NO: 17), or SSDTSYSTRV (SEQ ID NO: 23).

[0009] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises the following six CDRs: -V H CDR1: NNAMN (SEQ ID NO: 6), V H - CDR2: DISGSSRYIGYADFVKG (SEQ ID NO: 7), V H - CDR3: SSNYYDYNADAMDV (SEQ ID NO: 8), V L - CDR1:AGTSSDVGGNSYVS (SEQ ID NO: 9), V L CDR2: SDSSRPS (SEQ ID NO: 10), and V LCDR3: SSYTQYSTRV (SEQ ID NO: 11); or -V H CDR1: NYDMN (SEQ ID NO: 12), V H - CDR2: DISGSSSYIDYADFVKG (SEQ ID NO: 13), V H - CDR3: SSYYSGSGMDV (SEQ ID NO: 14), V L - CDR1:AGTSSDVGGGSSVS (SEQ ID NO: 15), V L CDR2: QDSYRPS (SEQ ID NO: 16), and V L CDR3: SSYTYYSTRV (SEQ ID NO: 17); or -V H CDR1: NYYMN (SEQ ID NO: 18), V H - CDR2: SISGSSRYIYYADFVKG (SEQ ID NO: 19), V H - CDR3: SSGSYFGNAMDV (SEQ ID NO: 20), V L - CDR1:AGTSSDVGGYYSVS (SEQ ID NO: 21), V L - CDR2: YDSSRPS (SEQ ID NO: 22), and VL-CDR3: SSDTSYSTRV (SEQ ID NO: 23).

[0010] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a variable region of a heavy chain (VH) comprising the sequence set forth in SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28, or a sequence with at least 80% identity to SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28. In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a variable region of a light chain (VL) comprising the sequence set forth in SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29, or a sequence with at least 80% identity to SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29.

[0011] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is - a VH comprising the sequence set forth in SEQ ID NO: 24 or a sequence having at least 80% identity to SEQ ID NO: 24, and a VL comprising the sequence set forth in SEQ ID NO: 25 or a sequence having at least 80% identity to SEQ ID NO: 25; or - a VH comprising the sequence set forth in SEQ ID NO: 26 or a sequence having at least 80% identity to SEQ ID NO: 26, and a VL comprising the sequence set forth in SEQ ID NO: 27 or a sequence having at least 80% identity to SEQ ID NO: 27; or - a VH comprising the sequence set forth in SEQ ID NO: 28 or a sequence which has at least 80% identity to SEQ ID NO: 28, and a VL comprising the sequence set forth in SEQ ID NO: 29 or a sequence which has at least 80% identity to SEQ ID NO: 29.

[0012] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is a CD8α-expressing cell-depleting antibody.

[0013] The present invention also relates to a fusion protein comprising the anti-CD8α antibody or antigen-binding fragment thereof, and a nucleic acid molecule encoding the anti-CD8α antibody or antigen-binding fragment thereof, or the fusion protein.

[0014] The present invention also relates to a pharmaceutical composition comprising the isolated anti-CD8α antibody or antigen-binding fragment thereof, the fusion protein and / or the nucleic acid molecule, and at least one pharmaceutically acceptable excipient.

[0015] The present invention also relates to the isolated anti-CD8α antibody or antigen-binding fragment thereof, the fusion protein, the nucleic acid molecule, or the pharmaceutical composition for use as a medicament.

[0016] The present invention also relates to the isolated anti-CD8α antibody or antigen-binding fragment thereof, the fusion protein, the nucleic acid molecule, or the pharmaceutical composition for use in treating a CD8-associated disease. In some embodiments, the CD8-associated disease is a cardiovascular disease.

[0017] The present invention also relates to an in vitro method for depleting CD8α-expressing cells in a sample, the method comprising contacting the sample with an isolated anti-CD8α antibody or antigen-binding fragment thereof described herein, or a fusion protein described herein. The present invention also relates to an in vitro method for detecting and / or quantitating CD8α in a sample, cell, tissue, or organ, the method comprising contacting the sample, cell, tissue, or organ with an isolated anti-CD8α antibody or antigen-binding fragment thereof as described herein, or a fusion protein as described herein.

[0018] definition In the present invention, the following terms have the following meanings:

[0019] The use of "about" before a number includes up to plus or minus 10% of the value of that number. It is to be understood that the value to which the term "about" refers is itself also specifically and preferably disclosed.

[0020] "Affinity" is used to define the strength of an antibody-antigen complex. Affinity measures the strength of the interaction between an antigen and an antibody or its antigen-binding fragment. It is defined as the affinity constant K A or the dissociation constant K D It can be represented by:

[0021] "Anti-CD8α" (sometimes referred to as "anti-CD8a"), when qualifying an antibody or antigen-binding fragment thereof, means that the antibody or antigen-binding fragment thereof specifically binds to or specifically recognizes CD8α (cluster of differentiation 8 alpha).

[0022] The terms "antibody (Ab)" and "immunoglobulin (Ig)" may be used interchangeably to refer to a protein having a combination of two heavy chains (H chains) and two light chains (L chains). In particular, the term "antibody" refers to such an aggregate having significant, known, specific immunoreactive activity against an antigen of interest (e.g., CD8α, particularly human CD8α). As explained elsewhere herein, "specificity" for human CD8α does not exclude cross-reactivity with orthologs of human CD8α, such as monkey or mouse CD8α. As noted above, antibodies and immunoglobulins comprise light and heavy chains, with or without interchain covalent bonds between them. The structure of basic immunoglobulins in vertebrate systems is relatively well understood. The general term "immunoglobulin" includes five biochemically distinct classes of immunoglobulins: IgG, IgM, IgA, IgD, and IgE. IgG immunoglobulins contain two identical light chains with a molecular weight of approximately 23 kDa and two identical heavy chains with a molecular weight of approximately 53-70 kDa. The four chains are held together by disulfide bonds in a "Y" configuration, in which the light chains sandwich the heavy chains, which begin at the mouth of the "Y" and continue through the variable region. Immunoglobulin light chains are classified as either kappa (κ) or lambda (λ). Each heavy chain class can bind either κ or λ light chains. Generally, when immunoglobulins are produced by either hybridomas, B cells, or genetically engineered host cells, 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 noncovalent bonds. In the heavy chains, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the base 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 each (e.g., γ1-γ4). It is the nature of the heavy chain that determines the "class" of an antibody: 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 confer functional specialization. The variable region of an antibody enables the antibody to selectively recognize and specifically bind to an antigen epitope. That is, the light chain variable region (VL) and heavy chain variable region (VH) of an antibody combine to form a variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure thus forms an antigen-binding site present at the end of each arm of the "Y." More specifically, the antigen-binding site is defined by three complementarity-determining regions (CDRs) of each of the VH and VL.

[0023] An "antigen-binding fragment" of an antibody is interchangeable with the term "antigen-binding domain" of an antibody and refers to a portion or region of an antibody or immunoglobulin that contains fewer amino acid residues than a whole antibody or immunoglobulin and is capable of binding to an antigen and / or competing with the whole antibody for antigen binding (e.g., for specific binding to CD8α). Examples of antigen-binding antibody fragments include, but are not limited to, Fab, Fab', F(ab'), Fv fragments, scFv fragments, disulfide-linked Fvs (sdFvs), Fd fragments consisting of the VH and CH1 domains, linear antibodies, single-domain antibodies such as sdAbs (either VL or VH), camelid VHH domains, multispecific antibodies formed from antibody fragments, such as bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, and isolated CDRs of an antibody. Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, a name reflecting their ability to crystallize readily. The Fab fragment consists of one entire L chain (the variable region of the L chain (VL) and the constant domain of the L chain (CL)) together with part of one H chain consisting of the variable region of the H chain (VH) and the first constant domain of the heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide-linked Fab fragments with divalent antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having additional few amino acid residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0024] As used herein, "antigen" or "Ag" refers to a protein (eg, CD8α) that is specifically recognized by an antibody or antibody-binding fragment thereof.

[0025] "CD8" or "cluster of differentiation 8" refers to a transmembrane glycoprotein predominantly expressed on cytotoxic T lymphocytes. The CD8 molecule functions as a co-receptor for the T cell receptor (TCR) on T cells, recognizing antigens presented by antigen-presenting cells in association with class I MHC molecules. CD8 functions as a homodimer composed of two CD8α chains or as a heterodimer composed of one CD8α chain and one CD8β chain. Both CD8α and CD8β are members of the immunoglobulin superfamily. Both the CD8α and CD8β chains contain a unique immunoglobulin-like domain that constitutes the extracellular portion of the protein, which is anchored to the cell membrane via a long glycosylated peptide chain. The two chains that make up a CD8 dimer (i.e., the heterodimer CD8α / CD8β or the homodimer CD8α / CD8α) associate via disulfide bonds. In the NCBI database (https: / / www.ncbi.nlm.nih.gov), the reference human CD8α gene sequence corresponds to NCBI gene ID: 925, updated on August 5, 2022. The human CD8α (hCD8α) gene consists of 10 exons on chromosome 2p11.2 and encodes a 235 amino acid protein for isoform 1 and a 198 amino acid protein for isoform 2. Human CD8α isoform 1 refers to the protein referenced in the NCBI database as NP_001139345.1, NP_001759.3, or NP_001369627.1, while isoform 2 refers to the protein referenced as NP_741969.1. Alternative names for CD8α include, by way of non-limiting example, "T cell surface glycoprotein CD8 alpha chain," "CD8 antigen," "alpha polypeptide," "Leu2 T lymphocyte antigen," "OKT8 T cell antigen," "T cell co-receptor," "T cell antigen Leu2," "T lymphocyte differentiation antigen T8," "T8 T cell antigen," "p32," "Leu-2," and "Leu2." The terms "CD8α" and "CD8" are used interchangeably herein. In the NCBI database, the reference human CD8β gene sequence corresponds to NCBI gene ID: 926, updated on August 5, 2022. The human CD8β gene consists of eight exons on chromosome 2p11.2 and encodes six isoforms by alternative splicing: a 246 amino acid protein for isoform 1, a 243 amino acid protein for isoform 2, a 221 amino acid protein for isoform 3, a 213 amino acid protein for isoform 4, a 210 amino acid protein for isoform 5, and a 198 amino acid protein for isoform 6. Human CD8β isoform 1 refers to the protein referenced in the NCBI database as XP_011531466.1, isoform 2 refers to the protein referenced as NP_757362.1, isoform 3 refers to the protein referenced as NP_742099.1, isoform 4 refers to the protein referenced as NP_742100.1, isoform 5 refers to the protein referenced as NP_004922.1, and isoform 6 refers to the protein referenced as NP_001171571.1. Alternative names for CD8β include, by way of non-limiting example, "LY3," "P37," "LEU2," "LYT3," "CD8B1," "T-cell surface glycoprotein CD8 beta chain," "CD8 antigen," "beta polypeptide 1," and "T-lymphocyte surface glycoprotein beta chain."

[0026] "CDR" or "complementarity-determining region" refers to the discontinuous antigen-binding sites found within both heavy and light chain variable regions. The precise amino acid sequence boundaries of a given CDR can be determined using any of several well-known schemes, including those described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (the "Kabat" numbering scheme); Al-Lazikani et al. (1997) JMB 273, 927-948 (the "Chothia" numbering scheme), or a combination thereof, such as the AbM definition, which is a compromise between the two used in Oxford Molecular's AbM antibody-decoding software. More recently, a universal numbering system, the ImMunoGeneTics (IMGT) Information System® (Lefranc et al., Nucleic Acids Res. 27:209-212 1999), has been developed and widely adopted. IMGT® is an integrated information system specialized for immunoglobulins (Ig), T cell receptors (TCR), and major histocompatibility complexes (MHC) of humans and other vertebrates. As used herein, CDRs are defined by their amino acid sequence and position within a light or heavy chain (e.g., VH -CDR1, VH -CDR2, VH -CDR3, VL -CDR1, VL -CDR2, VLThe "location" of CDRs within the structure of immunoglobulin variable regions is conserved across species and resides in structures called loops, so CDR and framework amino acid residues can be readily identified by using a numbering system that aligns variable region sequences according to structural features. This information can be used to graft and replace CDRs from one species of immunoglobulin onto an acceptor framework, typically derived from a human antibody. The agreement between Kabat numbering and the IMGT® specific numbering system is also well known to those skilled in the art (e.g., Lefranc et al., supra).

[0027] "Epitope" refers to a specific sequence of amino acids located on one or more proteins to which an antibody or antigen-binding fragment thereof specifically binds. Epitopes often consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be linear (or continuous) or conformational, i.e., involving two or more sequences of amino acids in different regions of the antigen, which may not necessarily be contiguous.

[0028] "Fc domain," "Fc portion," and "Fc region" may be used interchangeably and refer to the C-terminal fragment of an antibody heavy chain, e.g., from about amino acid (aa) 230 to about aa 450 of a human gamma heavy chain, or the corresponding sequences in other types of antibody heavy chains (e.g., α, δ, ε, and μ of human antibodies), or naturally occurring allotypes thereof.

[0029] "Framework regions" or "FR regions" or "non-CDR regions" include amino acid residues that are part of the variable region but not part of the CDRs (e.g., using the Kabat definition of a CDR, the Chothia definition of a CDR, or the IMGT® numbering definition of a CDR). Thus, variable region frameworks range in length from about 100 to 120 amino acids, but include only amino acids outside the CDRs. For specific examples of heavy chain variable regions (VH) and CDRs as defined by Kabat or Chothia: -FR1 may correspond to the domain of the variable region encompassing amino acids 1 to 25 according to the Chothia / AbM definition, or the last 5 amino acid residues according to the Kabat definition; -FR2 may correspond to the domain of the variable region encompassing amino acids 36 to 49; -FR3 may correspond to the domain of the variable region encompassing amino acids 67 to 98; and -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 light chain variable region (VL). In naturally occurring antibodies, the six CDRs present in each monomeric antibody are short, noncontiguous sequences of amino acids that are specifically positioned to form the antigen-binding site when the antibody assumes its three-dimensional configuration in an aqueous environment. As noted above, the remainder of the heavy and light variable regions, which exhibit less inter-molecular variability in amino acid sequence, correspond to framework regions. The framework regions primarily adopt a beta-sheet conformation, and the CDRs form loops that connect, and in some cases form part of, the beta-sheet structure. Thus, these framework regions act as a scaffold for correctly orienting the six CDRs through interchain noncovalent interactions. The antigen-binding site formed by the positioned CDRs defines a surface complementary to the epitope of the immunoreactive antigen. This complementary surface facilitates noncovalent binding of the antibody to the immunoreactive antigen epitope.

[0030] A "heavy chain region" comprises an amino acid sequence derived from the constant domain of an immunoglobulin heavy chain. A protein comprising a heavy chain region comprises at least one of a CH1 domain, a hinge region (e.g., an upper, middle, and / or lower hinge domain), a CH2 domain, a CH3 domain, or a variant or fragment thereof. In some embodiments, an antibody or antigen-binding fragment thereof described herein may comprise the Fc region of an immunoglobulin heavy chain (e.g., a hinge portion, a CH2 domain, and a CH3 domain). In some embodiments, an antibody or antigen-binding fragment thereof described herein lacks at least a region of the constant domain (e.g., all or a portion of the CH2 domain). In some embodiments, at least one, preferably all, of the constant domains is derived from a human immunoglobulin heavy chain. For example, in some embodiments, the heavy chain region comprises a fully human hinge domain. In some embodiments, the heavy chain region comprises a fully human Fc region (e.g., a hinge, a CH2, and a CH3 domain from a human immunoglobulin). In some embodiments, the constituent constant domains of the heavy chain region are derived from different immunoglobulin molecules. For example, the heavy chain region of the protein may comprise a CH2 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 or IgG4 molecule. In some embodiments, the constant domain is a chimeric domain comprising regions from different immunoglobulin molecules. For example, the hinge may comprise a first region derived from an IgG1 molecule and a second region derived from an IgG3 or IgG4 molecule. In some embodiments, the constant domain of the heavy chain region may be altered to differ in amino acid sequence from a naturally occurring (wild-type) immunoglobulin molecule. That is, the antibodies or antigen-binding fragments thereof described herein may comprise alterations or modifications to one or more heavy chain constant domains (CH1, hinge, CH2, or CH3) and / or light chain constant domains (CL). Exemplary modifications include addition, deletion, or substitution of one or more amino acids in one or more domains.

[0031] The "hinge region" comprises the region of the heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region contains approximately 25 amino acid residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains.

[0032] "Identity" or "identical," as used herein in the context of two or more polypeptide or two or more nucleic acid sequences, refers to the degree of sequence relatedness between the polypeptides or nucleic acids (respectively), as determined by the number of matches between two or more amino acid residues or strings of two or more nucleotides, respectively. "Identity" measures the percent of identical matches between the smaller of the two or more sequences, with gap alignment (if any) accommodated by a particular mathematical model or computer program (i.e., "algorithm"). The identity of related polypeptide or nucleic acid sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in "Computational Molecular Biology," Lesk, AM, ed., Oxford University Press, New York, 1988; "Biocomputing: Informatics and Genome Projects," Smith, DW, ed., Academic Press, New York, 1993; "Computer Analysis of Sequence Data," Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; "Sequence Analysis in Molecular Biology," von Heinje, G., Academic Press, 1987; "Sequence Analysis Primer," Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988). Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods to determine identity are described in publicly available computer programs.Preferred computer program methods for determining identity between two sequences include the GCG program package, which includes GAP (Devereux et al., Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387-95; Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. MoI. Biol. 215, 403-410(1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894; Altschul et al., J. MoI. Biol. 215, 403-410(1990)). Identity can also be determined using the well-known Smith Waterman algorithm.

[0033] "Isolated" or "non-naturally occurring" with respect to a biological component (such as an antibody or nucleic acid) refers to a biological component that has been altered or removed from its natural state. For example, an antibody or nucleic acid naturally present in a living animal is not "isolated," but the same antibody or nucleic acid partially or completely separated from the coexisting materials of its natural state is. An isolated antibody or nucleic acid can exist in a substantially purified form or can exist in a non-native environment, such as a host cell. Typically, an isolated antibody or nucleic acid preparation contains the antibody or nucleic acid at least about 80% pure, at least about 85% pure, at least about 90% pure, at least about 95% pure, more than 95% pure, more than about 96% pure, more than about 97% pure, more than about 98% pure, or more than about 99% pure. "Non-naturally occurring" or "isolated" nucleic acids and proteins, such as antibodies, include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in host cells as well as chemically synthesized nucleic acids.

[0034] A "monoclonal antibody (mAb)" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies within the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific because they are 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 (epitope) 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" should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies or antigen-binding fragments thereof as described herein can be prepared by well-known hybridoma methodologies or produced using recombinant DNA methods in bacterial, eukaryotic, animal, or plant cells. "Monoclonal antibodies" can also be isolated from phage antibody libraries using techniques commonly known in the art.

[0035] As used herein, the term "nucleic acid" or "polynucleotide" refers to a polymer of nucleotides covalently linked by phosphodiester bonds, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), in either single- or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues.

[0036] A "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to an excipient or carrier that does not produce adverse allergic reactions or other untoward reactions when administered to mammals, preferably humans. This includes, for example, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Thus, a pharmaceutically acceptable excipient or carrier refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary. For human administration, formulations should meet sterility, pyrogenicity, general safety, and purity standards required by regulatory authorities such as the FDA (U.S. Food and Drug Administration) or EMA (European Medicines Agency).

[0037] "Single-chain Fv", also abbreviated as "sFv" or "scFv", refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region (VL) and at least one antibody fragment comprising a heavy chain variable region (VH), wherein the light chain variable region and the heavy chain variable region are contiguously linked, for example, via a synthetic linker, such as a short, flexible polypeptide linker, and can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived.

[0038] "Subject" refers to a warm-blooded animal, more preferably a mammal. The term "mammal" as used herein refers to any mammal, including a human. Preferably, the mammal is a primate, more preferably a human. In some embodiments, the subject may be a "patient" who is awaiting or receiving medical care, or who has been / is the subject of or will be the subject of medical treatment, or who is being monitored for the development of a target disease or condition. In some embodiments, the subject is an adult (e.g., a subject over the age of 18). In some embodiments, the subject is a child (e.g., a subject under the age of 18). In some embodiments, the subject is male. In some embodiments, the subject is female.

[0039] "Variable", "variable region" or "variable domain" refers to a variable domain V H and V L This refers to the fact that certain regions of the VL and VH domains vary significantly in sequence between antibodies and are used for the binding and specificity of each particular antibody to its target antigen. However, variability is not uniformly distributed throughout the variable domains of antibodies. It is concentrated in three segments called "hypervariable loops" in each of the VL and VH domains that form part of the antigen-binding site. Each of the six hypervariable loops may contain a portion of the CDRs as defined above. DETAILED DESCRIPTION OF THE INVENTION

[0040] A first object of the present invention relates to an isolated antibody or antigen-binding fragment thereof that specifically binds to cluster of differentiation 8 alpha (CD8α). Accordingly, a first object of the present invention relates to an isolated anti-CD8α antibody or antigen-binding fragment thereof.

[0041] As used herein, an antibody or antigen-binding fragment thereof is said to be "specific," "immunospecific," or "specifically binds" to its cognate antigen (e.g., CD8α) if it reacts with said antigen at a detectable level. The binding properties of an antibody or antigen-binding fragment thereof to its cognate antigen (or cells or tissues expressing said antigen) can generally be determined and assessed using immunodetection methods well known in the art, including, for example, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and / or immunofluorescence-based assays such as fluorescence-activated cell sorting (FACS), or by surface plasmon resonance (SPR).

[0042] The affinity of an antibody or antigen-binding fragment thereof for its cognate antigen (e.g., CD8α) can be readily determined using conventional techniques, such as those described in Scatchard, 1949. Ann NY Acad Sci. 51:660-672. The affinity of an antibody or antigen-binding fragment thereof for its cognate antigen is generally determined by the equilibrium dissociation constant (K D) Thus, in some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is expressed as 10 -6 K below M D An antibody is said to be "immunospecific," "specific for," or "specifically binds" to the alloantigen CD8α if it reacts with that alloantigen at a detectable level.

[0043] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof described herein specifically binds to human CD8α (hCD8α or huCD8α). In other words, in some embodiments, the isolated antibody or antigen-binding fragment thereof is an anti-hCD8α antibody or anti-hCD8α antigen-binding fragment thereof.

[0044] Binding of an antibody or antigen-binding fragment thereof to its cognate antigen can also be assessed by determining the concentration of antibody or antigen-binding fragment thereof that confers half-maximal binding (i.e., EC50). For example, the EC50 of an antibody or antigen-binding fragment thereof can be determined by enzyme-linked immunosorbent assay (ELISA), particularly a dose-response ELISA. In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof binds to CD8α, preferably hCD8α, and has an EC50 of less than 10 nM, preferably less than 5 nM, and more preferably less than 2.5 nM, particularly when determined by dose-response ELISA.

[0045] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof as described herein specifically binds to human CD8α having the amino acid sequence as set forth in SEQ ID NO: 30, which corresponds to NCBI accession number NP_001759.3, last modified on June 12, 2022.

[0046] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof specifically binds to the extracellular domain of CD8α.

[0047] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof described herein specifically binds to human CD8α isoform 1 and isoform 2. In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof, as described herein, specifically binds to human CD8α isoform 1 and both the secreted and tethered secreted forms of isoform 2.

[0048] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof described herein specifically binds to CD8α, particularly human CD8α, present on the surface of white blood cells. In some embodiments, the white blood cells include CD8+ T lymphocytes (also known as CD8+ T cells), CD8α-expressing dendritic cells (also called CD8+ dendritic cells), and CD8α-expressing natural killer (NK) cells (also called CD8+ NK cells).

[0049] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof described herein can deplete CD8α-expressing cells (which may also be referred to as CD8+ cells). Thus, in some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is a depleting antibody, i.e., a CD8α-expressing cell-depleting antibody (which may also be referred to as a CD8+ cell-depleting antibody). The CD8α-expressing cells may be CD8α-expressing leukocytes, particularly CD8α-expressing T cells (also referred to as CD8+ T cells), CD8α-expressing NK cells (also referred to as CD8+ NK cells), and / or CD8α-expressing dendritic cells (also referred to as CD8+ dendritic cells). Thus, the isolated anti-CD8α antibody or antigen-binding fragment thereof described herein may be capable of depleting CD8+ T cells and / or CD8+ NK cells. Thus, in some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is a CD8α-expressing T cell-depleting antibody, which may also be referred to as a CD8+ T cell-depleting antibody.

[0050] An isolated anti-CD8α antibody or antigen-binding fragment thereof as described herein, upon contact with a sample, may be capable of inducing in vitro depletion of CD8α-expressing cells in the sample. An isolated anti-CD8α antibody or antigen-binding fragment thereof as described herein, upon administration to an organism or subject, may be capable of inducing in vivo depletion of CD8α-expressing cells in the organism or subject.

[0051] As used herein, the term "CD8α-expressing cell-depleting antibody" or "CD8+ cell-depleting antibody" refers to an antibody that binds to CD8α (i.e., CD8) expressed on the surface of CD8α-expressing cells (i.e., CD8+ cells) and, upon binding to CD8α (i.e., CD8), mediates the destruction or depletion of said cells.

[0052] As used herein, "deplete," "depleting," or "depletion," with respect to cells expressing CD8α described herein, refers to a measurable decrease or reduction in the number of CD8α-expressing cells in a sample, organism, or subject. For example, depletion with respect to cells expressing CD8α as described herein can refer to a measurable decrease or reduction in the number of circulating CD8α-expressing cells, and / or tissue-infiltrating CD8α-expressing cells, and / or CD8α-expressing cells present in the lymph nodes and / or spleen of an organism or subject.

[0053] The decrease or reduction may be at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, particularly with respect to the initial number of CD8α-expressing cells in a sample, organism, or subject. As used herein, the "initial number of CD8α-expressing cells" (or "number of CD8α-expressing cells at baseline") refers to the number of CD8α-expressing cells in a sample, organism, or subject prior to depletion induced by an isolated anti-CD8α antibody or antigen-binding fragment thereof as described herein (i.e., prior to administration of an isolated anti-CD8α antibody or antigen-binding fragment thereof as described herein). In some embodiments, the initial number of CD8α-expressing cells in an organism or subject is a normal number of CD8α-expressing cells, i.e., a number of CD8α-expressing cells within a range considered normal or normal for the organism or subject (i.e., the normal range). For example, a normal number of CD8+ T cells (particularly circulating CD8+ T cells) in a healthy human adult is between 150 and 1000 cells / mm 3 Thus, the normal average number of CD8+ T cells (particularly circulating CD8+ T cells) in a healthy adult human may be about 550 cells / μL. In some embodiments, the decrease or reduction may be at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, relative to the number of CD8α-expressing cells in said organism or subject within the normal range for said organism or subject.

[0054] In some embodiments, the terms "deplete," "depleting," and "depletion" in reference to cells expressing CD8α as described herein refer to a reduction or decrease in the number of CD8α-expressing cells in a sample, organism, or subject to an amount below the detectable limit.

[0055] As indicated above, the depletion of CD8α-expressing cells induced by an isolated anti-CD8α antibody or antigen-binding fragment thereof as described herein can be complete depletion. "Complete depletion" means that after depletion induced by the isolated anti-CD8α antibody or antigen-binding fragment thereof, CD8α-expressing cells are reduced by at least about 90%, preferably at least about 95%, 96%, 97%, 98%, or 99%, particularly relative to the initial number of CD8α-expressing cells in a sample, organism, or subject, preferably for at least about 1, 2, 3, 4, 5, 6, or 7 days.

[0056] The depletion of CD8α-expressing cells induced by an isolated anti-CD8α antibody or antigen-binding fragment thereof as described herein can be temporary depletion. By "temporary depletion" is meant that the depletion of CD8α-expressing cells induced by an isolated anti-CD8α antibody or antigen-binding fragment thereof is followed by repopulation of CD8α-expressing cells, preferably starting about 2, 3, 4, 5, 6, 7, or 8 weeks after the initial depletion.

[0057] As used herein, "repopulation" means an increase in the number of CD8α-expressing cells in a sample, organism, or subject following depletion of CD8α-expressing cells. The increase may be sufficient to reach at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, preferably at least about 55%, 60%, 65%, 70%, or 75%, more preferably at least about 80%, 85%, 90%, or 95% or more, particularly relative to the initial number of CD8α-expressing cells in the sample, organism, or subject. In some embodiments, the increase may be sufficient to reach at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%, preferably at least about 55%, 60%, 65%, 70%, or 75%, more preferably at least about 80%, 85%, 90%, or 95% or more, with respect to the number of CD8α-expressing cells in said organism or subject within the normal range for said organism or subject.

[0058] The isolated anti-CD8α antibody or antigen-binding fragment thereof may bind to CD8α and deplete CD8α-expressing cells as described herein by activating antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular phagocytosis (ADCP), and / or by inhibiting the proliferation of CD8α-expressing cells, and / or by inducing cell death (e.g., via apoptosis) of CD8α-expressing cells.

[0059] The isolated anti-CD8α antibody or antigen-binding fragment thereof may be capable of depleting CD8α-expressing cells as described herein via antibody-dependent cell-mediated cytotoxicity.

[0060] As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which antibodies bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, monocytes, and macrophages) enable these cytotoxic effector cells to specifically bind to antigen-bearing target cells (e.g., CD8-expressing cells described herein) and subsequently kill the target cells. To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337, can be performed.

[0061] The isolated anti-CD8α antibody or antigen-binding fragment thereof may be capable of depleting CD8α-expressing cells as described herein by complement-dependent cytotoxicity.

[0062] As used herein, 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 an antibody bound to its cognate antigen (e.g., CD8α). To assess complement activation, a CDC assay such as that described in Gazzano-Santoro et al., 1997. J Immunol Methods. 202(2):163-71 can be performed.

[0063] The isolated anti-CD8α antibody or antigen-binding fragment thereof may be capable of depleting CD8α-expressing cells as described herein through antibody-dependent phagocytosis.

[0064] As used herein, the term "antibody-dependent cellular phagocytosis" or "ADCP" refers to a form of phagocytosis in which antibodies bound to Fc receptors (FcRs) present on certain phagocytes (e.g., monocytes, macrophages, neutrophils, dendritic cells, and mast cells) enable these phagocytes to specifically bind to antigen-bearing target cells (e.g., CD8-expressing cells described herein) and subsequently phagocytize the target cells.

[0065] Methods for assessing whether a compound, such as an antibody or antigen-binding fragment thereof, can deplete CD8α-expressing cells are well known in the art and include, for example, the assays described in the Examples section below.

[0066] Assays for assessing whether an anti-CD8α antibody or antigen-binding fragment thereof depletes CD8α-expressing cells include flow cytometric detection and quantification of hCD8α-expressing cells in transgenic mice expressing an hCD8α transgene after injection of the anti-CD8α antibody or antigen-binding fragment thereof. In some embodiments, an anti-CD8α antibody or antigen-binding fragment thereof that can reduce the number of hCD8α-expressing cells, particularly compared to the baseline number of hCD8α-expressing cells (i.e., the number of hCD8α-expressing cells before injection of the anti-CD8α antibody or antigen-binding fragment thereof), is a CD8α-expressing cell-depleting antibody or antigen-binding fragment thereof. In some embodiments, the reduction in the number of hCD8α-expressing cells is assessed 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours after injection of the anti-CD8α antibody or antigen-binding fragment thereof. In some embodiments, the reduction in the number of hCD8α-expressing cells is assessed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after injection of the anti-CD8α antibody or antigen-binding fragment thereof. In some embodiments, the reduction in the number of hCD8α-expressing cells is assessed 1 week, 2 weeks, or 3 weeks after infusion of the anti-CD8α antibody or antigen-binding fragment thereof.

[0067] Assays for assessing whether an anti-CD8α antibody or antigen-binding fragment thereof depletes CD8α-expressing cells also include flow cytometric detection and quantification of CD8α-expressing cells in non-human primates, such as cynomolgus monkeys (Macaca fascicularis), after injection of the anti-CD8α antibody or antigen-binding fragment thereof. In some embodiments, an anti-CD8α antibody or antigen-binding fragment thereof that can reduce the number of CD8α-expressing cells compared to the baseline number of hCD8α-expressing cells (i.e., the number of hCD8α-expressing cells before injection of the anti-CD8α antibody or antigen-binding fragment thereof) is a CD8α-expressing cell-depleting antibody or antigen-binding fragment thereof. In some embodiments, the reduction in the number of hCD8α-expressing cells is assessed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours after injection of the anti-CD8α antibody or antigen-binding fragment thereof. In some embodiments, the reduction in the number of hCD8α-expressing cells is assessed 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, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 days after infusion of the anti-CD8α antibody or antigen-binding fragment thereof. In some embodiments, the reduction in the number of hCD8α-expressing cells is assessed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after infusion of the anti-CD8α antibody or antigen-binding fragment thereof.

[0068] In some embodiments, an isolated anti-CD8α antibody or antigen-binding fragment thereof as described herein can limit necrosis after myocardial infarction. In some embodiments, an isolated anti-CD8α antibody or antigen-binding fragment thereof as described herein can reduce infarct size after myocardial infarction. In some embodiments, an isolated anti-CD8α antibody or antigen-binding fragment thereof as described herein can improve cardiac function after myocardial infarction. In some embodiments, an isolated anti-CD8α antibody or antigen-binding fragment thereof as described herein can improve left ventricular ejection fraction after myocardial infarction.

[0069] Methods for assessing whether a compound, such as an antibody or antigen-binding fragment thereof, can limit necrosis, reduce infarct size, improve cardiac function, and / or improve left ventricular ejection fraction after myocardial infarction are well known in the art and include, for example, the assays described below in the Examples section.

[0070] An assay to evaluate whether an anti-CD8α antibody or its antigen-binding fragment can limit necrosis after myocardial infarction includes measuring the percentage of necrosis in sections of cardiac tissue from transgenic mice expressing the hCD8α transgene after injection of the anti-CD8α antibody or its antigen-binding fragment. In some embodiments, the percentage of necrosis is assessed 3 days after myocardial infarction. In some embodiments, cardiac tissue sections are stained with 2,3,5 triphenyltetrazolium chloride (TTC).

[0071] An assay to evaluate whether an anti-CD8α antibody or its antigen-binding fragment can reduce infarct size after myocardial infarction involves calculating infarct size by measuring the total tissue area and the area of ​​infarcted tissue in cardiac tissue sections from transgenic mice expressing the hCD8α transgene after injection of an anti-CD8α antibody or its antigen-binding fragment. In some embodiments, infarct size is assessed 3 days after myocardial infarction. In some embodiments, cardiac tissue sections are stained with 2,3,5 triphenyltetrazolium chloride (TTC).

[0072] Assays for evaluating whether an anti-CD8α antibody or its antigen-binding fragment can improve cardiac function and / or improve left ventricular ejection fraction after myocardial infarction include calculating the left ventricular end-diastolic volume (EDV) and end-systolic volume (ESV) in transgenic mice expressing a hCD8α transgene after injection of an anti-CD8α antibody or its antigen-binding fragment. In some embodiments, cardiac function is assessed 21 days after myocardial infarction. In some embodiments, the left ventricular end-diastolic volume (EDV) and end-systolic volume (ESV) are calculated 21 days after myocardial infarction. In some embodiments, cardiac function is recorded using an imaging system compatible with mouse cardiovascular imaging.

[0073] In some embodiments, "isolated," as in "isolated antibody or isolated antigen-binding fragment thereof," refers to an antibody or antigen-binding fragment thereof that is substantially free of other proteins or antibodies with different antigen specificities. Thus, an isolated anti-CD8α antibody or antigen-binding fragment thereof is substantially free of proteins or antibodies or antigen-binding fragments thereof that specifically bind to antigens other than CD8α, as described herein. However, an isolated antibody or antigen-binding fragment thereof that specifically binds to CD8α as described herein may have cross-reactivity to other related antigens, such as CD8α from other genera or species.

[0074] Furthermore, an isolated anti-CD8α antibody or antigen-binding fragment thereof as described herein may be substantially free of other cellular material and / or chemicals, particularly those that would interfere with any use (such as any therapeutic use) of the antibody or antigen-binding fragment thereof, including, but not limited to, enzymes, hormones and other proteinaceous or non-proteinaceous components.

[0075] In some embodiments, an isolated anti-CD8α antibody or antigen-binding fragment thereof as described herein is purified.

[0076] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is purified to a purity of greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% by weight of the antibody or antigen-binding fragment, preferably greater than about 95%, 96%, 97%, 98%, or 99% by weight of the antibody or antigen-binding fragment. In some embodiments, purity is determined by analytical size-exclusion chromatography (SEC), such as size-exclusion high-performance liquid chromatography (SE HPLC).

[0077] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof specifically binds to human CD8α and at least one ortholog of human CD8α. Thus, in some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof binds to human CD8α and at least one CD8α from another genus or species. In other words, in some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof exhibits cross-reactivity (cross-reacts). In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof binds to human CD8α and cynomolgus monkey CD8α (particularly cynomolgus monkey CD8α).

[0078] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof does not bind to mouse CD8α (particularly house mouse CD8α).

[0079] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof does not exhibit any off-target binding.

[0080] In some embodiments, the antigen-binding fragment of an anti-CD8α antibody is a molecule selected from the group comprising or consisting of a single-chain antibody or scFv, a dimeric single-chain antibody or di-scFv, Fv, Fab, Fab', Fab'-SH, and F(ab')2.

[0081] Antigen-binding fragments of antibodies can be obtained using standard methods. For example, Fab or F(ab')2 fragments can be produced by protease digestion of isolated antibodies according to conventional techniques. Alternatively, antigen-binding fragments of antibodies, such as Fab fragments, can be expressed as recombinant proteins.

[0082] In some embodiments, the isolated anti-CD8α antibody is a whole antibody. In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is selected from the group comprising or consisting of an Fc-silenced antibody or antigen-binding fragment thereof (i.e., an Fc-silenced antibody or antigen-binding fragment thereof), an antibody or antigen-binding fragment thereof with an engineered Fc, e.g., a defucosylated Fc (defucosylated antibody), and a bispecific antibody or antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment of the anti-CD8α antibody is a diabody, triabody, or tetrabody.

[0083] In some embodiments, the anti-CD8α antibody or antigen-binding fragment thereof is derived from the IgG class. Thus, the anti-CD8α antibody or antigen-binding fragment thereof can be derived from the IgG1, IgG2, IgG3, or IgG4 subclass.

[0084] In some embodiments, the anti-CD8α antibody or antigen-binding fragment thereof is derived from the IgG1 subclass, preferably from the human IgG1 subclass. Thus, in some embodiments, the anti-CD8α antibody or antigen-binding fragment thereof is an IgG1 antibody, preferably a human IgG1 antibody or a chimeric human IgG1 antibody.

[0085] In some embodiments, the anti-CD8α antibody or antigen-binding fragment thereof is derived from the IgG2 subclass, preferably from the human IgG2 subclass. Thus, in some embodiments, the anti-CD8α antibody or antigen-binding fragment thereof is an IgG2 antibody, preferably a human IgG2 antibody or a chimeric human IgG2 antibody. In particular, the IgG2 antibody may be an IgG2b antibody.

[0086] As used herein, a "chimeric antibody" refers to an antibody or antigen-binding fragment thereof that comprises a first amino acid sequence linked to a second amino acid sequence to which it is not naturally linked in nature. The amino acid sequences may normally reside in separate proteins that are combined in the chimeric (or fusion) protein, or may normally reside in the same protein, but are placed in a new arrangement in the chimeric (or fusion) protein. A chimeric protein can be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. The term "chimeric antibody" includes antibodies and antigen-binding fragments thereof, (a) the constant region or portion thereof is altered, substituted, or exchanged such that the variable region is linked to a constant region of a different or altered class, effector function, and / or species; or (b) the variable region or portion thereof is modified, substituted or exchanged with a variable region or portion thereof having a different or altered antigen specificity, or with the corresponding sequence from another species or another antibody class or subclass.

[0087] In the present invention, unless otherwise specified, the positions of the complementarity determining regions (CDRs) are determined using the Kabat nomenclature.

[0088] According to some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof as described herein comprises a heavy chain variable region (also referred to as heavy chain variable region or VH) comprising at least one, preferably at least two, and more preferably three, of the following CDRs: -V H -CDR1:NX1X2MN, in the formula, X1 is N or Y, X2 is D, A, or Y; -V H - CDR2: X3ISGSSX4YIX5YADFVKG (SEQ ID NO: 1), wherein X3 is D or S, X4 is S or R, X5 is D or G or Y, and / or -V H -CDR3:SSX6X7X8X9YX 10 X 11 X 12 X 13 MDV (SEQ ID NO: 2), wherein: X6 is N or does not contain an amino acid; X7 is Y or does not contain an amino acid; X8 is Y or G or does not contain an amino acid; X9 is D or S or does not contain an amino acid; X 10 is S, N or F, X 11 is A or G, X 12 is S or D or N, X 13 is A or G; The heavy chain variable region comprises:

[0089] V having an amino acid sequence corresponding to NX1X2MN as described above HExamples of CDR1 include, but are not limited to, NNAMN (SEQ ID NO: 6), NYDMN (SEQ ID NO: 12), and NYYMN (SEQ ID NO: 18). Thus, in some embodiments, a V having the amino acid sequence set forth in the sequence NX1X2MN is used. H - CDR1 comprises or is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 12, and SEQ ID NO: 18.

[0090] V having the amino acid sequence shown in SEQ ID NO: 1 H Examples of CDR2 include, but are not limited to, DISGSSRYIGYADFVKG (SEQ ID NO: 7), DISGSSSYIDYADFVKG (SEQ ID NO: 13), and SISGSSRYIYYADFVKG (SEQ ID NO: 19). Thus, in some embodiments, a V having the amino acid sequence set forth in SEQ ID NO: 1 is H - CDR2 comprises or is selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 13 and SEQ ID NO: 19.

[0091] V having the amino acid sequence shown in SEQ ID NO:2 as described above H Examples of CDR3 include, but are not limited to, SSNYYDYNADAMDV (SEQ ID NO: 8), SSYYSGSGMDV (SEQ ID NO: 14), and SSGSYFGNAMDV (SEQ ID NO: 20). Thus, in some embodiments, a V having the amino acid sequence set forth in SEQ ID NO: 2 is H - CDR3 is selected from the group comprising or consisting of SEQ ID NO:8, SEQ ID NO:14 and SEQ ID NO:20.

[0092] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a VH comprising the following CDRs, preferably at least one (e.g., 1, 2, or 3) of the following three CDRs: - V selected from the group comprising or consisting of NNAMN (SEQ ID NO: 6), NYDMN (SEQ ID NO: 12) and NYYMN (SEQ ID NO: 18) H -CDR1; - V selected from the group comprising or consisting of DISGSSRYIGYADFVKG (SEQ ID NO: 7), DISGSSSYIDYADFVKG (SEQ ID NO: 13) and SISGSSRYIYYADFVKG (SEQ ID NO: 19) H CDR2; and / or - a V selected from the group comprising or consisting of SSNYYDYNADAMDV (SEQ ID NO: 8), SSYYSGSGMDV (SEQ ID NO: 14), and SSGSYFGNAMDV (SEQ ID NO: 20); H - CDR3, including

[0093] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a VH comprising the following CDRs, preferably at least one (e.g., 1, 2, or 3) of the following three CDRs: -V H - CDR1: NNAMN (SEQ ID NO: 6); -V H CDR2: DISGSSRYIGYADFVKG (SEQ ID NO: 7); and / or -V H - CDR3: SSNYYDYNADAMDV (SEQ ID NO: 8).

[0094] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a VH comprising the following CDRs, preferably at least one (e.g., 1, 2, or 3) of the following three CDRs: -V H - CDR1: NYDMN (SEQ ID NO: 12); -V H CDR2: DISGSSSYIDYADFVKG (SEQ ID NO: 13); and / or -V H - CDR3: SSYYSGSGMDV (SEQ ID NO: 14).

[0095] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a VH comprising the following CDRs, preferably at least one (e.g., 1, 2, or 3) of the following three CDRs: -VH - CDR1: NYYMN (SEQ ID NO: 18); -V H CDR2: SISGSSRYIYYADFVKG (SEQ ID NO: 19); and / or -V H - CDR3: SSGSYFGNAMDV (SEQ ID NO: 20).

[0096] In some embodiments, V H -CDR1, V H CDR2 and / or V H - CDR3 has an amino acid sequence as set forth in any one of SEQ ID NOS: 6-8, 12-14, and 18-20, as described above, in which one, two, or three or more amino acids are substituted with different amino acids. For example, in some embodiments, an isolated anti-CD8α antibody or antigen-binding fragment thereof has a V having an amino acid sequence as set forth in SEQ ID NOS: 6, 12, or 18. H In some embodiments, the V-CDR1 comprises one, two, or three or more amino acids substituted with different amino acids. H -CDR1, V H CDR2 and / or V H - CDR3 has an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with a corresponding amino acid sequence as set forth in any one of SEQ ID NOs: 6-8, 12-14, and 18-20. For example, in some embodiments, an isolated anti-CD8α antibody or antigen-binding fragment thereof has an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 12, or SEQ ID NO: 18. H -Contains CDR1.

[0097] According to some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a light chain variable region (also referred to as light chain variable region or VL) comprising at least one, preferably at least two, and more preferably the following three CDRs: -V L -CDR1:AGTSSDVGGX 14 X 15 X 16 VS (SEQ ID NO: 3), wherein: X 14 is G or N or Y, X 15 is S or Y, X 16 is S or Y, -V L -CDR2:X 17 DSX 18 RPS (SEQ ID NO: 4), wherein: X 17 is Q or S or Y, X 18 is Y or S, and / or -V L -CDR3:SSX 19 TX 20 YSTRV (SEQ ID NO: 5), wherein: X 19 is Y or D, X 20 is Y or Q or S.

[0098] V having the amino acid sequence shown in SEQ ID NO:3 as described above L Examples of CDR1 include, but are not limited to, AGTSSDVGGNSYVS (SEQ ID NO: 9), AGTSSDVGGGSSVS (SEQ ID NO: 15), and AGTSSDVGGYYSVS (SEQ ID NO: 21). Thus, in some embodiments, a V having the amino acid sequence set forth in SEQ ID NO: 3 is L - CDR1 is selected from the group comprising or consisting of SEQ ID NO: 9, SEQ ID NO: 15 and SEQ ID NO: 21.

[0099] V having the amino acid sequence shown in SEQ ID NO: 4 as described aboveL Examples of CDR2 include, but are not limited to, SDSSRPS (SEQ ID NO: 10), QDSYRPS (SEQ ID NO: 16), and YDSSRPS (SEQ ID NO: 22). Thus, in some embodiments, a V having the amino acid sequence set forth in SEQ ID NO: 4. L - CDR2 comprises or is selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 16 and SEQ ID NO: 22.

[0100] V having the amino acid sequence shown in SEQ ID NO:5 as described above L Examples of CDR3 include, but are not limited to, SSYTQYSTRV (SEQ ID NO: 11), SSYTYYSTRV (SEQ ID NO: 17), and SSDTSYSTRV (SEQ ID NO: 23). Thus, in some embodiments, a V having the amino acid sequence set forth in SEQ ID NO: 5 is L - CDR3 comprises or is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 17 and SEQ ID NO: 23.

[0101] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a VL comprising the following CDRs, preferably at least one (e.g., 1, 2, or 3) of the following three CDRs: - V selected from the group comprising or consisting of AGTSSDVGGNSYVS (SEQ ID NO: 9), AGTSSDVGGGSSVS (SEQ ID NO: 15) and AGTSSDVGGYYSVS (SEQ ID NO: 21) L -CDR1; - V selected from the group comprising or consisting of SDSSRPS (SEQ ID NO: 10), QDSYRPS (SEQ ID NO: 16), and YDSSRPS (SEQ ID NO: 22) L CDR2; and / or - V selected from the group comprising or consisting of SSYTQYSTRV (SEQ ID NO: 11), SSYTYYSTRV (SEQ ID NO: 17), and SSDTSYSTRV (SEQ ID NO: 23) L - CDR3, including

[0102] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a VL comprising the following CDRs, preferably at least one (e.g., 1, 2, or 3) of the following three CDRs: -V L - CDR1:AGTSSDVGGNSYVS (SEQ ID NO: 9); -V L CDR2: SDSSRPS (SEQ ID NO: 10); and / or -V L - CDR3: SSYTQYSTRV (SEQ ID NO: 11).

[0103] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a VL comprising the following CDRs, preferably at least one (e.g., 1, 2, or 3) of the following three CDRs: -V L - CDR1:AGTSSDVGGGSSVS (SEQ ID NO: 15); V L CDR2: QDSYRPS (SEQ ID NO: 16); and / or -V L - CDR3: SSYTYYSTRV (SEQ ID NO: 17).

[0104] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a VL comprising the following CDRs, preferably at least one (e.g., 1, 2, or 3) of the following three CDRs: -V L - CDR1:AGTSSDVGGYYSVS (SEQ ID NO: 21); V L CDR2: YDSSRPS (SEQ ID NO: 22); and / or -V L - CDR3: SSDTSYSTRV (SEQ ID NO: 23).

[0105] In some embodiments, V L -CDR1, V L CDR2 and / or V L- CDR3 has the amino acid sequence set forth in any one of SEQ ID NOS: 9-11, 15-17, and 21-23, in which one, two, three, or more amino acids have been substituted with different amino acids. For example, in some embodiments, an isolated anti-CD8α antibody or antigen-binding fragment thereof has a V having the amino acid sequence set forth in SEQ ID NOS: 9, 15, or 21. L -CDR1, wherein one, two, three, or more amino acids are substituted with different amino acids. L -CDR1, V L CDR2 and / or V L - CDR3 has an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the corresponding amino acid sequence set forth in any one of SEQ ID NOs: 9-11, 15-17, and 21-23. For example, in some embodiments, an isolated anti-CD8α antibody or antigen-binding fragment thereof has an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the amino acid sequence set forth in SEQ ID NO: 9, SEQ ID NO: 15, or SEQ ID NO: 21. L -Contains CDR1.

[0106] In some embodiments, the isolated anti-CD8α antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) comprising at least one, preferably at least two, and more preferably three, of the following CDRs: -V H -CDR1:NX1X2MN, in the formula, X1 is N or Y, X2 is D, A, or Y; -V H - CDR2: X3ISGSSX4YIX5YADFVKG (SEQ ID NO: 1), wherein X3 is D or S, X4 is S or R, X5 is D or G or Y, and / or -V H -CDR3:SSX6X7X8X9YX 10 X 11 X 12 X 13 MDV (SEQ ID NO: 2), wherein: X6 is N or does not contain an amino acid; X7 is Y or does not contain an amino acid; X8 is Y or G or does not contain an amino acid; X9 is D or S or does not contain an amino acid; X 10 is S, N or F, X 11 is A or G, X 12 is S or D or N, X 13 is A or G; and A light chain variable region (VL) comprising at least one, preferably at least two, and more preferably three, of the following CDRs: -V L -CDR1:AGTSSDVGGX 14 X 15 X 16 VS (SEQ ID NO: 3), wherein: X 14 is G or N or Y, X 15 is S or Y, X 16 is S or Y, -V L -CDR2:X 17 DSX 18 RPS (SEQ ID NO: 4), wherein: X 17 is Q or S or Y, X 18 is Y or S, -V L -CDR3:SSX 19 TX 20 YSTRV (SEQ ID NO: 5), wherein: X 19 is Y or D, X 20is Y or Q or S, or an antigen-binding fragment thereof.

[0107] In some embodiments, the isolated anti-CD8α antibody, or antigen-binding fragment thereof, comprises a VH comprising the following three CDRs: -V H -CDR1:NX1X2MN, in the formula, X1 is N or Y, X2 is D, A, or Y; -V H - CDR2: X3ISGSSX4YIX5YADFVKG (SEQ ID NO: 1), wherein X3 is D or S, X4 is S or R, X5 is D or G or Y, and -V H -CDR3:SSX6X7X8X9YX 10 X 11 X 12 X 13 MDV (SEQ ID NO: 2), wherein: X6 is N or does not contain an amino acid; X7 is Y or does not contain an amino acid; X8 is Y or G or does not contain an amino acid; X9 is D or S or does not contain an amino acid; X 10 is S, N or F, X 11 is A or G, X 12 is S or D or N, X 13 is A or G; and VL containing the following three CDRs: -V L -CDR1:AGTSSDVGGX 14 X 15 X 16 VS (SEQ ID NO: 3), wherein: X 14 is G or N or Y, X 15is S or Y, X 16 is S or Y, -V L -CDR2:X 17 DSX 18 RPS (SEQ ID NO: 4), wherein: X 17 is Q or S or Y, X 18 is Y or S, -V L -CDR3:SSX 19 TX 20 YSTRV (SEQ ID NO: 5), wherein: X 19 is Y or D, X 20 is Y or Q or S, or an antigen-binding fragment thereof.

[0108] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is VH containing the following three CDRs: - V selected from the group comprising or consisting of NNAMN (SEQ ID NO: 6), NYDMN (SEQ ID NO: 12) and NYYMN (SEQ ID NO: 18) H -CDR1; - V selected from the group comprising or consisting of DISGSSRYIGYADFVKG (SEQ ID NO: 7), DISGSSSYIDYADFVKG (SEQ ID NO: 13) and SISGSSRYIYYADFVKG (SEQ ID NO: 19) H CDR2; and - a V selected from the group comprising or consisting of SSNYYDYNADAMDV (SEQ ID NO: 8), SSYYSGSGMDV (SEQ ID NO: 14), and SSGSYFGNAMDV (SEQ ID NO: 20); H CDR3; and VL containing the following three CDRs: - a V selected from the group comprising or consisting of: AGTSSDVGGNSYVS (SEQ ID NO: 9), AGTSSDVGGGSSVS (SEQ ID NO: 15), and AGTSSDVGGYYSVS (SEQ ID NO: 21);L -CDR1; - V selected from the group comprising or consisting of SDSSRPS (SEQ ID NO: 10), QDSYRPS (SEQ ID NO: 16), and YDSSRPS (SEQ ID NO: 22) L CDR2; and - V selected from the group comprising or consisting of SSYTQYSTRV (SEQ ID NO: 11), SSYTYYSTRV (SEQ ID NO: 17), and SSDTSYSTRV (SEQ ID NO: 23) L - CDR3, including

[0109] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises the following six CDRs: -V H CDR1: NNAMN (SEQ ID NO: 6), V H - CDR2: DISGSSRYIGYADFVKG (SEQ ID NO: 7), V H - CDR3: SSNYYDYNADAMDV (SEQ ID NO: 8), V L - CDR1:AGTSSDVGGNSYVS (SEQ ID NO: 9), V L CDR2: SDSSRPS (SEQ ID NO: 10), and V L CDR3: SSYTQYSTRV (SEQ ID NO: 11); or -V H CDR1: NYDMN (SEQ ID NO: 12), V H - CDR2: DISGSSSYIDYADFVKG (SEQ ID NO: 13), V H - CDR3: SSYYSGSGMDV (SEQ ID NO: 14), V L - CDR1:AGTSSDVGGGSSVS (SEQ ID NO: 15), V L CDR2: QDSYRPS (SEQ ID NO: 16), and V L CDR3: SSYTYYSTRV (SEQ ID NO: 17); or -V H CDR1: NYYMN (SEQ ID NO: 18), V H - CDR2: SISGSSRYIYYADFVKG (SEQ ID NO: 19), V H - CDR3: SSGSYFGNAMDV (SEQ ID NO: 20), V L- CDR1:AGTSSDVGGYYSVS (SEQ ID NO: 21), V L CDR2: YDSSRPS (SEQ ID NO: 22), and V L - CDR3: comprises SSDTSYSTRV (SEQ ID NO: 23).

[0110] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is VH containing the following three CDRs: -V H - CDR1: NNAMN (SEQ ID NO: 6), -V H CDR2: DISGSSRYIGYADFVKG (SEQ ID NO: 7), and -V H CDR3: SSNYYDYNADAMDV (SEQ ID NO: 8); and VL containing the following three CDRs: -V L - CDR1:AGTSSDVGGNSYVS (SEQ ID NO: 9), -V L CDR2: SDSSRPS (SEQ ID NO: 10), and -V L - CDR3: SSYTQYSTRV (SEQ ID NO: 11).

[0111] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a CDR having the amino acid sequence set forth in SEQ ID NOs: 6-11, as described above, in which one, two, three, or more amino acids have been replaced by different amino acids. In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises the above CDR having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the corresponding amino acid sequence set forth in SEQ ID NOs: 6-11.

[0112] V as shown in SEQ ID NOs: 6, 7 and 8, respectively H -CDR1, V H -CDR2 and V H- VH comprising CDR3 and V as shown in SEQ ID NOs: 9, 10 and 11, respectively L -CDR1, V L -CDR2 and V L An example of an anti-CD8α antibody comprising a VL comprising -CDR3 is hMP08-R3-F08.

[0113] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is VH containing the following three CDRs: -V H - CDR1: NYDMN (SEQ ID NO: 12), -V H CDR2: DISGSSSYIDYADFVKG (SEQ ID NO: 13), and -V H CDR3: SSYYSGSGMDV (SEQ ID NO: 14); and VL containing the following three CDRs: -V L - CDR1:AGTSSDVGGGSSVS (SEQ ID NO: 15), -V L CDR2: QDSYRPS (SEQ ID NO: 16), and -V L - CDR3: SSYTYYSTRV (SEQ ID NO: 17).

[0114] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a CDR having the amino acid sequence set forth in SEQ ID NOs: 12-17, as described above, in which one, two, three, or more amino acids have been substituted with different amino acids. In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises the above CDR having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the corresponding amino acid sequence set forth in SEQ ID NOs: 12-17.

[0115] V as shown in SEQ ID NOs: 12, 13 and 14, respectively H -CDR1, V H-CDR2 and V H - VH comprising CDR3 and V as shown in SEQ ID NOs: 15, 16 and 17, respectively L -CDR1, V L -CDR2 and V L An example of an anti-CD8α antibody comprising a VL comprising -CDR3 is hMP08-R3-C11.

[0116] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is VH containing the following three CDRs: -V H - CDR1: NYYMN (SEQ ID NO: 18), -V H CDR2: SISGSSRYIYYADFVKG (SEQ ID NO: 19), and -V H CDR3: SSGSYFGNAMDV (SEQ ID NO: 20); and VL containing the following three CDRs: -V L - CDR1:AGTSSDVGGYYSVS (SEQ ID NO: 21), -V L CDR2: YDSSRPS (SEQ ID NO: 22), and -V L - CDR3: SSDTSYSTRV (SEQ ID NO: 23).

[0117] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a CDR having the amino acid sequence set forth in SEQ ID NOs: 18-23, as described above, in which one, two, three, or more amino acids have been substituted with different amino acids. In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises the above-described CDR having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the corresponding amino acid sequence set forth in SEQ ID NOs: 18-23.

[0118] V as shown in SEQ ID NOs: 18, 19 and 20, respectivelyH -CDR1, V H -CDR2 and V H - VH comprising CDR3 and V as shown in SEQ ID NOs: 21, 22 and 23, respectively L -CDR1, V L -CDR2 and V L An example of an anti-CD8α antibody comprising a VL comprising -CDR3 is hMP09-R3-D03.

[0119] According to some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a variable region of a heavy chain (VH) comprising, or consisting of, a sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28.

[0120] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a VH comprising, or consisting of, a sequence selected from the group comprising or consisting of SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:28, with 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 or more amino acids substituted with different amino acids. In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a VH comprising, or consisting of, a sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:28, with 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 or more amino acids substituted with a different amino acid, wherein the amino acid substitutions are within the three CDRs ( VH -CDR1, VH CDR2, andVH -CDR3).

[0121] In some embodiments, the isolated anti-CD8α antibody, or antigen-binding fragment thereof, comprises a VH comprising or consisting of an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28. In some embodiments, the isolated anti-CD8α antibody, or antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence of framework regions (i.e., non-CDR regions) that share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the amino acid sequence of the framework regions (i.e., non-CDR regions) of ... VH -CDR1, VH -CDR2 and VH -CDR3), and a VH comprising an amino acid sequence of a framework region (i.e., a non-CDR region) that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the amino acid sequence of the framework region (i.e., a non-CDR region) of SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28.

[0122] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a VH comprising or consisting of a sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO:26 and SEQ ID NO:28.

[0123] According to some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a variable region of a light chain (VL) comprising, or consisting of, a sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29.

[0124] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a VL comprising, or consisting of, a sequence selected from the group comprising or consisting of SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:29, with 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 or more amino acids substituted with a different amino acid. In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a VL comprising, or consisting of, a sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:29, with 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 or more amino acids substituted with a different amino acid, wherein the amino acid substitutions are within the three CDRs ( VL -CDR1, VL CDR2, and VL -CDR3).

[0125] In some embodiments, the isolated anti-CD8α antibody, or antigen-binding fragment thereof, comprises a VL comprising or consisting of an amino acid sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the amino acid sequence set forth in SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29. In some embodiments, the isolated anti-CD8α antibody, or antigen-binding fragment thereof, comprises a VL comprising an amino acid sequence of framework regions (i.e., non-CDR regions) sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the amino acid sequence of the framework regions (i.e., non-CDR regions) of ... VL -CDR1, VL -CDR2 and VL -CDR3), and a VL comprising an amino acid sequence of a framework region (i.e., a non-CDR region) that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the amino acid sequence of the framework region (i.e., a non-CDR region) of SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29.

[0126] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a VL comprising or consisting of a sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:27 and SEQ ID NO:29.

[0127] According to some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises: - a variable region of the heavy chain (VH) comprising or consisting of a sequence selected from the group comprising or consisting of SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28; and - a light chain variable region (VL) comprising or consisting of a sequence selected from the group comprising or consisting of SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29.

[0128] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is -The above three CDRs ( VH -CDR1, VH -CDR2 and VH - a VH comprising a VH1, VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH11, VH12, VH13, VH14, VH15, VH16, VH17, VH18, VH19, VH20, VH21, VH22, VH23, VH24, VH25, VH36, VH26, VH27, VH37, VH28, VH29, VH38, VH39, VH40, VH41, VH42, VH43, VH44, VH45, VH46, VH47, VH48, VH49, VH50, VH51, VH52, VH53, VH54, VH55, VH56, VH57, VH58, VH59, VH60, VH61, VH62, VH63, VH64, VH65, VH66, VH67, VH68, VH69, VH70, VH71, VH72, VH73, VH74, VH75, VH76, VH77, VH78, VH79, VH79, VH79, VH79, VH79, VH79, VH71, VH72, VH75, VH76, VH77, VH78, VH79 ... -The above three CDRs ( VL -CDR1, VL -CDR2 and VL -CDR3) and a VL comprising the amino acid sequences of the framework regions (i.e., non-CDR regions) of SEQ ID NO:25, SEQ ID NO:27 or SEQ ID NO:29, and the amino acid sequences of the framework regions (i.e., non-CDR regions) that share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity.

[0129] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is - a VH comprising or consisting of a sequence set forth in SEQ ID NO: 24 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO: 24; and - a VL comprising or consisting of a sequence selected from the group comprising or consisting of SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29.

[0130] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is -The above three CDRs ( VH -CDR1, VH -CDR2 and VH - a VH comprising a VH-CDR3) and an amino acid sequence of a framework region (i.e., a non-CDR region) that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the amino acid sequence of the framework region (i.e., a non-CDR region) of SEQ ID NO: 24; and -The above three CDRs ( VL -CDR1, VL -CDR2 and VL -CDR3) and a VL comprising the amino acid sequences of the framework regions (i.e., non-CDR regions) of SEQ ID NO:25, SEQ ID NO:27 or SEQ ID NO:29, and the amino acid sequences of the framework regions (i.e., non-CDR regions) that share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity.

[0131] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is - a VH comprising or consisting of a sequence set forth in SEQ ID NO: 26, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO: 26; and - a VL comprising or consisting of a sequence selected from the group comprising or consisting of SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29.

[0132] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is -The above three CDRs ( VH -CDR1, VH -CDR2 and VH - a VH comprising a VH-CDR3) and an amino acid sequence of a framework region (i.e., a non-CDR region) that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the amino acid sequence of the framework region (i.e., a non-CDR region) of SEQ ID NO: 26; and -The above three CDRs ( VL -CDR1, VL -CDR2 and VL -CDR3) and a VL comprising the amino acid sequences of the framework regions (i.e., non-CDR regions) of SEQ ID NO:25, SEQ ID NO:27 or SEQ ID NO:29, and the amino acid sequences of the framework regions (i.e., non-CDR regions) that share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity.

[0133] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is - a VH comprising or consisting of a sequence set forth in SEQ ID NO: 28 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO: 28; and - a VL comprising or consisting of a sequence selected from the group comprising or consisting of SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29.

[0134] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is -The above three CDRs ( VH -CDR1, VH -CDR2 and VH - a VH comprising a VH-CDR3) and an amino acid sequence of a framework region (i.e., a non-CDR region) that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the amino acid sequence of the framework region (i.e., a non-CDR region) of SEQ ID NO: 28; and -The above three CDRs ( VL -CDR1, VL -CDR2 and VL -CDR3) and a VL comprising the amino acid sequences of the framework regions (i.e., non-CDR regions) of SEQ ID NO:25, SEQ ID NO:27 or SEQ ID NO:29, and the amino acid sequences of the framework regions (i.e., non-CDR regions) that share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity.

[0135] According to some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises: - a VH comprising or consisting of SEQ ID NO: 24, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO: 24, and a VL comprising or consisting of SEQ ID NO: 25, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO: 25, or - a VH comprising or consisting of SEQ ID NO: 26 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO: 26, and a VL comprising or consisting of SEQ ID NO: 27 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO: 27; or - a VH comprising or consisting of SEQ ID NO: 28 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO: 28, and a VL comprising or consisting of SEQ ID NO: 29 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO: 29.

[0136] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is - a VH comprising or consisting of SEQ ID NO: 24 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO: 24, and - comprises a VL comprising or consisting of SEQ ID NO: 25 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO: 25.

[0137] An example of an anti-CD8α antibody comprising the VH shown in SEQ ID NO: 24 and the VL shown in SEQ ID NO: 25 is hMP08-R3-F08.

[0138] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is - a VH comprising or consisting of SEQ ID NO: 26 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO: 26, and - comprises a VL comprising or consisting of SEQ ID NO: 27 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO: 27.

[0139] An example of an anti-CD8α antibody comprising the VH shown in SEQ ID NO: 26 and the VL shown in SEQ ID NO: 27 is hMP08-R3-C11.

[0140] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof is - a VH comprising or consisting of SEQ ID NO: 28 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO: 28, and - comprises a VL comprising or consisting of SEQ ID NO:29 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO:29.

[0141] An example of an anti-CD8α antibody comprising the VH set forth in SEQ ID NO: 28 and the VL set forth in SEQ ID NO: 29 is hMP09-R3-D03.

[0142] Another object of the present invention is a fusion protein comprising an anti-CD8α antibody or antigen-binding fragment thereof as described herein.

[0143] Another object of the invention is a nucleic acid encoding an anti-CD8α antibody or antigen-binding fragment thereof as described herein. Another object of the invention is a nucleic acid encoding a fusion protein as described herein.

[0144] In some embodiments, the nucleic acid is an isolated nucleic acid. In some embodiments, "isolated nucleic acid" refers to a nucleic acid that is substantially separated from other nucleic acid sequences and proteins or complexes that naturally accompany the natural sequence, such as ribosomes and polymerases. This term encompasses nucleic acid sequences that have been removed from their natural environment, including recombinant or cloned nucleic acids, chemically synthesized analogs, and biologically synthesized analogs in heterologous systems. A substantially pure nucleic acid includes a nucleic acid in isolated form. This refers to the nucleic acid that was originally isolated, and does not exclude nucleic acids or sequences that are later added to the isolated nucleic acid by the hand of man.

[0145] In some embodiments, the isolated nucleic acid is purified.

[0146] In some embodiments, the isolated nucleic acid is purified to (i) greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% or more by weight of the nucleic acid, most preferably greater than about 96%, 97%, 98%, or 99% by weight, as determined by absorbance or fluorescence methods (e.g., by measuring the ratio of absorbance at 260 and 280 nm (A260 / 280)), or (ii) homogeneity as demonstrated by agarose gel electrophoresis and the use of an intercalating agent, e.g., ethidium bromide, SYBR Green, GelGreen.

[0147] In some embodiments, the nucleic acid encodes at least the heavy chain variable region (VH) and / or light chain variable region (VL) of an anti-CD8α antibody or antigen-binding fragment thereof as described herein. In some embodiments, the nucleic acid encodes at least the VH and VL of an anti-CD8α antibody or antigen-binding fragment thereof described herein on separate nucleic acid molecules or the same nucleic acid molecule. The nucleic acid may encode the variable and constant regions of an anti-CD8α antibody or antigen-binding fragment thereof as described herein. The nucleic acid may encode the heavy and light chains of an anti-CD8α antibody or antigen-binding fragment thereof on separate nucleic acid molecules or the same nucleic acid molecule.

[0148] According to some embodiments, the nucleic acid comprises or consists of a sequence encoding the VH or antigen-binding fragment thereof of an anti-CD8α antibody, as described above.

[0149] According to some embodiments, the nucleic acid comprises or consists of a sequence encoding the VL of an anti-CD8α antibody, or an antigen-binding fragment thereof, as described above.

[0150] According to some embodiments, the nucleic acid is a sequence encoding the VH of an anti-CD8α antibody or an antigen-binding fragment thereof, as described above; and / or - comprising or consisting of a sequence encoding the VL or an antigen-binding fragment thereof of an anti-CD8α antibody, as described above.

[0151] According to some embodiments, the nucleic acid is a sequence encoding a VH or antigen-binding fragment thereof of an anti-CD8α antibody, wherein said sequence is any one of SEQ ID NOs: 31, 33 or 35, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity with SEQ ID NO: 31, 33 or 35; and / or - comprising or consisting of a sequence encoding the VL or antigen-binding fragment of an anti-CD8α antibody, wherein said sequence is any one of SEQ ID NOs: 32, 34 or 36, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity with SEQ ID NO: 32, 34 or 36.

[0152] According to some embodiments, the nucleic acid is a sequence encoding a VH or antigen-binding fragment thereof of an anti-CD8α antibody, said sequence being SEQ ID NO: 31 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 31, and / or a sequence encoding a VL or antigen-binding fragment thereof of an anti-CD8α antibody, said sequence being SEQ ID NO: 32 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 32; or a sequence encoding a VH or antigen-binding fragment thereof of an anti-CD8α antibody, said sequence being SEQ ID NO: 33 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 33, and / or a sequence encoding a VL or antigen-binding fragment thereof of an anti-CD8α antibody, said sequence being SEQ ID NO: 34 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 34; or - comprising or consisting of a sequence encoding the VH or antigen-binding fragment of an anti-CD8α antibody, said sequence being SEQ ID NO: 35 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 35, and / or a sequence encoding the VL or antigen-binding fragment of an anti-CD8α antibody, said sequence being SEQ ID NO: 36 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 36.

[0153] Typically, the nucleic acids described herein are DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) molecules, which may be contained in any suitable vector.

[0154] Another object of the invention is a vector, such as an expression vector or a cloning vector, comprising a nucleic acid encoding an anti-CD8α antibody or antigen-binding fragment thereof, or a fusion protein as described herein.

[0155] The terms "vector," "cloning vector," and "expression vector" refer to a vehicle by which a nucleic acid sequence (e.g., a DNA molecule) encoding an anti-CD8α antibody, or antigen-binding fragment thereof, or a fusion protein can be introduced into a host cell to transform or transfect the host cell and promote expression (e.g., transcription and / or translation) of the introduced nucleic acid sequence.

[0156] In some embodiments, the vector, cloning vector or expression vector is a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

[0157] Examples of plasmids include replicative plasmids that contain replication origins, or integrative plasmids such as pUC, pcDNA and pBR.Examples of viral vectors include adenovirus, adeno-associated virus (AAV), retrovirus, and herpes virus vectors.Such viral vectors can be produced by techniques known in the art, for example, by transfecting packaging cells, or by transient transfection with helper plasmids or viruses.

[0158] Any expression vector for animal cells can be used as long as it can insert and express a nucleic acid encoding an anti-CD8α antibody or an antigen-binding fragment thereof, or a fusion protein described herein.

[0159] The vectors described herein may contain regulatory elements such as promoters, enhancers, and / or terminators to cause or direct expression of an anti-CD8α antibody or antigen-binding fragment thereof, or a fusion protein, for example, when introduced into a host cell or administered to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include, but are not limited to, the SV40 early promoter and enhancer, the Moloney murine leukemia virus LTR promoter and enhancer, and the immunoglobulin heavy chain promoter and enhancer.

[0160] In some embodiments, the vectors described herein comprise a nucleic acid sequence encoding the variable region of the heavy chain (VH) or antigen-binding fragment thereof of the anti-CD8α antibody described above operably linked to a regulatory element. In some embodiments, the vectors described above comprise a sequence encoding the variable region of the light chain (VL) or antigen-binding fragment thereof of the anti-CD8α antibody described above operably linked to a regulatory element.

[0161] In some embodiments, the vector is monocistronic. "Monocistronic" means that a single nucleic acid is expressed in a single vector. In some embodiments, the vector is polycistronic. "Polycistronic" means that at least two or more nucleic acid sequences are expressed in a single vector.

[0162] Another object of the present invention is a host cell comprising the vector described herein. In some embodiments, the host cell is an isolated host cell. The host cell can be used for the recombinant production of an anti-CD8α antibody, or antigen-binding fragment thereof, or a fusion protein described herein.

[0163] Host cells can be prokaryotic, or eukaryotic, such as yeast or mammalian cells.

[0164] It should be noted that with respect to animal and human cells, the term "host cell" generally refers to cells of a cultured cell line. Animals and humans into which a vector encoding an anti-CD8α antibody or antigen-binding fragment thereof, or fusion protein described herein has been introduced are expressly excluded from the definition of "host cell."

[0165] Another object of the present invention is a method for producing and optionally purifying an isolated anti-CD8α antibody or antigen-binding fragment thereof as described herein.

[0166] In some embodiments, the method comprises: - introducing a nucleic acid or vector described herein into a host cell in vitro or ex vivo (i.e., transforming or transfecting a host cell with a nucleic acid or vector described herein); - culturing in vitro or ex vivo host cells transformed or transfected with the nucleic acid or vector under conditions suitable for expression of the anti-CD8α antibody or antigen-binding fragment thereof; - optionally selecting a host cell that expresses and / or secretes an anti-CD8α antibody or antigen-binding fragment thereof; and and recovering the expressed and / or secreted anti-CD8α antibody, or the expressed and / or secreted antigen-binding fragment thereof.

[0167] Such methods are well known in the art and can be used for large-scale production of antibodies or antigen-binding fragments thereof, including monoclonal antibodies, intended for in vitro, ex vivo and / or in vivo use, e.g., therapeutic use.

[0168] In some embodiments, the recovered anti-CD8α antibody or recovered antigen-binding fragment thereof is further purified. Methods for purifying antibodies or antigen-binding fragments thereof are well known in the art and include, but are not limited to, the use of anti-CH1 antibodies, protein A-Sepharose, gel electrophoresis, and chromatography, particularly affinity chromatography.

[0169] Another object of the present invention is to provide at least one anti-CD8α antibody or antigen-binding fragment thereof as described herein, at least one fusion protein as described herein, at least one nucleic acid encoding an anti-CD8α antibody, or antigen-binding fragment thereof, or a fusion protein described herein; at least one vector comprising such a nucleic acid molecule, or - a composition comprising, consisting essentially of, or consisting of at least one host cell containing such a vector.

[0170] As used herein, "consisting essentially of" with respect to a composition means that at least one anti-CD8α antibody, or antigen-binding fragment thereof, fusion protein nucleic acid, vector, or host cell is the only active, therapeutic, or agent having biological activity within said composition.

[0171] Another object of the present invention is to provide at least one anti-CD8α antibody or antigen-binding fragment thereof as described herein, at least one fusion protein as described herein, at least one nucleic acid encoding an anti-CD8α antibody, or antigen-binding fragment thereof, or a fusion protein described herein; at least one vector comprising such a nucleic acid molecule, or at least one host cell containing such a vector, and at least one pharmaceutically acceptable excipient.

[0172] Pharmaceutically acceptable excipients that may be used in pharmaceutical 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.

[0173] Another object of the present invention is to provide at least one anti-CD8α antibody or antigen-binding fragment thereof as described herein, at least one fusion protein as described herein, at least one nucleic acid encoding an anti-CD8α antibody, or antigen-binding fragment thereof, or a fusion protein described herein; at least one vector comprising such a nucleic acid molecule, or - a drug comprising, consisting essentially of or consisting of at least one host cell containing such a vector.

[0174] In some embodiments, the compositions, pharmaceutical compositions, or medicaments described herein are formulated for administration to a subject.

[0175] The compositions, pharmaceutical compositions or drugs described herein can be administered or formulated for administration systemically, orally, parenterally, by injection, topically, by inhalation spray, rectally, nasally, or via an implanted reservoir. The term administration as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0176] In some embodiments, the compositions, pharmaceutical compositions, or drugs described herein are formulated for administration by injection, e.g., subcutaneous injection, or infusion, e.g., intravenous infusion, etc. Examples of forms suitable for injection include, but are not limited to, solutions, e.g., sterile aqueous solutions, gels, dispersions, emulsions, suspensions, solid forms suitable for use in preparing a solution or suspension by addition of a liquid prior to use, e.g., powders, liposomal forms, etc.

[0177] In some embodiments, the regimen or dosage used for administering a composition, pharmaceutical composition, or drug can be adapted as a function of various parameters, particularly the mode of administration used, the associated pathology, or the desired duration of treatment. For example, it is well within the skill of one of ordinary skill in the art to start with a dose lower than that required to achieve the desired therapeutic effect and gradually increase the dose of an anti-CD8α antibody, antigen-binding fragment thereof, or fusion protein described herein until the desired effect is achieved. Doses of the anti-CD8α antibody, antigen-binding fragment thereof, or fusion protein described herein can vary over a wide range, from 0.01 to 10,000 mg per day for an adult. A composition, pharmaceutical composition, or drug typically contains, for example, about 0.01 mg to about 10,000 mg, preferably about 0.1 mg to about 5,000 mg, of an anti-CD8α antibody, antigen-binding fragment thereof, or fusion protein described herein.

[0178] Another object of the present invention is an anti-CD8α antibody or antigen-binding fragment thereof as described herein for use as a medicament. Another object of the present invention is a fusion protein as described herein for use as a medicament.

[0179] Another object of the present invention is a nucleic acid encoding an anti-CD8α antibody or antigen-binding fragment thereof, or a fusion protein as described herein, or a vector comprising such a nucleic acid molecule, or a host cell comprising such a vector, for use as a medicament.

[0180] Another object of the present invention is an anti-CD8α antibody or antigen-binding fragment thereof, as described herein, for use in treating a CD8-associated disease.

[0181] As used herein, "CD8-associated disease" refers to a disease, disorder, or condition caused or enhanced by an increase in the proportion of cells expressing CD8, particularly cells expressing CD8α, in a subject and / or an increase in the expression level of CD8, particularly CD8α, in the cells of the subject.

[0182] Another object of the present invention is a fusion protein as described herein for use in the treatment of a CD8-associated disease.

[0183] Another object of the invention is a nucleic acid encoding an anti-CD8α antibody or antigen-binding fragment thereof, or a fusion protein as described herein, or a vector comprising such a nucleic acid molecule, or a host cell comprising such a vector, for use in treating a CD8-related disease.

[0184] In some embodiments, the CD8-associated disease is cardiovascular disease. In some embodiments, the cardiovascular disease is myocardial infarction or acute myocardial infarction.

[0185] Examples of cardiovascular diseases include, but are not limited to, myocardial infarction, acute myocardial infarction, cardiac remodeling after myocardial infarction, heart failure after myocardial infarction, stroke, coronary artery disease (i.e., atherosclerosis), acute coronary syndrome, myocarditis, and atrial fibrillation.

[0186] In some embodiments, the cardiovascular disease is selected from myocardial infarction, acute myocardial infarction, cardiac remodeling after myocardial infarction, heart failure after myocardial infarction, stroke, coronary artery disease, acute coronary syndrome, myocarditis, and atrial fibrillation.

[0187] Another object of the present invention is a method of treating a disease as described herein in a subject in need thereof, said method comprising administering to the subject: an anti-CD8α antibody or antigen-binding fragment thereof as described herein, a fusion protein as described herein, a nucleic acid as described herein encoding an anti-CD8α antibody, antigen-binding fragment thereof, or fusion protein; a vector as described herein comprising such a nucleic acid molecule, or - administering at least one of the host cells described herein containing such a vector.

[0188] As mentioned above, in some embodiments, the disease to be treated is a CD8-related disease. In some embodiments, the disease to be treated is a cardiovascular disease, particularly myocardial infarction or acute myocardial infarction. In some embodiments, the cardiovascular disease is selected from myocardial infarction, acute myocardial infarction, cardiac remodeling after myocardial infarction, heart failure after myocardial infarction, stroke, coronary artery disease, acute coronary syndrome, myocarditis and atrial fibrillation.

[0189] In some embodiments, the method comprises administering a therapeutically effective dose of an anti-CD8α antibody or antigen-binding fragment thereof as described herein, a fusion protein described herein, a nucleic acid described herein encoding an anti-CD8α antibody, antigen-binding fragment thereof, or fusion protein, a vector described herein comprising such a nucleic acid molecule, or a host cell described herein comprising such a vector.

[0190] A therapeutically effective dose may correspond to, for example, a dose ranging from 0.0001 mg / kg to about 100 mg / kg of body weight per day, preferably from 0.001 mg / kg to about 50 mg / kg of body weight per day. It will be understood that these doses are exemplary and that the optimal dosage can be adapted taking into account the affinity and tolerability of the anti-CD8α antibody, antigen-binding fragment, or fusion protein in the composition, pharmaceutical composition, or drug.

[0191] The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disease and severity of the disease being treated; the activity of the isolated anti-CD8α antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition, or drug used; the subject's age, weight, general health, sex, and diet; the time of administration, route of administration, and excretion rate of the specific anti-CD8α antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition, or drug used; the duration of treatment; drugs used in combination with or simultaneously with the specific isolated anti-CD8α antibody or antigen-binding fragment thereof, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition, or drug used; and similar factors well known in the medical arts. For example, it is well within the skill of the art to start administering a compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. The total dose required for each treatment may be given in multiple administrations or in a single administration.

[0192] Another object of the present invention is a pharmaceutical composition for treating or for use in treating a disease as described herein in a subject in need thereof, said pharmaceutical composition comprising: an anti-CD8α antibody or antigen-binding fragment thereof as described herein, a fusion protein as described herein, a nucleic acid as described herein encoding an anti-CD8α antibody, antigen-binding fragment thereof, or fusion protein; a vector as described herein comprising such a nucleic acid molecule, or a host cell as described herein comprising such a vector, and at least one pharmaceutically acceptable excipient.

[0193] Another object of the present invention is to provide a method for the manufacture of a medicament for the treatment of a disease as described herein in a subject in need thereof, comprising: an anti-CD8α antibody or antigen-binding fragment thereof as described herein, a fusion protein as described herein, a nucleic acid as described herein encoding an anti-CD8α antibody, antigen-binding fragment thereof, or fusion protein; a vector as described herein comprising such a nucleic acid molecule, or - using at least one of the host cells described herein containing such a vector.

[0194] As mentioned above, in some embodiments, the disease to be treated is a CD8-related disease. In some embodiments, the disease to be treated is a cardiovascular disease, particularly myocardial infarction or acute myocardial infarction. In some embodiments, the cardiovascular disease is selected from myocardial infarction, acute myocardial infarction, cardiac remodeling after myocardial infarction, heart failure after myocardial infarction, stroke, coronary artery disease, acute coronary syndrome, myocarditis and atrial fibrillation.

[0195] The present invention further relates to the use of an isolated anti-CD8α antibody, or antigen-binding fragment thereof, or fusion protein, as described herein, for depleting CD8α-expressing cells in a sample, and to an in vitro method for depleting CD8α-expressing cells in a sample, comprising contacting the sample with an isolated anti-CD8α antibody, or antigen-binding fragment thereof, or fusion protein, as described herein.

[0196] As used herein, "sample" refers to any biological material obtained from a subject by an appropriate method known to those skilled in the art. Samples can be collected in a clinically acceptable manner, for example, in a manner that preserves cells, nucleic acids (such as DNA and RNA), proteins, and / or extracellular vesicles. A "sample" can be body tissue and / or body fluid, preferably body fluid. Examples of body fluids include, but are not limited to, blood, plasma, serum, lymph, ascites, cyst fluid, urine, bile, nipple exudate, vomit, breast milk, tears, wound drainage, feces, vaginal secretions, synovial fluid, bronchoalveolar lavage fluid, sputum, amniotic fluid, ascites, cerebrospinal fluid, pleural effusion, pericardial fluid, semen, saliva, sweat, and alveolar macrophages, tissue lysates, biopsies, and extracts prepared from diseased tissues.

[0197] In some embodiments, the sample has been previously collected or retrieved from the subject, i.e., the methods described herein do not include the active step of retrieving a sample from the subject. Thus, according to some embodiments, the methods described herein are non-invasive methods, i.e., the methods of the present invention are in vitro methods.

[0198] The present invention further relates to the use of an isolated anti-CD8α antibody or antigen-binding fragment thereof, or fusion protein described herein for detecting or quantifying CD8α in a sample, cell, tissue, organ, organism, or subject; and to a method for detecting and / or quantifying CD8α in a sample, cell, tissue, organ, organism, or subject, comprising contacting the sample, cell, tissue, or organ, or administering to the organism or subject, an isolated anti-CD8α antibody or antigen-binding fragment thereof, or fusion protein described herein.

[0199] The uses and methods for detecting and / or quantifying CD8α can be in vitro or in vivo.

[0200] In some embodiments, the isolated anti-CD8α antibody or antigen-binding fragment thereof described herein is labeled for detection or diagnostic purposes.

[0201] Assays suitable for detecting or quantifying CD8α using the isolated anti-CD8α antibodies or antigen-binding fragments or fusion proteins thereof described herein are well known in the art and include ELISA, sandwich ELISA, RIA, FACS, tissue immunohistochemistry, Western blot and immunoprecipitation. Sequence Listing [Table 1] JPEG2025538489000002.jpg212159JPEG2025538489000003.jpg217159JPEG2025538489000004.jpg217159 JPEG2025538489000005.jpg217159JPEG2025538489000006.jpg218159JPEG2025538489000007.jpg206159 [Brief explanation of the drawings]

[0202] [Figure 1] Figures 1A-C are a composite of graphs showing dose-response ELISAs of three antibody clones (i.e., MP08-R3-F08, MP08-R3-C11, and MP09-R3-D03) in human IgG1 format for immobilized recombinant human CD8α, mouse CD8α, and human CD4. Twelve different concentrations were tested (75, 25, 8.3, 2.8, 0.93, 0.31, 0.103, 0.034, 0.011, 0.0038, 0.0013, and 0.00042 μg / mL). All antibodies had an average molecular weight of approximately 150 kDa. An unrelated human IgG1 antibody designated 13R4 was included as a negative control. Figure 1A is human CD8α. Figure 1B is mouse CD8α, and Figure 1C is human CD4.

[0203] [Figure 2] Figures 2A-C are a composite of graphs showing dose-response ELISAs of three antibody clones (i.e., MP08-R3-F08, MP08-R3-C11, and MP09-R3-D03) in rat IgG2b format for immobilized recombinant human CD8α, mouse CD8α, and human CD4. Twelve different concentrations were tested (75, 25, 8.3, 2.8, 0.93, 0.31, 0.103, 0.034, 0.011, 0.0038, 0.0013, and 0.00042 μg / mL). All antibodies had an average molecular weight of approximately 150 kDa. Figure 2A is for human CD8α. Figure 2B is for mouse CD8α, and Figure 2C is for human CD4.

[0204] [Figure 3] Figures 3A-E are a combination of flow cytometry plots showing the binding of three antibody clones in the human IgG1 format (i.e., MP08-R3-F08, MP08-R3-C11, and MP09-R3-D03) to CD8+ cells (HPB-ALL cells). An irrelevant human IgG1 13R4 antibody was used as a negative control, and a commercially available mouse anti-human CD8α antibody was used as a positive control. Antibodies were used at a concentration of 3.3 μg / mL. Light gray represents cells alone (background signal), while darker gray represents cells with one of the indicated antibodies (binding signal). Figure 3A represents the MP08-R3-F08 human IgG1 clone. Figure 3B represents the MP08-R3-C11 human IgG1 clone. Figure 3C represents the MP09-R3-D03 human IgG1 clone. Figure 3D represents the irrelevant human IgG1 13R4 antibody control. FIG. 3E depicts a commercially available mouse anti-human CD8α antibody.

[0205] [Figure 4] Figures 4A-E are a combination of flow cytometry plots showing the binding of three antibody clones (i.e., MP08-R3-F08, MP08-R3-C11, and MP09-R3-D03) in a human IgG1 format onto CD8- cells (H1299 cells). An irrelevant human IgG1 13R4 antibody was used as a negative control, and a commercially available mouse anti-human CD8α antibody was used as a positive control. Antibodies were used at a concentration of 3.3 μg / mL. Light gray represents cells alone (background signal), while darker gray represents cells with one of the indicated antibodies (binding signal). Figure 4A represents the MP08-R3-F08 human IgG1 clone. Figure 4B represents the MP08-R3-C11 human IgG1 clone. Figure 4C represents the MP09-R3-D03 human IgG1 clone. Figure 4D represents the irrelevant human IgG1 13R4 antibody control. FIG. 4E depicts a commercially available mouse anti-human CD8α antibody.

[0206] [Figure 5]Figures 5A-E are a combination of flow cytometry plots showing the binding of three antibody clones in the human IgG1 format (i.e., MP08-R3-F08, MP08-R3-C11, and MP09-R3-D03) to human primary CD8+ T cells. An irrelevant human IgG1 13R4 antibody was used as a negative control, and a commercially available mouse anti-human CD8α antibody was used as a positive control. Antibodies were used at a concentration of 1.1 μg / mL. Light gray represents cells alone (background signal), while darker gray represents cells with one of the indicated antibodies (binding signal). Figure 5A represents the MP08-R3-F08 human IgG1 clone. Figure 5B represents the MP08-R3-C11 human IgG1 clone. Figure 5C represents the MP09-R3-D03 human IgG1 clone. Figure 5D represents the irrelevant human IgG1 13R4 antibody control. FIG. 5E depicts a commercially available mouse anti-human CD8α antibody.

[0207] [Figure 6] Figure 6A-B is a combination of flow cytometry plots showing the percentage of cells expressing the human CD8α transgene from CD45+ cells in control WT mice (Figure 6A) and transgenic mice expressing the human CD8α transgene (Figure 6B).

[0208] [Figure 7] Figures 7A-C are a combination of graphs showing the depletion and subsequent repopulation of human CD8α-expressing cells in mice after injection with antibody MP08-R3-F08 (Figure 7A), MP08-R3-C11 (Figure 7B), or MP09-R3-D03 (Figure 7C) at doses of 50 μg, 100 μg, or 200 μg, as indicated.

[0209] [Figure 8]Figures 8A-C are a combination of histograms showing the depletion and subsequent repopulation of human CD8α-expressing cells in mice after injection with antibodies MP08-R3-F08, MP08-R3-C11, or MP09-R3-D03 at doses of 200 μg (Figure 8A), 100 μg (Figure 8B), or 50 μg (Figure 8C), as indicated.

[0210] [Figure 9] Figures 9A-E are flow cytometry plots showing the percentage of murine CD8α+ cells in control C56Bl6 mice after injection with a control IgG1 isotype antibody (Figure 9A), MP08-R3-C11 (Figure 9B), MP08-R3-F08 (Figure 9C), or MP09-R3-D03 (Figure 9D), paired with a histogram showing quantification 1 hour and 7 days after injection (Figure 9E).

[0211] [Figure 10] Figures 10A-C are composites of fluorescent images showing confirmation screening of anti-CD8α antibody clone MP08-R3-F08 (Figures 10A and 10C) and control antibody rituximab (Figures 10B and 10C) in fixed (Figures 10A-B) and unfixed (Figure 10C) cells. Each spot corresponds to a cell transfected with an expression vector expressing the protein, as indicated.

[0212] [Figure 11] Figures 11A-B are a combination of histograms showing the depletion of CD8α-expressing cells in cynomolgus monkeys after injection of anti-CD8α antibodies. Figure 10A shows the percentage of CD8+ cells within CD45+ cells in cynomolgus monkeys administered 20 mg / kg of body weight of anti-CD8α antibody clone MP08-R3-F08, MP08-R3-C11, or MP09-R3-D03, as indicated. Figure 10B shows the percentage of CD8+ cells within CD45+ cells in cynomolgus monkeys administered 7.5 mg / kg or 20 mg / kg of body weight of anti-CD8α antibody clone MP08-R3-F08, as indicated.

[0213] [Figure 12] Figures 12A-C show flow cytometry plots showing the percentage of CD45+ hCD8α+ cells in the blood of control transgenic mice expressing the human CD8α transgene treated with saline (Figure 12A) and transgenic mice expressing the human CD8α transgene treated with the anti-CD8α antibody clone MP08-R3-F08 (Figure 12B) 3 days after injection, combined with a histogram showing quantification (Figure 12C). Males are represented by filled circles and females by filled inverted triangles. Control: n=9 vs. MP08-R3-F08: n=7. (***p<0.001).

[0214] [Figure 13] Figures 13A-C are flow cytometry plots showing the percentage of CD45+ hCD8α+ cells in the spleens of control transgenic mice expressing the human CD8α transgene treated with saline (Figure 12A) and transgenic mice expressing the human CD8α transgene treated with the anti-CD8α antibody clone MP08-R3-F08 (Figure 12B) 3 days after injection, combined with a histogram showing quantification (Figure 12C). Males are represented by filled circles and females by filled inverted triangles. Control = n=9 vs. MP08-R3-F08 = n=7. (***p<0.001).

[0215] [Figure 14] Histogram showing the percentage of necrosis measured by staining with 2,3,5 triphenyltetrazolium chloride (TTC) in cardiac sections from control transgenic mice expressing the human CD8α transgene that received saline or the anti-CD8α antibody clone MP08-R3-F08 3 days after induction of myocardial infarction. Males are represented by filled circles and females by filled inverted triangles. Control, n=9 vs. MP08-R3-F08, n=7. (**p<0.01).

[0216] Example The present invention is further illustrated by the following examples. [Table 2] Table 1: Example antibody sequences

[0217] Example 1: Antibody production material and method Three antibody clones (i.e., MP08-R3-F08, MP08-R3-C11, and MP09-R3-D03) were produced and purified in both human IgG1 lambda and rat IgG2b kappa formats. For IgG expression, VH and VL chain fragments were cloned into an IgG expression system encoding the heavy and light chains in separate pcDNA3.4 plasmids. Constructs were sequenced to verify correct insertion of the VH and VL sequences.

[0218] The plasmids were transiently transfected into ExpiCHO cells, and the IgG-containing supernatant was filtered (0.22 μm) and purified on a Protein A column. Antibodies that did not reach 98% purity were subjected to a second purification step by preparative SEC (size exclusion chromatography). A horseshoe crab hemocyte lysate test was performed to measure endotoxin levels in the purified IgG.

[0219] result An overview of antibody production, purification, and quality control is shown in Table 2.

[0220] All three antibody clones, both in human IgG1 format, showed high expression yields and were recovered in large quantities after Protein A purification (up to 400 mg / L culture). In rat IgG2b format, clone MP08-R3-F08 expressed at similarly high yields, while clones MP08-R3-C11 and MP09-R3-D3 expressed at moderate yields, with 6.3 and 7.5 mg of purified IgG recovered from 100 mL of culture, respectively.

[0221] The six antibodies were analyzed by SDS-PAGE: 1.3 μg of each antibody clone was loaded per well under reducing and non-reducing conditions. All six antibodies showed a standard migration profile with a 50 kDa band and another 25 kDa band corresponding to the heavy and light chains, respectively (data not shown). [Table 3] Table 2: Summary of production, purification, and quality control of three antibody clones in both human IgG1 and rat IgG2b formats.

[0222] Example 2: Antibody binding to recombinant CD8α protein material and method Nunc Maxisorp 96-well plates (ThermoFisher, #442404) were coated with 5 μg / mL streptavidin at 100 μL / well and incubated at room temperature for 1 hour. Biotinylated human CD8α (Fisher Scientific, #17196181) and mouse CD8α (Sino biological, #50389-M08H-B) were added at 0.5 μg / mL in 100 μL / well and incubated overnight at 4°C. Human CD4 (Sino biological, #10400-H08H) was passively coated at 1 μg / mL in 100 μL / well and incubated overnight at 4°C. Plates were then blocked with 200 μL of PBS containing 4% nonfat milk for 1 hour at room temperature.

[0223] Three antibody clones (both human IgG1 lambda and rat IgG2b kappa formats) were diluted in milk-PBS and tested at different concentrations (75, 25, 8.3, 2.8, 0.93, 0.31, 0.103, 0.034, 0.011, 0.0038, 0.0013, and 0.00042 μg / mL). 100 μL / well was incubated at room temperature for 1 hour.

[0224] Human IgG was detected with anti-hFab-HRP (Sigma, #A0293, 1:4000 dilution), and rat IgG was detected with anti-rat-HRP (Sigma, #A9037, 1:10000 dilution) at 100 μL / well and incubated for 1 hour at room temperature.

[0225] TMB ((3,3',5,5' tetramethylbenzidine) Thermo Scientific, #12617087) was added for a 3 minute incubation. The reaction was stopped with H2SO4 and the absorbance at 450 nm was read on a microplate reader (Pherastar FS, BMG Labtech).

[0226] result All three tested antibody clones were able to bind to human CD8α, with EC50s ranging from 0.28 nM to 2.0 nM for the human IgG1-format clones (Figure 1A and Table 3) and from 0.20 nM to 0.99 nM for the rat IgG2b-format clones (Figure 2A and Table 3). No cross-reactivity was observed with mouse CD8α (Figure 1B for the human IgG1 clones and Figure 2B for the rat IgG2b clones).

[0227] Binding specificity tested with human CD4 showed that none of the three antibody clones bound to this protein, neither in the human IgG1 format nor in the rat IgG2b format (Fig. 1C for the human IgG1 clone and Fig. 2C for the rat IgG2b clone).

[0228] These results demonstrate that the three antibody clones (i.e., MP08-R3-F08, MP08-R3-C11, and MP09-R3-D03) have strong binding and good specificity for human CD8α. Furthermore, none of the three antibody clones exhibits binding to human CD4 or cross-reactivity with mouse CD8α. [Table 4] Table 3: Summary of EC50, CI95% and coefficient of determination for three antibody clones in human IgG1 and rat IgG2b formats.

[0229] Example 3: Antibody binding to CD8+ and non-binding to CD8- cells material and method Two hundred thousand HPB-ALL cells (expressing CD8α, also called CD8+) or 1299 cells (not expressing CD8α, also called CD8-) were incubated with a primary antibody: mouse anti-CD8α (Sino Biological, #10980-MM28, diluted 1:50) or one of three antibody clones (i.e., MP08-R3-F08, MP08-R3-C11, and MP09-R3-D03) in human IgG1 or rat IgG2b format at concentrations of 10, 3.3, 1.1, or 0.37 μg / mL. HPB-ALL is a human lymphocyte cell line derived from T-cell leukemia. 1299 (or H1299) is a human epithelial cell line derived from non-small cell lung carcinoma.

[0230] Human antibodies were detected using anti-human F(ab')2-AF647 antibody (Jackson, #109-605-006, 1:100 dilution), rat antibody with anti-rat FITC antibody (Sigma, #F1763, 1:320 dilution), and mouse anti-human CD8α antibody with anti-mouse FITC antibody (Sigma, #12-506, 1:100 dilution). Results were acquired on a CytoFLEX cytometer (Beckman Coulter) and analyzed using FlowJo.

[0231] result The cells were verified by flow cytometry using control experiments (data not shown). Background signals from the secondary antibody were determined for both cell lines (data not shown). An irrelevant human IgG1 called "13R4" showed no binding to either cell line (Figure 3D for HPB-ALL cells and Figure 4D for H1299 cells). CD8α expression was also assessed using a commercially available anti-CD8α antibody, and as expected, showed a strong signal in HPB-ALL cells (CD8+ cells) but no signal in H1299 cells (CD8- cells) (Figure 3E for HPB-ALL cells and Figure 4E for H1299 cells).

[0232] Antibody binding to both cell lines was assessed by flow cytometry. All three antibody clones in the human IgG1 format bound to HPB-ALL CD8+ cells (Figures 3A-C show a concentration of 3.3 μg / mL), but no binding was observed to H1299 CD8- cells (Figures 4A-C show a concentration of 3.3 μg / mL).

[0233] At the lowest concentration (0.37 μg / mL), clones MP08-R3-C11 and MP08-R3-F08 showed the strongest signals (data not shown), suggesting higher affinity.

[0234] Similar results were obtained in the rat IgG2b format, with clones MP08-R3-C11 and MP08-R3-F08 providing the strongest signal at 0.37 μg / mL (data not shown).

[0235] These results confirm those obtained with recombinant human CD8α protein and demonstrate that the three antibody clones (i.e., MP08-R3-F08, MP08-R3-C11, and MP09-R3-D03) have strong binding and good specificity for human CD8α.

[0236] Example 4: Antibody binding to primary CD8+ T cells material and method Human PBMCs (peripheral blood mononuclear cells) were prepared from buffy coats using a density gradient. The buffy coat was diluted 1 / 2 with 2% PBS-SVF, and 30 mL was loaded into 15 mL of Lymphoprep (Fisher Scientific, #17171036). The tube was centrifuged at 2000 rpm without the brake for 20 minutes. PBMCs were collected at the lymphoprep / plasma interface, washed with PBS-SVF, and centrifuged at 1200 rpm for 5 minutes. CD8α + CD8+ T cell isolation was performed using a T Cell Isolation Kit (Miltenyi Biotec, #130-104-075) according to the manufacturer's instructions.

[0237] Cells were incubated with primary antibodies: mouse anti-CD8α (Sino Biological, #10980-MM28, diluted 1:50) or one of three antibody clones (i.e., MP08-R3-F08, MP08-R3-C11, and MP09-R3-D03) in human IgG1 or rat IgG2b formats at concentrations of 10, 3.3, 1.1, or 0.37 μg / mL. Human antibodies were detected with anti-human F(ab')2-AF647 (Jackson, #109-605-006, diluted 1:100), rat anti-rat FITC-containing antibody (Sigma, #F1763, diluted 1:320), and mouse anti-human CD8α (Sigma, #12-506, diluted 1:100). Results were acquired on a CytoFLEX cytometer (Beckman Coulter) and analyzed using FlowJo.

[0238] result The cells were validated by flow cytometry using control experiments (data not shown). The background signal from the secondary antibody was determined (data not shown). An irrelevant human IgG1 called "13R4" showed no binding to fresh CD8+ T cells (Figure 5D). CD8α expression was also assessed using a commercially available anti-CD8α antibody, which showed a strong signal on purified primary CD8+ T cells (Figure 5E).

[0239] Antibody binding to fresh primary CD8+ T cells was assessed by flow cytometry and demonstrated binding of all three antibody clones in the human IgG1 format (Figure 5A-C).

[0240] Similar to the HPB-ALL CD8+ cell line, at the lowest concentration (0.37 μg / mL), clones MP08-R3-C11 and MP08-R3-F08 again showed the strongest signals (data not shown), suggesting a higher affinity for these two clones.

[0241] Similar results were obtained in the rat IgG2b format, with clones MP08-R3-C11 and MP08-R3-F08 providing the strongest signal at 0.37 μg / mL (data not shown).

[0242] These results confirm those obtained with recombinant human CD8α protein and CD8+ / CD8− cell lines and demonstrate that the three antibody clones (i.e., MP08-R3-F08, MP08-R3-C11, and MP09-R3-D03) exhibit good binding to human CD8α.

[0243] Example 5: In vivo human CD8 depletion material and method mouse We used B6;SJL-Tg(CD8aCD8b)57Scr / J mice (Jackson Laboratory, #003202, LaFace et al. Human CD8 transgene regulation of HLA recognition by murine T cells. J Exp Med. 1995 Nov 1;182(5):1315-25), which were subsequently engineered as hCD8 mice. These hCD8 mice were cloned using mouse p56 lck It expresses a transgene containing the human CD8 (hCD8) α and β chains expressed under the control of a proximal promoter.

[0244] Preliminary experiments confirmed the absence of human CD8α (i.e., hCD8α) expression in control C57B16 mice (designated control WT mice, Figure 6A) and strong human CD8α expression in transgenic mice expressing the hCD8α transgene (Figure 6B). However, this hCD8α expression is not restricted to CD8 T cells.

[0245] Mice were maintained in the same animal facility throughout the experiment under the same standard conditions, including housing, regular care, and a regular chow diet (food and water available ad libitum, 12-hour light / dark cycle). Mice were 9-12 weeks old at the start of the experiment. Due to the availability of mice, only males were used, except for the hCD8 100 μg depletion group, which was half female.

[0246] Antibody injection After isoflurane anesthesia, human IgG1 antibodies (i.e., MP08-R3-F08, MP08-R3-C11, and MP09-R3-D03) were injected intravenously through the retro-orbital sinus using a 30-gauge insulin syringe. After full recovery from anesthesia, mice were returned to their home cages.

[0247] blood sample Blood samples were collected from the submandibular vein at the required time points. After isoflurane anesthesia, the left submandibular vein was punctured using a 25-gauge needle. Several drops of blood, equivalent to approximately 100 μL, were collected into a collection tube containing 5 μL of heparin. After sample collection, a sterile gauge was applied to stop bleeding. After complete recovery from anesthesia, the mice were returned to their home cages.

[0248] Flow cytometry For each sample, 50 μL of blood was incubated with 10 μL of FACS antibody solution for 30 minutes at room temperature.

[0249] Blood samples were stained and then lysed for red blood cells using BD FACS lysing solution (BD Biosciences). Acquisition was performed on an LSR Fortessa cytometer, followed by analysis using FlowJO v10.8. Forward scatter (FSC) and side scatter (SSC) parameters were used to gate on live cells, excluding red blood cells, debris, and cell aggregates. Cells were then selected based on CD45 expression. For hCD8 transgenic mice, hCD8α expression levels were assessed in total CD45+ cells. In control mouse experiments, mCD8α expression was assessed in CD45+CD11b-CD3+ cells.

[0250] Representation and normalization of data The number of hCD8α+ cells was assessed within the total CD45+ cell population and then normalized to the mean proportion of hCD8α+ in control non-depleted mice to assess hCD8α+ cell repopulation.

[0251] result To evaluate the efficiency and duration of hCD8α-expressing cell (hCD8+ cell) depletion, three antibody clones (i.e., MP08-R3-F08, MP08-R3-C11, and MP09-R3-D03) were injected into mice at three different doses (50 μg, 100 μg, and 200 μg), with three mice per group. Immediate confirmation of depletion was performed by flow cytometry analysis 1 h after antibody injection. hCD8α cell repopulation was followed weekly for the two lower doses, and on days 7, 14, and 42 for the higher dose. One animal in the MP08-R3-C11 50 μg group was excluded due to injection of the wrong antibody.

[0252] Depletion of hCD8α-expressing cells was complete 1 h after injection for all clones at 200 μg, but nearly complete for the 50 μg and 100 μg doses (Figures 7A-C and 8A-C). At 7 days post-injection, depletion of hCD8α was complete for all clones and doses (Figures 7A-C and 8A-C).

[0253] Subsequent time points showed progressive hCD8α+ repopulation starting from the 50 μg dose, with all clones at 50 μg achieving the 50% repopulation threshold by day 28, with very similar trajectories (Figure 8C). At higher doses, only clones MP08-R3-C11 (Figure 7B) and MP09-R3-D03 (Figure 7C) reached 50% repopulation at 100 μg, and only MP09-R3-D03 reached 50% repopulation at 200 μg by day 42. Repopulation within the 100 μg and 200 μg groups followed very similar trends for clones MP08-R3-F08 (Figure 7A) and MP09-R3-D03 (Figure 7C).

[0254] To examine the cross-reactivity of the three human CD8α antibody clones with mouse CD8α, we injected each of the three antibody clones into control C57Bl6 mice and assessed changes in the blood CD8 T cell population. As shown in the flow cytometry plots (Figure 9A-D) and quantification (Figure 9E), none of the three anti-human CD8α antibody clones was able to deplete mouse CD8 T cells in control mice, either 1 hour or 7 days after injection. These results demonstrate that all three antibody clones do not cross-react with mouse CD8α.

[0255] conclusion These results demonstrate that all three antibody clones (i.e., MP08-R3-F08, MP08-R3-C11, and MP09-R3-D03) can strongly bind to human CD8α recombinant protein, as well as to human CD8α expressed on the surface of CD8α-expressing cell lines and primary CD8+ T cells. Furthermore, all three antibody clones have good specificity for human CD8α, do not bind to human CD4, and have no cross-reactivity with mouse CD8α.

[0256] Furthermore, all three antibody clones are capable of depleting CD8α-expressing cells in vivo, and all three clones have good specificity for human CD8α and no cross-reactivity to mouse CD8α in vivo.

[0257] All clones induce complete or near-complete depletion of CD8α-expressing cells within 1 hour after injection in human CD8α-expressing mice, depending on the dose. Furthermore, depletion is complete for all clones and doses within at least 7 days. After 7 days of depletion, regrowth of hCD8α-expressing cells begins gradually. At the 50 μg dose, regrowth reaches 50% by day 28 for all clones, whereas only clones MP08-R3-C11 and MP09-R3-D03 reach 50% by day 42 at the 100 μg dose, and only clone MP09-R3-D03 at the 200 μg dose.

[0258] Thus, depletion of CD8α-expressing cells by the anti-CD8α antibodies of the present invention is rapid, nearly complete, if not complete, and transient. Indeed, repopulation of CD8α-expressing cells after depletion is controlled and dependent on the dose of antibody used.

[0259] Taken together, these results indicate that anti-CD8α antibodies, such as those described herein, may be a valuable therapeutic tool for transiently depleting CD8α-expressing cells in a controlled manner.

[0260] Example 6: Antibody specificity material and method The Retrogenix Cell Microarray Technology platform was used to screen for specific off-target binding interactions of the anti-CD8α antibody clone MP08-R3-F08.

[0261] Pre-screening We assessed the level of background binding of the anti-CD8α antibody clone MP08-R3-F08 to fixed, untransfected HEK293 cells and cells overexpressing CD8α (isoform 1, secreted, or tethered secreted isoform 2). Binding to untransfected and target-expressing cells was assessed using an AlexaFluor 647-conjugated anti-human IgG Fc detection antibody, followed by fluorescence imaging.

[0262] Library screening The binding level of the anti-CD8α antibody clone MP08-R3-F08 was screened using fixed human HEK293 cells individually overexpressing 6105 full-length human plasma membrane proteins, human secreted proteins, and cell surface-tethered human secreted proteins, as well as an additional 400 human heterodimers.

[0263] Binding detection was performed using the same AlexaFluor 647-labeled anti-human IgG Fc detection antibody as above, followed by fluorescent imaging. Anti-CD8α antibody clone MP08-R3-F08 was tested in duplicate. Fluorescent images were analyzed and quantified using Image Quant software.

[0264] Protein interactions were defined as double spots showing elevated signals compared to background levels, and interactions were classified as strong, moderate, weak, or very weak depending on the intensity of the double spots.

[0265] Confirmatory Screening All proteins identified in the library screening process as interacting with the anti-CD8α antibody clone MP08-R3-F08 were re-expressed in HEK293 cells. The binding levels of the anti-CD8α antibody clone MP08-R3-F08 were assessed in both fixed and unfixed cells using fluorescence imaging in the same manner as in the library screening, and compared with those of rituximab (an anti-CD20 antibody) used as a control antibody. The objective was to determine which interactions were reproducible and specific to the anti-CD8α antibody clone MP08-R3-F08.

[0266] result Pre-screening The anti-CD8α antibody clone MP08-R3-F08 showed low levels of background binding to fixed, untransfected HEK293 cells at all three concentrations tested (i.e., 2 μg / mL, 5 μg / mL, and 20 μg / mL). The anti-CD8α antibody clone MP08-R3-F08 showed binding to overexpressed CD8α (both secreted and tethered secreted isoform 2) at all three concentrations, whereas binding to overexpressed CD8α (isoform 1) was only observed at 5 and 20 μg / mL. Based on these results, further screening was performed at 20 μg / mL on fixed cells.

[0267] Library screening After screening 6105 full-length human plasma membrane proteins, human secreted proteins, and cell surface-tethered human secreted proteins, as well as an additional 400 human heterodimers overexpressed in HEK293 cells using 20 μg / mL of the anti-CD8α antibody clone MP08-R3-F08, 23 interactions were identified and further analyzed.

[0268] Confirmatory Screening For confirmation screening, 23 proteins identified in the library screening process as interacting with the anti-CD8α antibody clone MP08-R3-F08, as well as CD8α (isoform 1) and two control receptors (CD20 and EGFR), were overexpressed in HEK293 cells. The cells were used either fixed or unfixed to evaluate interactions with the anti-CD8α antibody clone MP08-R3-F08 and compare them with those of a control antibody (i.e., rituximab).

[0269] In fixed cell microarrays, 22 of the 23 proteins identified as interacting with the anti-CD8α antibody clone MP08-R3-F08 were reproducibly observed to interact with the anti-CD8α antibody clone MP08-R3-F08 (Fig. 10A).

[0270] The exception was FCGR3B, which showed very weak intensity in the library screening, and the lack of reproducibility in the confirmation screening indicated that the interaction was not real. Seventeen of the 23 identified proteins bound both the anti-CD8α antibody clone MP08-R3-F08 and the control antibody, and were therefore classified as nonspecific interactions (Figure 10A-B). These included Fc gamma receptors, likely mediated by the Fc domain, and IGHG3, which was directly bound by the detection antibody.

[0271] Three more of the 23 proteins, as well as CD8α (isoform 1), appeared to be specific for the anti-CD8α antibody clone MP08-R3-F08, but the signal intensity was very weak, close to background levels, and classified as insignificant.

[0272] After filtering out irreproducible, nonspecific, and nonsignificant interactions, only two remained that were significant and specific for the anti-CD8α antibody clone MP08-R3-F08: the secreted and tethered forms of CD8α isoform 2. In unfixed cells, the interaction of the anti-CD8α antibody clone MP08-R3-F08 with both the secreted and tethered forms of CD8α isoform 2 was confirmed (Figure 10C).

[0273] In conclusion, the anti-CD8α antibody clone MP08-R3-F08 showed significant specific interactions with CD8α (both secreted and tethered secreted isoform 2) in both fixed and live cell microarrays. No other specific interactions were identified, demonstrating the high specificity of the anti-CD8α antibody clone MP08-R3-F08 for its target, CD8α isoform 2.

[0274] Example 7: CD8 depletion in non-human primates material and method animal Four naive, healthy male cynomolgus monkeys, aged 2–4 years, were used for this study. The animals were housed in stainless steel cages, one monkey per cage. Room temperature was maintained at 20–29°C with a relative humidity of 40–70%. Fluorescent lighting was provided on a 12-hour light (08:00–20:00) and 12-hour dark cycle. The monkeys had unlimited access to water and food.

[0275] Antibody injection Each monkey was intravenously injected via the saphenous vein with either (1) 20 mg / kg body weight of anti-CD8α antibody clone MP08-R3-F08, (2) 20 mg / kg body weight of anti-CD8α antibody clone MP08-R3-C11, (3) 20 mg / kg body weight of anti-CD8α antibody clone MP09-R3-D3, or (4) 7.5 mg / kg body weight of anti-CD8α antibody clone MP08-R3-F08. The antibody infusion was completed within 30 minutes.

[0276] Sample collection Whole blood was collected into potassium (K2) EDTA tubes from each animal on days −1, 0 (1 hour), 0 (6 hours), 0 (24 hours), 5, 10, 15, and 22 post-injection for the three monkeys that received the 20 mg / kg dose of antibody, and on days −1, 0 (3 hours), 0 (6 hours), 0 (24 hours), 7, 14, 28, 48, and 62 post-injection for the monkeys that received the 7.5 mg / kg dose of antibody.

[0277] Flow cytometry From whole blood samples, red blood cells were lysed using BD FACS lysing solution (BD Biosciences catalog number 555898). After lysis, pelleted cells were washed twice with PBS 1% FBS. Cells were resuspended in PBS 1% FBS and incubated in FcR blocking buffer for 10 minutes before staining with the antibodies detailed in Table 4 below at 4°C for 1 hour. After staining, cells were washed twice with PBS 1% FBS, fixed for 10 minutes with fixation buffer, washed again, resuspended in PBS, and then acquired on a flow cytometer. Forward scatter (FSC) and side scatter (SSC) parameters were used to gate on live cells, excluding red blood cells, debris, and cell aggregates. Cells were subsequently selected based on CD45 expression. CD8+ cells were identified as CD45+CD11b-CD3+CD4-CD8+ cells. [Table 5] Table 4: Antibodies used in flow cytometry in non-human primate experiments

[0278] Representation in the data The number of CD8α+ cells was assessed within the total CD45+ cell population and expressed as a percentage of CD45+ cells.

[0279] result To evaluate the efficiency of CD8α-expressing cell depletion, three antibody clones (i.e., MP08-R3-F08, MP08-R3-C11, and MP09-R3-D03) were injected into monkeys at a dose of 20 mg / kg body weight, with one monkey per group. Immediate confirmation of depletion was performed by flow cytometry analysis 1 and 6 hours after antibody injection. Follow-up of CD8α-expressing cell depletion was performed 24 hours, and on days 5, 10, 15, and 22 after antibody injection.

[0280] Depletion of CD8α-expressing cells was complete 1 hour after injection of clone MP08-R3-F08, and the percentage of CD8+ cells remained at 0% until day 22 (Figure 10A). Antibody clones MP08-R3-C11 and MP09-R3-D03 induced a significant decrease in CD8α-expressing cells, which persisted until day 22 (Figure 10A). However, the depletion of CD8α-expressing cells induced by antibody clones MP08-R3-C11 and MP09-R3-D03 was not complete.

[0281] Because antibody clone MP08-R3-F08 induced the most efficient depletion of CD8α-expressing cells, additional doses were tested. To evaluate the depletion efficiency of CD8α-expressing cells, antibody clone MP08-R3-F08 was injected into one additional monkey at a dose of 7.5 mg / kg body weight. Immediate confirmation of depletion was performed by flow cytometry analysis 1 and 6 hours after antibody injection. Follow-up of CD8α-expressing cell depletion was performed 24 hours, and on days 7, 14, 28, 48, and 62 after antibody injection.

[0282] Even at a lower dose of 7.5 mg / kg body weight, antibody clone MP08-R3-F08 induced complete depletion of CD8α-expressing cells within 1 hour after injection. Furthermore, the percentage of CD8+ cells remained at 0% until day 14 after injection. From day 14, CD8α-expressing cells began to re-increase very gradually, reaching 2.5% by day 62 (Figure 10B).

[0283] Example 8: Effect of anti-CD8α antibody in a mouse model of myocardial infarction material and method mouse B6;SJL-Tg(CD8aCD8b)57Scr / J were used (Jackson Laboratory strain, #003202) and subsequently engineered as hCD8 mice, as described in Example 5 above.

[0284] Mice were maintained in the same animal facility throughout the experiment under identical standard conditions, including housing, routine care, and a regular chow diet (food and water available ad libitum, 12-hour light / dark cycle). Mice were 9-15 weeks old at the start of the experiment. Both males and females were used.

[0285] Myocardial infarction Myocardial infarction (MI) was induced by permanent ligation of the left anterior descending artery in its proximal third, as previously described (Zouggari et al., Nature Medicine, 2013). Mice were anesthetized by peritoneal injection of ketamine (100 mg per kg body weight; Ketamine 1000, Vibrac) and xylazine (10 mg per kg body weight; Xylazine 2%, Bayer), intubated, and ventilated with air using a small animal ventilator (SAR 830 / AP Ventilator, CWE Incorporated, Ardmore, MD, USA). Body temperature was maintained at 37°C using a homeothermic blanket system (Harvard Apparatus). The chest wall was shaved, and a thoracotomy was performed at the fourth left intercostal space. The left ventricle was localized, and the pericardial sac was opened. The left anterior descending artery was permanently ligated using a 7 / 0 PVDF monofilament suture (Peters Surgical, France) 5 mm below its point of emergence from the left atrium. Persistent blanching of the myocardium below the ligation was considered to indicate successful coronary artery occlusion. Lidocaine (20 mg / mL lidocaine, Aguettant) was applied topically, and the chest and skin were closed with a 6 / 0 PVDF monofilament suture (Peters Surgical, France). Once spontaneous breathing resumed, the endotracheal tube was removed, and the mouse was placed in a cage on a heating pad maintained at 37°C until fully recovered. To alleviate pain, buprenorphine (0.1 mg / kg) was injected before surgery, 6 hours after the start of surgery, and twice daily for 48 to 72 hours.

[0286] Antibody injection One hour after surgery, mice were intravenously injected with either anti-CD8α antibody clone MP08-R3-F08 or sterile saline. Using a 29-gauge insulin syringe, 100 μL of a solution containing either 100 μg of anti-CD8α antibody clone MP08-R3-F08 or saline was slowly injected into the retroorbital sinus over a 10-second period. Mice were then placed in a cage on a warm pad maintained at 37°C. After complete recovery from anesthesia, they were returned to their home cage.

[0287] organ harvesting Mice were euthanized using 150 mg / kg Eutasol in 250 μL of saline injected intraperitoneally under isoflurane sedation. Blood was collected from the vena cava using a 27-gauge needle. The spleen and heart were weighed and rinsed with PBS for further analysis.

[0288] Preparation of cell suspensions and flow cytometry Mice were sacrificed on days 3 or 21 post-MI. Peripheral blood was collected via the inferior vena cava using a syringe equipped with a 27-gauge needle primed with heparin solution. For blood staining, red blood cells were lysed using BD FACS lysing solution (BD Biosciences). The spleen was surgically removed and dissociated to obtain a single-cell suspension, which was filtered through a 40 μm nylon mesh (BD Biosciences). The cell suspension was centrifuged at 400 g for 15 minutes at 4°C. Red blood cells were removed using red blood cell lysis buffer (Sigma-Aldrich), and the cells were washed with PBS and resuspended in 5 mL of PBS supplemented with 3% FBS.

[0289] The general characteristics of the antibodies are summarized in Table 5 below. Surface marker staining was performed by incubating cell suspensions (50 μL of blood or 50 μL of splenocyte suspension) with 10 μL of antibody cocktail for 30 minutes at 4°C, followed by washing with PBS. Forward scatter (FSC) and side scatter (SSC) parameters were used to gate live cells, excluding red blood cells, debris, and cell aggregates. Cells were analyzed using a BD LSR Fortessa flow cytometer (BD Biosciences), and data were analyzed using FlowJo v10.9 software (BD Biosciences).

[0290] The expression level of hCD8α was compared with that of total CD45 + was evaluated in cells. [Table 6] Table 5: Antibodies used for flow cytometry in mouse models of MI experiments

[0291] Assessment of infarct size on day 3 by TTC staining To assess infarct size at an early time point (3 days after MI), 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich) was used. Hearts were harvested and, while semi-frozen, divided into four 1.5- to 2-mm-thick sections from the apex. The sections were then incubated in PBS-1% TTC solution at 37°C for 40 minutes. The sections were then rinsed twice with PBS at room temperature for 5 minutes. The sections were photographed, and infarct size was blindly calculated using ImageJ by measuring the total tissue area and the area of ​​infarcted tissue in each tissue section.

[0292] Echocardiography Cardiac function was assessed 21 days after MI. The chest wall was shaved using depilatory cream. During isoflurane anesthesia (2.5% isoflurane and 2 L / min oxygen for induction; 2% isoflurane and 1 L / min oxygen for echocardiography), cardiac function was recorded using a VEVO2100 imaging system and an MS400 transducer (18-38 MHz) (VisualSonics, Canada), suitable for mouse cardiovascular imaging. Data were analyzed using the cardiac package in VEVO-Lab (VisualSonics, Canada). Left ventricular end-diastolic volume (EDV) and end-systolic volume (ESV) were calculated on cine loops using the two-dimensional area-length method, allowing for calculation of left ventricular ejection fraction (EF) as follows: EF (%) = [(EDV - ESV) / ​​EDV] × 100.

[0293] Histopathological analysis Cardiac remodeling after MI was assessed on day 21. Hearts were harvested and rinsed with PBS. The atria and right ventricles were excised, and the remaining left ventricle (LV) was embedded in Tissue-TEK OCT compound (Sakura), frozen in liquid nitrogen-cooled isopentane, and stored at -80°C. Hearts were cut along their length into 8-μm-thick myocardial cryosections (CM 3050S, Leica). Consecutive sections were mounted on microscope slides, with each section separated by 500 μm. Eight sections were thus cut, allowing for analysis of the entire LV tissue. Masson's trichrome and Sirius red staining were performed to assess infarct size and interstitial fibrosis, respectively. Infarct size was calculated as the ratio of the infarct area to the total LV area. Collagen volume fraction was calculated as the ratio of the total interstitial fibrosis area to the total cardiac area in the entire field of view of the section.

[0294] result To evaluate the effect of the anti-CD8α antibody of the present invention on myocardial infarction, the anti-CD8α antibody clone MP08-R3-F08 was injected into transgenic mice expressing human CD8α one hour after induction of myocardial infarction.

[0295] First, we assessed the depletion of hCD8 + cells in the blood and spleen of hCD8α-expressing transgenic mice that received either saline (control) or anti-CD8α antibody clone MP08-R3-F08 3 days after induction of myocardial infarction and injection of saline or anti-CD8α antibody clone MP08-R3-F08.

[0296] As shown in Figures 12A-C and 13A-C, depletion of CD45+ hCD8α+ cells was complete in the blood of mice treated with anti-CD8α antibody clone MP08-R3-F08 (Figures 12B and 12C) compared to control mice (Figures 12A and 12C), while there was more than 90% depletion in the spleens of mice treated with anti-CD8α antibody clone MP08-R3-F08 (Figures 13B and 13C) compared to control mice (Figures 13A and 13C).

[0297] Furthermore, 3 days after induction of myocardial infarction and injection of saline or anti-CD8α antibody clone MP08-R3-F08, hearts were harvested from mice, sectioned, and stained with 2,3,5 triphenyltetrazolium chloride (TTC) to measure necrosis induced after myocardial infarction.

[0298] As shown in Figure 14, the necrotic area was significantly reduced by 20% in transgenic mice treated with the anti-CD8α antibody clone MP08-R3-F08 compared to control transgenic mice treated with saline.

[0299] conclusion These results demonstrate that the anti-CD8α antibody clone MP08-R3-F08 has strong specificity for its target CD8α isoform 2 (both the secreted and tethered secreted forms) and does not exhibit any off-target binding.

[0300] Furthermore, all three anti-CD8α antibody clones (i.e., MP08-R3-F08, MP08-R3-C11, and MP09-R3-D03) are capable of depleting CD8α-expressing cells in vivo in non-human primates. All anti-CD8α antibody clones (i.e., MP08-R3-F08, MP08-R3-C11, and MP09-R3-D03) induce potent or even complete depletion of CD8α-expressing cells within 1 hour of antibody injection in non-human primates. Furthermore, depletion with anti-CD8α antibody clone MP08-R3-F08 is complete for at least 14 days. After 14 days of depletion, repopulation of CD8α-expressing cells begins slowly and gradually at a dose of 7.5 mg / kg body weight, reaching only 2.5% at 62 days post-injection. Thus, depletion of CD8α-expressing cells by the anti-CD8α antibodies of the invention is rapid, potent, if not complete, and transient. Repopulation of CD8α-expressing cells after depletion is slow and progressive.

[0301] Furthermore, the anti-CD8α antibody clone MP08-R3-F08 can limit the necrotic area induced after myocardial infarction in hCD8α-expressing transgenic mice.

[0302] Taken together, these results indicate that anti-CD8α antibodies such as those described herein may be a valuable therapeutic tool for transiently depleting CD8α-expressing cells in a controlled manner and for treating cardiovascular diseases such as myocardial infarction.

Claims

1. 1. An isolated anti-CD8α antibody, or antigen-binding fragment thereof, comprising: a) the variable region (VH) of the heavy chain of the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises the following three CDRs: -V Ｈ -CDR1:NX １ X ２ MN, formula X １ is N or Y, X ２ is D or A or Y, -V Ｈ - CDR2: X ３ ISGSSX ４ YIX ５ YADFVKG (SEQ ID NO: 1), wherein: X ３ is D or S, X ４ is S or R, X ５ is D or G or Y, -V Ｈ - CDR3: SSX ６ X ７ X ８ X ９ YX １０ X １１ X １２ X １３ MDV (SEQ ID NO: 2), wherein: X ６ is N or does not contain an amino acid, X ７ is Y or contains no amino acids, X ８ is Y or G or does not contain any amino acid, X ９ is D or S or does not contain any amino acids, X １０ is S or N or F, X １１ is A or G, X １２ is S or D or N, X １３ is A or G; b) the variable region of the light chain (VL) of the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises the following three CDRs: -V Ｌ -CDR1:AGTSSDVGGX １４ X １５ X １６ VS (SEQ ID NO: 3), wherein: X １４ is G or N or Y, X １５ is S or Y, X １６ is S or Y, -V Ｌ - CDR2: X １７ DSX １８ RPS (SEQ ID NO: 4), wherein: X １７ is Q or S or Y, X １８ is Y or S, -V Ｌ - CDR3: SSX １９ TX ２０ YSTRV (SEQ ID NO: 5), wherein: X １９ is Y or D, X ２０ is Y or Q or S; An isolated anti-CD8α antibody, or antigen-binding fragment thereof.

2. a) the VH or antigen-binding fragment thereof of the isolated anti-CD8α antibody comprises the following three CDRs: -V Ｈ CDR1: NNAMN (SEQ ID NO: 6), NYDMN (SEQ ID NO: 12), or NYYMN (SEQ ID NO: 18); -V Ｈ CDR2: DISGSSRYIGYADFVKG (SEQ ID NO: 7), DISGSSSYIDYADFVKG (SEQ ID NO: 13), or SISGSSRYIYYADFVKG (SEQ ID NO: 19); -V Ｈ CDR3: SSNYYDYNADAMDV (SEQ ID NO: 8), SSYYSGSGMDV (SEQ ID NO: 14), or SSGSYFGNAMDV (SEQ ID NO: 20); b) the VL or antigen-binding fragment thereof of the isolated anti-CD8α antibody comprises the following three CDRs: -V Ｌ CDR1: AGTSSDVGGNSYVS (SEQ ID NO: 9), AGTSSDVGGGSSVS (SEQ ID NO: 15), or AGTSSDVGGYYSVS (SEQ ID NO: 21); -V Ｌ CDR2: SDSSRPS (SEQ ID NO: 10), QDSYRPS (SEQ ID NO: 16), or YDSSRPS (SEQ ID NO: 22) -V Ｌ CDR3: SSYTQYSTRV (SEQ ID NO: 11), SSYTYYSTRV (SEQ ID NO: 17), or SSDTSYSTRV (SEQ ID NO: 23), The isolated anti-CD8α antibody or antigen-binding fragment thereof of claim 1.

3. The isolated anti-CD8α antibody or antigen-binding fragment thereof comprises the following six CDRs: -V Ｈ - CDR1: NNAMN (SEQ ID NO: 6), V Ｈ - CDR2: DISGSSRYIGYADFVKG (SEQ ID NO: 7), V Ｈ - CDR3: SSNYYDYNADAMDV (SEQ ID NO: 8), V Ｌ - CDR1: AGTSSDVGGNSYVS (SEQ ID NO: 9), V Ｌ - CDR2: SDSSRPS (SEQ ID NO: 10), and V Ｌ CDR3: SSYTQYSTRV (SEQ ID NO: 11); or -V Ｈ - CDR1: NYDMN (SEQ ID NO: 12), V Ｈ - CDR2: DISGSSSYIDYADFVKG (SEQ ID NO: 13), V Ｈ - CDR3: SSYYSGSGMDV (SEQ ID NO: 14), V Ｌ - CDR1: AGTSSDVGGGSSVS (SEQ ID NO: 15), V Ｌ - CDR2: QDSYRPS (SEQ ID NO: 16), and V Ｌ CDR3: SSYTYYSTRV (SEQ ID NO: 17); or -V Ｈ - CDR1: NYYMN (SEQ ID NO: 18), V Ｈ - CDR2: SISGSSRYIYYADFVKG (SEQ ID NO: 19), V Ｈ - CDR3: SSGSYFGNAMDV (SEQ ID NO: 20), V Ｌ - CDR1: AGTSSDVGGYYSVS (SEQ ID NO: 21), V Ｌ - CDR2: YDSSRPS (SEQ ID NO: 22), and VL-CDR3: SSDTSYSTRV (SEQ ID NO: 23) 3. The isolated anti-CD8α antibody or antigen-binding fragment thereof of claim 1 or 2, comprising:

4. The isolated anti-CD8α antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28, or a sequence having at least 80% identity to SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:

28.

5. The isolated anti-CD8α antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the isolated anti-CD8α antibody or antigen-binding fragment thereof comprises a variable region of a light chain (VL) comprising the sequence set forth in SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29, or a sequence having at least 80% identity to SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:

29.

6. the isolated anti-CD8α antibody or antigen-binding fragment thereof a VH comprising the sequence shown in SEQ ID NO: 24 or a sequence having at least 80% identity with SEQ ID NO: 24, and a VL comprising the sequence shown in SEQ ID NO: 25 or a sequence having at least 80% identity with SEQ ID NO: 25; or a VH comprising the sequence shown in SEQ ID NO: 26 or a sequence having at least 80% identity with SEQ ID NO: 26, and a VL comprising the sequence shown in SEQ ID NO: 27 or a sequence having at least 80% identity with SEQ ID NO: 27; or a VH comprising the sequence shown in SEQ ID NO: 28 or a sequence having at least 80% identity with SEQ ID NO: 28, and a VL comprising the sequence shown in SEQ ID NO: 29 or a sequence having at least 80% identity with SEQ ID NO: 29; The isolated anti-CD8α antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, comprising:

7. The isolated anti-CD8α antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the isolated anti-CD8α antibody or antigen-binding fragment thereof is a CD8α-expressing cell-depleting antibody.

8. A fusion protein comprising the anti-CD8α antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. A nucleic acid molecule encoding the anti-CD8α antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or the fusion protein according to claim 8.

10. A pharmaceutical composition comprising an isolated anti-CD8α antibody or antigen-binding fragment thereof described in any one of claims 1 to 7, a fusion protein described in claim 8, or a nucleic acid molecule described in claim 9, and at least one pharmaceutically acceptable excipient.

11. 11. The isolated anti-CD8α antibody or antigen-binding fragment thereof of any one of claims 1 to 7, the fusion protein of claim 8, the nucleic acid molecule of claim 9, or the pharmaceutical composition of claim 10, for use as a medicament.

12. 11. The isolated anti-CD8α antibody or antigen-binding fragment thereof of any one of claims 1 to 7, the fusion protein of claim 8, the nucleic acid molecule of claim 9, or the pharmaceutical composition of claim 10, for use in treating a CD8-related disease.

13. The isolated anti-CD8α antibody or antigen-binding fragment thereof, fusion protein, nucleic acid molecule, or pharmaceutical composition for use according to claim 12, wherein the CD8-related disease is cardiovascular disease.

14. 10. An in vitro method for depleting CD8α-expressing cells in a sample, comprising contacting the sample with the isolated anti-CD8α antibody or antigen-binding fragment thereof of any one of claims 1 to 7, or the fusion protein of claim 8.

15. 10. An in vitro method for detecting and / or quantifying CD8α in a sample, cell, tissue or organ, comprising contacting the sample, cell, tissue or organ with an isolated anti-CD8α antibody or antigen-binding fragment thereof of any one of claims 1 to 7, or a fusion protein of claim 8.

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