Antibodies to block cleavage of CD95L by metalloproteases

Abstract

We hypothesized that monoclonal antibodies targeting the Metalloprotease-Cleavage Sites of CD95L (MCSC), which include amino acid residues 103 to 145, could block access to metalloproteases and inhibit CD95L shedding. We therefore generated antibodies corresponding to various epitopes in MCSC, two of which (i.e., 3C7 and 9F5) effectively blocked CD95L cleavage by MMP2 and MMP3. This result indicates that these MCSC-targeted mAbs could compete with MMPs and represent an original and attractive therapeutic option to inhibit the release of inflammatory s-CD95L.

Description

[Technical field]

[0001] The present invention is in the fields of medicine, particularly immunology and oncology. [Background technology]

[0002] CD95L (FasL) belongs to the tumor necrosis factor (TNF) family and is the ligand for the death receptor CD95 (also known as Fas). Whereas CD95 is widely expressed on healthy cells, CD95L shows a restricted expression pattern and is detected mainly on the surface of lymphocytes, where it plays a central role in the elimination of infected and transformed cells (Strasser, A.; Jost, PJ; Nagata, S. The many roles of FAS receptor signaling in the immune system. Immunity. 2009, 30, 180-192). CD95L is a transmembrane glycoprotein (m-CD95L) that acts locally through cell-cell contact and mediates the synthesis of metalloproteases such as MMP3 (Matsuno, H. et al. J Rheumatol. 2001, 28, 22-28), MMP7 (Vargo-Gogola, T et al. Arch Biochem Biophys. 2002, 408, 155-161), MMP9 (Kiaei, M. et al. Exp Neurol. 2007, 205, 74-81), or A Disintegrin And Metalloproteinase 10 (ADAM-10) (Kirkin, V. et al. The Cell Death After cleavage by NF-κB and other receptors, soluble CD95L (termed s-CD95L or cleaved CD95L, cl-CD95L) is released into the bloodstream. This soluble ligand contributes to exacerbation of inflammation in chronic inflammatory diseases such as systemic lupus erythematosus (SLE) by inducing non-apoptotic signaling pathways such as NF-κB and PI3K (Tauzin, S. et al. PLoS Biol. 2011, 9, e1001090), and may exert pro-oncogenic functions by promoting survival in ovarian and liver cancer, and chemoresistance in lung cancer. The CD95L receptor, called CD95 or Fas, has an intracellular conserved portion, the death domain (DD), which functions as a docking platform to induce cell death.Binding of membrane-bound hexameric CD95L to CD95 leads to recruitment of the adaptor protein FADD (Fas-associated death domain) through homotypic interaction via the respective DDs (Holler, N. et al., Mol Cell Biol. 2003, 23, 1428-1440). FADD then aggregates the initiators caspase-8 and caspase-10. The CD95 / FADD / caspase complex is called the death-inducing signaling complex (DISC) and leads to the elimination of cancer cells by an apoptotic mechanism (Kischkel, FC et al. Embo J. 1995, 14, 5579-5588). In contrast, homotrimeric cl-CD95L does not induce DISC formation but instead upregulates Ca. 2+ It induces the formation of a non-apoptotic complex called the motility-inducing signaling complex (MISC) that responds to the IL-1 receptor (Tauzin, S. et al. PLoS Biol. 2011, 9, e1001090). Recent data have revealed that cl-CD95L induces calcium responses by inducing a direct interaction between CD95 and PLCγ1 (Poissonnier, A. et al. Immunity. 2016, 45, 209-223). Indeed, in the presence of cl-CD95L, the juxtamembrane region of CD95, called the calcium induction domain (CID), recruits PLCγ1 to induce endothelial transmigration of Th17 cells in SLE (Poissonnier, A. et al. Immunity. 2016, 45, 209-223). Moreover, a peptide consisting of CID conjugated to the cell-permeable domain of TAT, i.e., TAT-CID, binds PLCγ1 and blocks its recruitment to CD95. Surprisingly, in lupus-prone mice (MRL Lpr / + Repeated injection of TAT-CID into mice inhibits endothelial transmigration of Th17 cells and alleviates clinical symptoms (Poissonnier, A. et al. Immunity. 2016, 45, 209-223). Summary of the Invention

[0003] The present invention is defined by the claims. In particular, the present invention relates to antibodies for blocking cleavage of CD95L by metalloproteases. [Brief description of the drawings]

[0004] [Figure 1A-C] Figure 1 shows the mapping of cleavage sites and metalloproteases involved in CD95L. A shows that full-length CD95L has been reported in the literature to be cleaved at various sites. Arrows represent the putative cleavage sites reported in the literature. B shows the top of the histogram, in which a 43-mer corresponding to the stalk region of CD95L was incubated with the same concentrations of the indicated MMPs and ADAMs. The cleavage efficiency of the 43-mer was assessed by measuring degradation using LC-MS / MS. The percentage of degradation estimated from the decrease in the peak area corresponding to the starting 43-mer was related to each single protease. C shows that from the same experiment, privileged cleavage sites (113EL114 for MMPs and 129KQ130 for ADAMs) were determined from MS / MS sequencing of the resulting cleavage products. [Figure 2A-D]FIG. 2 shows monoclonal antibodies targeting the metalloprotease cleavage site (MCSC) of CD95L. In A, the left panel shows that the extracellular region of CD95L, including amino acid residues 102 to 281, can be cleaved at various sites by metalloproteases. The arrows represent the putative cleavage sites in the CD95L sequence. The right panel shows that three peptides (peptides 1, 2, and 3) corresponding to the majority of the MCSC of CD95L were used to generate monoclonal antibodies. The indicated peptides were injected into mice to generate monoclonal antibodies, and three different hybridomas were selected for their ability to bind CD95L. According to their affinity for the indicated peptides (assessed using ELISA), the 3C7 epitope is believed to correspond to the HTASSLEK sequence (SEQ ID NO: 24). B shows that recombinant Ig-CD95L (100 ng) was preincubated with the indicated antibodies (100 μg / mL) for 15 min, followed by treatment with the indicated MMPs for 1 h. Metalloprotease-cleaved Ig-CD95L fragments (CD95L lacking the released Ig domain of gp190) were monitored by immunoblotting. C, CD95L-expressing leukemic T cells 1A12 were incubated with the indicated antibodies (10 μg / mL) or the pan-MMP inhibitor GM6001 (10 μM) for 24 h, and the expression levels of membrane-bound CD95L were assessed by flow cytometry. D, Supernatants were harvested from cells treated in C, and metalloprotease-cleaved CD95L was measured by ELISA (Diaclone). The concentration of s-CD95L is significantly reduced in 1A12 cells treated with GM6001 or 3C7 for 24 h compared to control cells treated with isotype (anti-myc9E10 mAb). [Figure 3A-B]Figure 3 shows that antibodies targeting the metalloprotease cleavage site (MCSC) of CD95L inhibit the non-apoptotic functions of CD95L without affecting its apoptotic function. A, WR19L (CD95L-) or 1A12 (CD95L+) cells were placed in the lower chamber of a Boyden chamber assay in the presence of the indicated mAbs (10 μg / mL). Triple-negative breast cancer cells MDA-MB-231 were added to the upper chamber, and cell migration through the gradient of metalloprotease-cleaved CD95L was assessed 24 h later by fixing and staining (Giemsa) the migrating cells covering the underside of the porous membrane. The left panel shows Giemsa stained MDA-MB-231 cells migrated to the underside of the membrane with isotype (9E10) or anti-MCSC (3C7) monoclonal antibodies in the presence of WR19L (CD95L-) or 1A12 (CD95L+) cells. The right panel shows that Giemsa stained MDA-MB-231 cells were lysed and absorbance was measured at 560 nm. Values ​​represent the mean and SEM of three independent experiments. **p<0.01 was calculated using a two-tailed Mann-Whitney test. In B, the upper panel shows that Jurkat cells are sensitive to CD95-mediated apoptotic signals. Jurkat cells were stained with DIOC6 (measurement of mitochondrial membrane potential) and then incubated with 1A12 (CD95L+) or WR19L (CD95L-) cells at the indicated ratios for 4 h. Cell death was monitored by measuring the decrease in mitochondrial transmembrane potential (Δψm). The lower panel shows that 1A12 cells were pretreated with the indicated mAbs at 10 μg / mL for 24 h and then washed. The cells were then incubated with DIOC6 stained Jurkat cells at the indicated ratios for 4 h. Cell death was monitored by measuring the decrease in mitochondrial transmembrane potential (Δψm). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0005] CD95L is a transmembrane glycoprotein that acts locally through cell-cell contact. After being cleaved by metalloproteases, soluble CD95L (cleaved CD95L or cl-CD95L) is released into the bloodstream. This soluble ligand may exert a pro-oncogenic function by contributing to exacerbation of inflammation in chronic inflammatory diseases such as systemic lupus erythematosus (SLE) and promoting survival in ovarian and liver cancer, as well as chemoresistance in lung cancer. We hypothesized that monoclonal antibodies targeting the metalloprotease-cleavage sites of CD95L (MCSC), also called the stalk region and including amino acid residues 103 to 145, may block access to metalloproteases and thereby inhibit shedding of CD95L. Therefore, we generated antibodies corresponding to various epitopes in MCSCs, two of which (i.e., 3C7 and 9F5) effectively blocked the cleavage of CD95L by MMP2 and MMP3. This result indicates that these MCSC-targeted mAbs can compete with MMPs and may be an original and attractive therapeutic option to inhibit the release of inflammatory s-CD95L.

[0006] Thus, a first object of the present invention relates to an antibody that binds to the extracellular domain of CD95L, wherein said antibody prevents cleavage of CD95L by at least one metalloprotease, and wherein the epitope of said antibody comprises at least one amino acid residue in the amino acid sequence ranging from amino acid residue 110 to amino acid residue 130 in SEQ ID NO:1.

[0007] The present invention also relates to an antibody that binds to the extracellular domain of CD95L, wherein the antibody prevents cleavage of CD95L by at least one metalloprotease, and the epitope of the antibody comprises at least one amino acid residue in the amino acid sequence ranging from the 103rd amino acid residue to the 130th amino acid residue in SEQ ID NO:1.

[0008] As used herein, the term "CD95L" has its general meaning in the art and refers to the cognate ligand of the transmembrane protein CD95. This term is also known as APT1LG1, FASL, and TNFSF6. An exemplary amino acid sequence of CD95L is shown in SEQ ID NO:1. The extracellular domain typically consists of the amino acid sequence ranging from amino acid residue 103 to amino acid residue 281 in SEQ ID NO:1. SEQ ID NO:1>sp|P48023|TNFL6_HUMAN Tumor necrosis factor ligand superfamily member 6 OS=Homo sapiens OX=9606 GN=FASLG PE=1 SV=1. The extracellular domain is underlined in the sequence. The location of the epitope is indicated in bold. [ka]

[0009] As used herein, the term "soluble CD95L" has its general meaning in the art and refers to the soluble ligand produced by cleavage of transmembrane CD95L by metalloproteases. The terms "serum CD95L", "soluble CD95L", "metalloprotease-cleaved CD95L", and "cl-CD95L" have the same meaning herein.

[0010] As used herein, the term "metalloprotease" or "MMP" has its general meaning in the art and refers to an enzyme that uses metal-activated water, such as zinc-activated water, to catalyze the hydrolysis of proteins or polypeptides into smaller amino acid polymers. Examples of MMPs in the present invention include MMP-2, MMP-3, MMP-7, MMP12, and MMP13. In particular, the monoclonal antibodies of the present invention block the cleavage of CD95L by MMP-2 and / or MMP-3.

[0011] As used herein, the terms "antibody" or "immunoglobulin" have the same meaning and may be used equally in the present invention. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds to an antigen. Thus, the term antibody includes not only whole antibody molecules, but also antibody fragments and variants (including derivatives) of antibodies and antibody fragments. In natural antibodies, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to one light chain by a disulfide bond. There are two types of light chains: lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes), IgM, IgD, IgG, IgA, and IgE, which determine the functional activity of the antibody molecule. Each chain contains a specific sequence domain. The light chain contains two domains: a variable domain (VL) and a constant domain (CL). Heavy chains contain four (α, δ, γ) to five (μ, ε) domains, a variable domain (VH) and three to four constant domains (CH1, CH2, CH3, and CH4, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity to the antigen. The constant region domains of the light chain (CL) and the heavy chain (CH) confer important biological properties such as antibody chain association, secretion, transplacental transfer, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of an antibody resides in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is mainly composed of residues from the hypervariable regions or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) may participate in the antibody binding site or may affect the overall domain structure, i.e., the combining site. CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site.The light and heavy chains of immunoglobulins each have three CDRs, called L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3. Thus, an antigen-binding site typically comprises six CDRs, including a set of CDRs from each of the heavy and light chain V regions. Framework region (FR) refers to the amino acid sequence located between the CDRs. In the present invention, the amino acid residues in the variable domain, complementarity determining region (CDR) and framework region (FR) of the antibody of the present invention are identified using the Immunogenetics (IMGT) database (http: / / imgt.cines.fr) (Lefranc et al. (2003) Dev Comp Immunol. 27(1):55-77). The IMGT database was developed using sequence information from immunoglobulin (IgG), T-cell receptor (TcR), and major histocompatibility complex (MHC) molecules, with uniform numbering across antibody lambda and kappa light chains, heavy chains, and T-cell receptor chains, and no insertion codes except for rare length insertions. IMGT also takes into account and combines the definition of framework regions (FR) and complementarity determining regions (CDR) by Kabat et al., the characterization of hypervariable loops by Chothia et al., and structural data from X-ray diffraction studies.

[0012] As used herein, the term "specificity" refers to the ability of an antibody to detect and bind to a target molecule (e.g., an epitope presented on an antigen) while having relatively little detectable reactivity to other target molecules.Specificity can be measured relatively, for example, by binding or competitive binding assay using a Biacore instrument, as described elsewhere herein.Specificity can be shown, for example, by the ratio of affinity / avidity of binding to specific antigen to non-specific binding to other unrelated molecules of about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1 or more.

[0013] The term "affinity" as used herein means the strength of binding of an antibody to a target molecule (e.g., an epitope). The affinity of a binding protein is given by the dissociation constant Kd. For an antibody, the above Kd is defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant Ka is defined by 1 / Kd. Preferred methods for determining the affinity of binding proteins can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are incorporated herein by reference in their entireties. One preferred standard method, well known in the art, for determining the affinity of binding proteins is the use of a Biacore instrument.

[0014] As used herein, the term "binding" refers to a direct association between two molecules, for example, by covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges. In particular, as used herein, the term "binding" in the context of an antibody binding to a given target molecule (e.g., an antigen or epitope) typically refers to a binding that occurs within about 10 -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or less, about 10 -10 M or less, or about 10 more -11 K below M D The binding occurs with an affinity corresponding to

[0015] As used herein, the term "epitope" refers to a specific arrangement of amino acids located in a protein that antibody binds to.Epitope is often composed of a chemically active surface group of molecules such as amino acids or sugar side chains, and has specific three-dimensional structural characteristics and specific charge characteristics.Epitope can be linear or conformational, i.e., it involves two or more amino acid sequences in different regions of antigen that are not necessarily adjacent.

[0016] In some embodiments, an antibody of the invention binds to an epitope comprising at least one amino acid residue in the amino acid sequence ranging from amino acid residue 110 to amino acid residue 129 of SEQ ID NO:1.

[0017] In some embodiments, an antibody of the invention binds to an epitope comprising, or consisting of, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acid residues from amino acid residues 110 to 130 of SEQ ID NO:1.

[0018] In some embodiments, an antibody of the invention binds to an epitope comprising, or consisting of, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acid residues from amino acid residues 103 to 130 of SEQ ID NO:1.

[0019] In some embodiments, an antibody of the invention binds to an epitope comprising or consisting of an amino acid sequence ranging from amino acid residue 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, or 123 to amino acid residue 129 or 130 of SEQ ID NO:1.

[0020] In some embodiments, the antibodies of the invention bind to an epitope comprising or consisting of an amino acid sequence ranging from amino acid residue 122 to amino acid residue 129 in SEQ ID NO: 1. In other words, in some embodiments, the antibodies of the invention bind to an epitope comprising or consisting of the sequence shown as HTASSLEK (SEQ ID NO: 24).

[0021] In some embodiments, the antibodies of the invention bind to a linear epitope.

[0022] In some embodiments, the antibodies of the invention bind to a conformational epitope.

[0023] In some embodiments, an antibody of the invention binds to a linear or conformational epitope comprising, or based on, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acid residues from amino acid residues 110 to 130 of SEQ ID NO:1.

[0024] In some embodiments, the antibodies of the invention bind to a linear or conformational epitope comprising or consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acid residues from amino acid residues 103 to 130 of SEQ ID NO:1.

[0025] In some embodiments, the antibodies of the invention bind to a linear or conformational epitope comprising or consisting of an amino acid sequence ranging from amino acid residue 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, or 123 to amino acid residue 129 or 130 of SEQ ID NO:1.

[0026] In some embodiments, the antibodies of the invention bind to a linear or conformational epitope comprising or consisting of the amino acid sequence ranging from amino acid residue 122 to amino acid residue 129 of SEQ ID NO:1.

[0027] In some embodiments, the antibodies of the invention bind to a peptide comprising or consisting of the sequence set forth as HTASSLEK (SEQ ID NO:24).

[0028] In some embodiments, an antibody of the invention binds to a peptide comprising or consisting of the sequence set forth as LAELRESTSQMHTASSLEK (SEQ ID NO:22).

[0029] In some embodiments, an antibody of the invention binds to a peptide comprising or consisting of the sequence set forth as LAELRESTSQMHTASSLEK (SEQ ID NO: 22) and / or HTASSLEKQIGHPSP (SEQ ID NO: 23).

[0030] In some embodiments, the antibody of the present invention is a monoclonal antibody. The terms "monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", or "mAb", as used herein, refer to a preparation of antibody molecules of single molecular composition. Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in minor amounts. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. For example, monoclonal antibodies can be made using the method of Kohler and Milstein (Nature, 256:495, 1975). To make monoclonal antibodies useful in the present invention, mice or other suitable host animals can be immunized with a suitable form of antigen (i.e., CD95L polypeptide) at appropriate intervals (e.g., twice weekly, weekly, twice monthly, or monthly). The animals may be given a final "booster" with antigen within one week before being sacrificed. In many cases, it is desirable to use an immune adjuvant during immunization. Suitable immune adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or QuilA, or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are well known in the art. Animals may be immunized subcutaneously, intraperitoneally, intramuscularly, intravenously, intranasally, or by other routes. A given animal may be immunized with multiple forms of antigen by multiple routes. Monoclonal antibodies useful in the present invention may also be prepared from non-immunized animals or humans. However, the modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies may also be prepared by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic, or plant cells (see, e.g., U.S. Pat. No. 4,816,567).The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature, 352:624-628 (1991), and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0031] In some embodiments, the antibody of the present invention is a chimeric antibody. As used herein, the term "chimeric antibody" refers to an antibody that contains the VH and VL domains of a non-human antibody and the CH and CL domains of a human antibody.

[0032] In some embodiments, the antibody of the present invention is a humanized antibody. The term "humanized antibody" refers to an antibody that has variable region framework and constant regions from a human antibody, but retains the CDRs of a previous non-human antibody. In some embodiments, a humanized antibody contains minimal non-human immunoglobulin-derived sequences. In many cases, humanized antibodies and antibody fragments thereof can be human immunoglobulins (recipient antibodies or antibody fragments) in which the residues of the recipient complementarity determining regions (CDRs) are replaced with the residues of the CDRs of a non-human species (donor antibody), such as mouse, rat, or rabbit, that has the desired specificity, affinity, and capacity.

[0033] In some embodiments, the antibody of the present invention is a human antibody. As used herein, the term "human monoclonal antibody" is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. The human antibody of the present invention may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). More specifically, the term "human monoclonal antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as mouse, are grafted onto human framework sequences.

[0034] In some embodiments, the antibody of the present invention is an antibody fragment. As used herein, the term "antibody fragment" refers to at least a portion of an intact antibody, preferably an antigen-binding region or variable region of an intact antibody, that retains the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, single-chain antibody molecules, particularly scFv antibody fragments, disulfide-linked Fv (sdFv), Fd fragments consisting of VH and CHI domains, linear antibodies, single-domain antibodies, e.g., sdAb (either VL or VH), camelid VHH domains, multispecific antibodies formed from antibody fragments such as bivalent fragments comprising two Fab fragments linked at the hinge region by a disulfide bridge, and isolated CDRs, or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto polypeptide-based scaffolds such as fibronectin type III (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies). Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments and a remaining "Fc" fragment, a name that reflects the ability to crystallize readily.

[0035] Fragments and derivatives of the antibodies of this invention (included in the term "antibody" as used herein unless otherwise specified or clearly contradicted by the context) can be produced by techniques known in the art. A "fragment" includes a portion of an intact antibody, i.e., generally, an antigen-binding site or a variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments, diabodies, any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as "single-chain antibody fragments" or "single-chain polypeptides"), including, but not limited to, (1) a single-chain Fv molecule, (2) a single-chain polypeptide comprising only one light chain variable domain without associated heavy chain portions, or a fragment thereof comprising the three CDRs of the light chain variable domain, and (3) a single-chain polypeptide comprising only one heavy chain variable region without associated light chain portions, or a fragment thereof comprising the three CDRs of the heavy chain variable region, as well as multispecific antibodies formed from antibody fragments. Fragments of this antibody can be obtained using standard methods.

[0036] For example, Fab or F(ab')2 fragments can be produced by protease digestion of isolated antibodies according to conventional techniques. It is understood that immunoreactive fragments can be modified using known methods, for example, to delay clearance in vivo to obtain a more desirable pharmacokinetic profile, and fragments can be modified with polyethylene glycol (PEG). Methods for coupling PEG to Fab' fragments for site-specific conjugation are described, for example, in Leong et al., Cytokines 16(3):106-119 (2001) and Delgado et al., Br.J.Cancer 5 73(2):175-182 (1996), the disclosures of which are incorporated herein by reference.

[0037] In some embodiments, the antibody is a nanobody. As used herein, the term "nanobody" has its general meaning in the art and refers to an antibody-derived therapeutic protein that contains the unique structural and functional properties of naturally occurring heavy chain antibodies. These heavy chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). As used herein, the term "derived from" refers to the relationship between a first molecule and a second molecule. It generally refers to the structural similarity between the first molecule and the second molecule, and does not imply or include a limitation on the process or source of the first molecule derived from the second molecule.

[0038] In some embodiments, an antibody of the invention comprises a light chain that comprises at least one or at least two of the following CDRs: L-CDR1: QSLLSSR-X8-QK-X 11 -Y, where X8 is T or N, and X 11 is H or T (SEQ ID NO: 2) L-CDR2: FTS (SEQ ID NO: 3) L-CDR3: X1-QHYNTPWT, where X1 is K or Q (SEQ ID NO: 4)

[0039] In some embodiments, an antibody of the invention comprises a heavy chain that comprises at least one or at least two of the following CDRs: H-CDR1: GYTFT-X6-YW, where X6 is S or T (SEQ ID NO:5) H-CDR2: INP-X4-X5-GYT, where X4 is N or R and X5 is T or N (SEQ ID NO: 6) H-CDR3: AVDLGY (SEQ ID NO: 7)

[0040] In some embodiments, an antibody of the invention comprises a light chain comprising at least one CDR, (i) a VL-CDR1 as set forth in SEQ ID NO: 8 (QSLLSSRTQKHY), (ii) a VL-CDR2 as set forth in SEQ ID NO: 3 (FTS), and (iii) a VL-CDR3 as set forth in SEQ ID NO: 9 (KQHYNTPWT), and / or a heavy chain comprising at least one CDR, (i) a VH-CDR1 as set forth in SEQ ID NO: 10 (GYTFTSYW), (ii) a VH-CDR2 as set forth in SEQ ID NO: 11 (INPNTGYT), and (iii) a VH-CDR3 as set forth in SEQ ID NO: 7 (AVDLGY).

[0041] In some embodiments, an antibody of the invention comprises a light chain comprising at least one CDR, (i) a VL-CDR1 as set forth in SEQ ID NO: 12 (QSLLSSRNQKTY), (ii) a VL-CDR2 as set forth in SEQ ID NO: 3 (FTS), and (iii) a VL-CDR3 as set forth in SEQ ID NO: 13 (QQHYNTPWT), and / or a heavy chain comprising at least one CDR, (i) a VH-CDR1 as set forth in SEQ ID NO: 14 (GYTFTTYW), (ii) a VH-CDR2 as set forth in SEQ ID NO: 15 (INPRNGYT), and (iii) a VH-CDR3 as set forth in SEQ ID NO: 7 (AVDLGY).

[0042] In some embodiments, an antibody of the invention comprises a light chain comprising CDRs: (i) a VL-CDR1 set forth in SEQ ID NO: 8 (QSLLSSRTQKHY), (ii) a VL-CDR2 set forth in SEQ ID NO: 3 (FTS), and (iii) a VL-CDR3 set forth in SEQ ID NO: 9 (KQHYNTPWT), and a heavy chain comprising CDRs: (i) a VH-CDR1 set forth in SEQ ID NO: 10 (GYTFTSYW), (ii) a VH-CDR2 set forth in SEQ ID NO: 11 (INPNTGYT), and (iii) a VH-CDR3 set forth in SEQ ID NO: 7 (AVDLGY) (the "3C7 antibody").

[0043] In some embodiments, an antibody of the invention comprises a light chain comprising CDRs: (i) a VL-CDR1 set forth in SEQ ID NO: 12 (QSLLSSRNQKTY), (ii) a VL-CDR2 set forth in SEQ ID NO: 3 (FTS), and (iii) a VL-CDR3 set forth in SEQ ID NO: 13 (QQHYNTPWT), and a heavy chain comprising CDRs: (i) a VH-CDR1 set forth in SEQ ID NO: 14 (GYTFTTYW), (ii) a VH-CDR2 set forth in SEQ ID NO: 15 (INPRNGYT), and (iii) a VH-CDR3 set forth in SEQ ID NO: 7 (AVDLGY) ("9F5 antibody").

[0044] In some embodiments, an antibody of the invention comprises a light chain having 70% identity to SEQ ID NO:16 or SEQ ID NO:18, and / or a heavy chain having 70% identity to SEQ ID NO:17 or SEQ ID NO:19.

[0045] In some embodiments, an antibody of the invention comprises a light chain having 70% identity to SEQ ID NO: 16 and / or a heavy chain having 70% identity to SEQ ID NO: 17 (the "3C7 antibody").

[0046] In some embodiments, an antibody of the invention comprises a light chain having 70% identity to SEQ ID NO: 16 and comprising CDRs (i) VL-CDR1 set forth in SEQ ID NO: 8 (QSLLSSRTQKHY), (ii) VL-CDR2 set forth in SEQ ID NO: 3 (FTS), and (iii) VL-CDR3 set forth in SEQ ID NO: 9 (KQHYNTPWT), and / or a heavy chain having 70% identity to SEQ ID NO: 17 and comprising CDRs (i) VH-CDR1 set forth in SEQ ID NO: 10 (GYTFTSYW), (ii) VH-CDR2 set forth in SEQ ID NO: 11 (INPNTGYT), and (iii) VH-CDR3 set forth in SEQ ID NO: 7 (AVDLGY) (the "3C7 antibody").

[0047] In some embodiments, an antibody of the invention comprises a light chain having the amino acid sequence set forth in SEQ ID NO: 16 and / or a heavy chain having the amino acid sequence set forth in SEQ ID NO: 17 (the "3C7 antibody").

[0048] In some embodiments, an antibody of the invention comprises a light chain having 70% identity to SEQ ID NO: 18 and / or a heavy chain having 70% identity to SEQ ID NO: 19 (the "9F5 antibody").

[0049] In some embodiments, an antibody of the invention comprises a light chain having 70% identity to SEQ ID NO: 18 and comprising CDRs (i) VL-CDR1 set forth in SEQ ID NO: 12 (QSLLSSRNQKTY), (ii) VL-CDR2 set forth in SEQ ID NO: 3 (FTS), and (iii) VL-CDR3 set forth in SEQ ID NO: 13 (QQHYNTPWT), and / or a heavy chain having 70% identity to SEQ ID NO: 19 and comprising CDRs (i) VH-CDR1 set forth in SEQ ID NO: 14 (GYTFTTYW), (ii) VH-CDR2 set forth in SEQ ID NO: 15 (INPRNGYT), and (iii) VH-CDR3 set forth in SEQ ID NO: 7 (AVDLGY) ("9F5 antibody").

[0050] In some embodiments, an antibody of the invention comprises a light chain having the amino acid sequence set forth in SEQ ID NO: 18 and / or a heavy chain having the amino acid sequence set forth in SEQ ID NO: 19 (the "9F5 antibody").

[0051] [ka]

[0052] In the present invention, a first amino acid sequence having at least 70% identity to a second amino acid sequence means that the first sequence has 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the second amino acid sequence. Sequence identity is often measured as a percentage of identity (or similarity or homology), with the higher the percentage, the more similar the two sequences are. Methods of aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444, 1988; Higgins and Sharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS, 5:151-153, 1989; Corpet et al., Nuc. Acids Res., 16:10881-10890, 1988; Huang et al., Comp. Appls. Biosci., 8:155-165, 1992, and Pearson et al., Meth. Mol. Biol., 24:307-31, 1994. Altschul et al., Nat. Genet., 6:119-129, 1994, provide a detailed discussion of sequence alignment methods and homology calculations. As an example, the alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17, 1989) or LFASTA (Pearson and Lipman, 1988) can be used to perform sequence comparisons (Internet Program, 1996, WR Pearson and the University of Virginia, fasta20u63 version 2.0u63, published December 1996). ALIGN compares entire sequences to each other, while LFASTA compares regions of local similarity.These alignment tools and their respective tutorials are available on the Internet, for example, at the NCSA website. Alternatively, for comparison of amino acid sequences of more than about 30 amino acids, the Blast2 sequences function can be used with the default BLOSUM62 matrix set to default parameters (gap existence cost 11, gap cost per residue 1). When aligning short peptides (fewer than about 30 amino acids), the alignment should be performed using the Blast2 sequences function with the PAM30 matrix set to default parameters (start gap penalty 9, extension gap penalty 1). The BLAST sequence comparison system is available, for example, from the NCBI website; see also Altschul et al., J. Mol. Biol., 215:403-410, 1990; Gish and States, Nature Genet., 3:266-272, 1993; Madden et al., Meth. Enzymol., 266:131-141, 1996; Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997; and Zhang and Madden, Genome Res., 7:649-656, 1997.

[0053] In some embodiments, the heavy and / or light chains of the antibodies of the present invention contain conservative sequence modifications. The term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter the biological function of the protein that contains the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into proteins by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A "conservative substitution" is one in which an amino acid is replaced with another amino acid having similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydrophobic / hydrophilic properties of the polypeptide to remain substantially unchanged. Thus, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, such as, for example, their hydrophobicity, hydrophilicity, charge, and size. Exemplary substitutions that take into account the various above-mentioned characteristics are well known to those of skill in the art and include arginine for lysine, glutamic acid for aspartic acid, serine for threonine, glutamine for asparagine, and valine for leucine for isoleucine. Amino acid substitutions can also be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids containing uncharged polar head groups with similar hydrophilicity values ​​include leucine, isoleucine, and valine, glycine and alanine, asparagine and glutamine, and serine, threonine, phenylalanine, and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr, (2) cys, ser, tyr, thr, (3) val, ile, leu, met, ala, phe, (4) lys, arg, his, and (5) phe, tyr, trp, his. Families of other amino acid residues with similar side chains have been defined in the art.These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), polar uncharged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within an antibody of the invention can be substituted with other amino acid residues from the same side chain family, and the modified antibodies can be tested for binding to CD95L.

[0054] In some embodiments, the monoclonal antibodies of the invention cross-compete for binding to a CD95L isoform with a monoclonal antibody comprising a light chain comprising CDRs: (i) VL-CDR1 as set forth in SEQ ID NO: 8 (QSLLSSRTQKHY), (ii) VL-CDR2 as set forth in SEQ ID NO: 3 (FTS), and (iii) VL-CDR3 as set forth in SEQ ID NO: 9 (KQHYNTPWT), and a heavy chain comprising CDRs: (i) VH-CDR1 as set forth in SEQ ID NO: 10 (GYTFTSYW), (ii) VH-CDR2 as set forth in SEQ ID NO: 11 (INPNTGYT), and (iii) VH-CDR3 as set forth in SEQ ID NO: 7 (AVDLGY).

[0055] In some embodiments, the monoclonal antibodies of the invention cross-compete for binding to a CD95L isoform with a monoclonal antibody comprising a light chain comprising CDRs: (i) VL-CDR1 as set forth in SEQ ID NO: 12 (QSLLSSRNQKTY), (ii) VL-CDR2 as set forth in SEQ ID NO: 3 (FTS), and (iii) VL-CDR3 as set forth in SEQ ID NO: 13 (QQHYNTPWT), and a heavy chain comprising CDRs: (i) VH-CDR1 as set forth in SEQ ID NO: 14 (GYTFTTYW), (ii) VH-CDR2 as set forth in SEQ ID NO: 15 (INPRNGYT), and (iii) VH-CDR3 as set forth in SEQ ID NO: 7 (AVDLGY).

[0056] As used herein, the term "cross-competing" refers to monoclonal antibodies that share the ability to bind to a specific region of an antigen. In the present disclosure, a "cross-competing" monoclonal antibody has the ability to block the binding of other monoclonal antibodies to an antigen in a standard competitive binding assay. Such a monoclonal antibody may bind to the same, related, or nearby (e.g., structurally similar or spatially close) epitope as the competing antibody, in a non-limiting theory. Cross-competition exists when antibody A reduces the binding of antibody B by at least 60%, specifically at least 70%, and more specifically at least 80%, compared to a positive control that does not have one of the antibodies, and vice versa. As one skilled in the art will appreciate, competition can be evaluated in a variety of assay settings. One suitable assay involves the use of Biacore technology (e.g., by using a BIAcore3000 instrument (Biacore, Uppsala, Sweden)), which can measure the degree of interaction using surface plasmon resonance technology. Other assays for measuring cross-competition use ELISA-based techniques. Furthermore, a high throughput process for "binning" antibodies based on their cross-competitiveness is described in WO 2003 / 48731.

[0057] In the present invention, the cross-competing antibody as described above retains the activity of a monoclonal antibody comprising a light chain comprising CDRs which are (i) VL-CDR1 shown in SEQ ID NO: 8 (QSLLSSRTQKHY), (ii) VL-CDR2 shown in SEQ ID NO: 3 (FTS), and (iii) VL-CDR3 shown in SEQ ID NO: 9 (KQHYNTPWT), and a heavy chain comprising CDRs which are (i) VH-CDR1 shown in SEQ ID NO: 10 (GYTFTSYW), (ii) VH-CDR2 shown in SEQ ID NO: 11 (INPNTGYT), and (iii) VH-CDR3 shown in SEQ ID NO: 7 (AVDLGY).

[0058] In the present invention, the cross-competing antibody as described above retains the activity of a monoclonal antibody comprising a light chain comprising CDRs which are (i) VL-CDR1 shown in SEQ ID NO: 12 (QSLLSSRNQKTY), (ii) VL-CDR2 shown in SEQ ID NO: 3 (FTS), and (iii) VL-CDR3 shown in SEQ ID NO: 13 (QQHYNTPWT), and a heavy chain comprising CDRs which are (i) VH-CDR1 shown in SEQ ID NO: 14 (GYTFTTYW), (ii) VH-CDR2 shown in SEQ ID NO: 15 (INPRNGYT), and (iii) VH-CDR3 shown in SEQ ID NO: 7 (AVDLGY).

[0059] To determine whether a cross-competing antibody retains the desired activity, any assay known in the art may be suitable. For example, the assay described in the Examples, based on determining the ability to block the cleavage of CD95L by metalloproteases such as MMP-2 or MMP-3, may be suitable for determining whether an antibody retains the above-mentioned ability.

[0060] The antibody of the present invention is produced by any technique known in the art, including, but not limited to, any chemical, biological, genetic, or enzymatic technique, alone or in combination. Typically, one skilled in the art can easily produce the above-mentioned antibody by standard techniques for the production of polypeptides, knowing the amino acid sequence of the desired sequence. For example, it can be synthesized using the well-known solid-phase method, preferably using a commercially available peptide synthesizer, such as those manufactured by Applied Biosystems (Foster City, California), following the manufacturer's instructions. Alternatively, the antibody of the present invention can be synthesized by recombinant DNA techniques well known in the art. For example, the antibody can be obtained as a DNA expression product after incorporating a DNA sequence encoding the antibody into an expression vector and introducing such a vector into a suitable eukaryotic or prokaryotic host that is capable of expressing the desired antibody and from which the antibody can then be isolated using well-known methods.

[0061] A further object of the invention therefore relates to a nucleic acid molecule encoding an antibody according to the invention, more particularly, said nucleic acid molecule encoding the heavy and / or light chain of said antibody according to the invention.

[0062] As used herein, the term "encode" refers to the unique property of a particular sequence of nucleotides in a polynucleotide, e.g., a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the biological properties derived therefrom. Thus, a gene, cDNA, or RNA encodes a protein when it produces the protein in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually shown in the sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA, can be referred to as encoding a protein or other product of the gene or cDNA. Unless specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Also, the phrase "nucleotide sequence or RNA encoding a protein" can include introns to the extent that a nucleotide sequence encoding a protein may include introns in some types.

[0063] Typically, the above-mentioned nucleic acid is a DNA or RNA molecule, which can be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector. As used herein, the terms "vector", "cloning vector", and "expression vector" refer to a vehicle that can introduce a DNA or RNA sequence (e.g., a foreign gene) into a host cell to transform the host and enhance the expression (e.g., transcription and translation) of the introduced sequence. Thus, a further object of the present invention relates to a vector comprising the nucleic acid of the present invention. Such a vector can include regulatory elements such as promoters, enhancers, and terminators to express or induce the expression of the above-mentioned antibody upon administration to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include the SV40 early promoter and enhancer, the Moloney murine leukemia virus LTR promoter and enhancer, and the immunoglobulin H chain promoter and enhancer, etc. Any expression vector for animal cells can be used as long as it can insert and express a gene encoding a human antibody C region. Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, and pSG1βd2-4, etc. Examples of other plasmids include replicative plasmids that contain an origin of replication, or integrative plasmids, such as pUC, pcDNA, and pBR. Examples of other viral vectors include adenovirus, retrovirus, herpes virus, and AAV vectors. Such recombinant viruses can be produced by methods known in the art, such as by transfecting packaging cells, or by transiently transfecting helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc.Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO 95 / 14785, WO 96 / 22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056, and WO 94 / 19478.

[0064] The term "promoter / regulatory sequence" refers to a nucleic acid sequence (e.g., a DNA sequence) that is recognized by the synthetic machinery of a cell or introduced synthetic machinery required to initiate specific transcription of a polynucleotide sequence, thereby allowing expression of a gene product that is operably linked to the promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses a gene product in a tissue-specific manner.

[0065] The term "operably linked" or "transcriptional control" refers to the functional linkage of a heterologous nucleic acid sequence with a regulatory sequence that results in the expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when the promoter affects the transcription or expression of the coding sequence. The operably linked DNA sequences can be close to each other, and can be in the same reading frame, for example, when two protein coding regions need to be joined.

[0066] A further object of the present invention relates to host cells transfected, infected or transformed with the nucleic acids and / or vectors according to the invention.

[0067] In certain embodiments, the host cell comprises the nucleic acid and / or vector of the invention. As used herein, the term "transformation" refers to the introduction of a "foreign" (i.e., exogenous or extracellular) gene, DNA, or RNA sequence into a host cell, such that the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme, encoded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA is "transformed."

[0068] The nucleic acids of the invention can be used to produce the antibodies of the invention in a suitable expression system. The term "expression system" refers to a host cell and a corresponding vector under suitable conditions for the expression of a protein encoded by, for example, the foreign DNA carried by the vector and introduced into the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, yeasts of Kluyveromyces or Saccharomyces, mammalian cell lines (such as Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (such as those made from lymphoblast cells, fibroblast cells, embryonic cells, epithelial cells, neuronal cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") (Urlaub G et al; 1980), and rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells"). The present invention also relates to a method for producing a recombinant host cell expressing an antibody of the present invention, the method comprising the steps of (i) introducing a recombinant nucleic acid or vector as described above into a competent host cell in vitro or ex vivo, (ii) culturing the resulting recombinant host cell in vitro or ex vivo, and (iii) optionally selecting cells expressing and / or secreting the antibody described above. Such recombinant host cells can be used to produce the antibody of the present invention.

[0069] Examples of vectors include all those known in the art, including, without limitation, cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating the recombinant polynucleotide.

[0070] The antibodies of the invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0071] In some embodiments, the antibodies of the invention (preferably monoclonal antibodies) do not comprise an Fc region that mediates antibody-dependent cell-mediated cytotoxicity and therefore do not comprise an Fc moiety that induces antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the antibodies of the invention (preferably monoclonal antibodies) do not comprise an Fc region that induces complement-dependent cytotoxicity (CDC) or antibody-dependent phagocytosis. In some embodiments, the antibodies of the invention (preferably monoclonal antibodies) do not directly or indirectly result in depletion of cells expressing a CD95L polypeptide (e.g., CD95L +In some embodiments, the antibodies (preferably monoclonal antibodies) of the invention do not comprise an Fc domain capable of substantially binding to an FcγRIIIA (CD16) polypeptide. In some embodiments, the antibodies (preferably monoclonal antibodies) of the invention do not comprise an Fc domain (e.g., no CH2 and / or CH3 domain) or comprise an Fc domain of an IgG2 or IgG4 isotype. In some embodiments, the antibodies (preferably monoclonal antibodies) of the invention comprise an Fc domain (e.g., of an IgG1) with an altered glycosylation profile, eliminating ADCC activity of the antibody. In some embodiments, the antibodies (preferably monoclonal antibodies) of the invention consist of or comprise a Fab, Fab', Fab'-SH, F(ab')2, Fv, diabody, single chain antibody fragment, or a multispecific antibody comprising multiple different antibody fragments. In some embodiments, the antibodies (preferably monoclonal antibodies) of the invention are not linked to a toxic moiety. In some embodiments, one or more selected amino acids from the amino acid residues can be replaced with a different amino acid residue such that the antibody has altered C2q binding and / or reduced or eliminated CDC. This approach is described in further detail in U.S. Patent No. 6,194,551 by Idusogie et al.

[0072] As used herein, the term "Fc region" includes the polypeptides that contain the constant region of an antibody other than the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the N-terminal movable hinges of these domains. In IgA and IgM, Fc can include the J chain. In IgG, Fc includes the immunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3), and the hinge between Cgamma1 (Cγ1) and Cgamma2 (Cγ2). Although the boundaries of the Fc region may vary, the Fc region of a human IgG heavy chain is usually defined to include residues C226 or P230 to its carboxyl terminus, with numbering according to the EU index as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). "EU index as described by Kabat" refers to the residue numbering of the EU antibody of human IgG1 as described by Kabat et al., supra. Fc may refer to this region in isolation or to this region in the case of an antibody, antibody fragment, or Fc fusion protein. An Fc variant protein can be an antibody, an Fc fusion, or any protein or protein domain that includes an Fc region. Particularly preferred are proteins that include variant Fc regions that are non-naturally occurring variants of the Fc region. The amino acid sequence of a non-naturally occurring Fc region (also referred to herein as a "variant Fc region") includes at least one amino acid residue substitution, insertion, and / or deletion compared to the wild-type amino acid sequence. Any new amino acid residues that appear in the sequence of the variant Fc region as a result of insertion or substitution may be referred to as non-naturally occurring amino acid residues. Of note, polymorphisms have been observed at a number of Fc positions, including but not limited to Kabat 270, 272, 312, 315, 356, and 358, and therefore slight differences between the present and prior art sequences may exist.

[0073] The term "Fc receptor" or "FcR" is used to refer to a receptor that binds to the Fc region of an antibody. Primary cells that mediate ADCC express FcγRIII, and monocytes express FcγRI, FcγRII, FcγRIII, and / or FcγRIV. FcR expression on hematopoietic cells is described in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991). To assess the ADCC activity of a molecule, an in vitro ADCC assay such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337 can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of the molecule of interest can be evaluated in vivo, for example in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA), 95:652-656 (1998). As used herein, the term "effector cell" refers to a leukocyte that expresses one or more FcRs and exhibits effector function. The aforementioned cells express at least FcγRI, FcγRII, FcγRIII, and / or FcγRIV and exhibit ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils.

[0074] Thus, a further object of the present invention relates to an antibody of the present invention for use as a medicament. More specifically, the present invention provides a method of treatment in a patient in need of treatment, said method comprising administering to the patient a therapeutically effective amount of an antibody of the present invention.

[0075] As used herein, "treatment" or "treating" is an approach to obtain beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from a disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the progression of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or slowing the progression of the disease, ameliorating the disease state, remission (partially or completely) of the disease, reducing the dose of one or more other medicines required to treat the disease, slowing the progression of the disease, improving quality of life, and / or prolonging survival. "Treatment" also encompasses the alleviation of the pathological consequences of cancer. The methods of the present invention contemplate any one or more of these aspects of treatment. In some embodiments, the term "treat" or "treatment" refers to therapeutic treatment and preventative or prophylactic measures, the purpose of which is to prevent or slow (alleviate) the targeted disease. Thus, in some embodiments, those in need of treatment can include those already with the disorder as well as those prone to have the disorder or those in which the disorder is to be prevented.

[0076] In some embodiments, the antibody of the present invention is specifically for the treatment of cancer in a subject in need of such treatment.As used herein, the term "cancer" has its general meaning in the art, including, but not limited to, solid tumors and blood-borne tumors.The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood, and blood vessels.The term "cancer" further includes both primary and metastatic cancers.Examples of cancers that can be treated by the methods and compositions of the present invention include, but are not limited to, cancer cells of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, skin, stomach, testis, tongue, or uterus. Further, the cancer may specifically be malignant neoplasm, carcinoma, undifferentiated carcinoma, giant cell and spindle cell carcinoma, small cell carcinoma, papillary carcinoma, squamous cell carcinoma, lymphoepithelial carcinoma, basal cell carcinoma, hair matrix carcinoma, transitional cell carcinoma, papillary transitional cell carcinoma, adenocarcinoma, malignant gastrinoma, cholangiocarcinoma, hepatocellular carcinoma, mixed hepatocellular carcinoma and cholangiocarcinoma, trabecular adenocarcinoma, adenoid cystic carcinoma, adenocarcinoma in adenomatous polyps, familial polyposis coli adenocarcinoma, solid tumor, malignant carcinoid tumor, branchio-alveolar adenocarcinoma, papillary adenocarcinoma, chromophobe carcinoma, eosinophilic carcinoma, acidophilic adenocarcinoma, basophilic carcinoma, clear cell adenocarcinoma, granular cell carcinoma, follicular adenocarcinoma, papillary and follicular adenocarcinoma, nonencapsulated sclerosing carcinoma, adrenal cortical carcinoma, endometrioid carcinoma,carcinoma), skin adnexal carcinoma, apocrine gland carcinoma, sebaceous gland carcinoma, earwax gland carcinoma, mucinous epidermoid carcinoma, cystadenocarcinoma, papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, mucinous cystadenocarcinoma, mucinous adenocarcinoma, signet ring cell carcinoma, invasive ductal carcinoma, medullary carcinoma, lobular carcinoma, inflammatory carcinoma, Paget's disease of the breast, acinic cell carcinoma, adenosquamous carcinoma, adenocarcinoma with squamous metaplasia, malignant thymoma, malignant ovarian stromal tumor, malignant thecal tumor, malignant granulosa cell tumor, Malignant neuroblastoma (roblastoma), Sertoli cell carcinoma, malignant Leydig cell tumor, malignant lipocytoma, malignant paraganglioma, malignant extramammary paraganglioma, pheochromocytoma, glomus angiosarcoma, malignant melanoma, amelanotic melanoma, superficial spreading melanoma, malignant melanoma in giant pigmented nevus, epithelioid cell melanoma, malignant blue nevus, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, myxosarcoma, liposarcoma tumor, leiomyosarcoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, stromal sarcoma, malignant mixed tumor, mixed Müllerian tumor, nephroblastoma, hepatoblastoma, carcinosarcoma, malignant mesenchymoma, malignant Brenner tumor, malignant phyllodes tumor, synovial sarcoma, malignant mesothelioma, dysgerminoma, embryonal carcinoma, malignant teratoma, malignant ovarian thyroid tumor, choriocarcinoma, malignant mesothelioma, angiosarcoma, malignant hemangioendothelioma, Kaposi's sarcoma, malignant hemangiopericytoma, lymphangiosarcoma, bone Sarcoma, Parosteal Osteosarcoma, Chondrosarcoma, Malignant Chondroblastoma, Mesenchymal Chondrosarcoma, Giant Cell Tumor of Bone, Ewing's Sarcoma, Malignant Odontogenic Tumor, Ameloblastic Odontosarcoma, Malignant Ameloblastic Tumor, Ameloblastic Fibrosarcoma, Malignant Pinealoma, Chordoma, Malignant Glioma, Ependymoma, Astrocytoma, Protoplasmic Astrocytoma, Fibrous Astrocytoma, Astroblastoma, Glioblastoma, Oligodendroglioma, Primitive Neuroectodermal Tumorneuroectodermal), cerebellar sarcoma, ganglioneuroblastoma, neuroblastoma, retinoblastoma, olfactory neurogenic tumor, malignant meningioma, neurofibrosarcoma, malignant schwannoma, malignant granular cell tumor, malignant lymphoma, Hodgkin's disease, Hodgkin's lymphoma, lateral granuloma, malignant small lymphocytic lymphoma, malignant diffuse large cell lymphoma, malignant follicular lymphoma, mycosis fungoides, other specified non-Hodgkin's disease The histology may be, but is not limited to, lymphoma, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative small intestinal disease, leukemia, lymphocytic leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma cell leukemia, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myeloid sarcoma, and hairy cell leukemia.

[0077] In some embodiments, the subject is afflicted with a cancer selected from the group consisting of breast cancer, colon cancer, lung cancer, prostate cancer, testicular cancer, brain cancer, skin cancer, rectal cancer, gastric cancer, esophageal cancer, sarcoma, tracheal cancer, head and neck cancer, pancreatic cancer, liver cancer, ovarian cancer, lymphatic cancer, cervical cancer, vulvar cancer, melanoma, mesothelioma, kidney cancer, bladder cancer, thyroid cancer, bone cancer, carcinoma, sarcoma, and soft tissue cancer.

[0078] In some embodiments, the antibody of the present invention is specifically for treating triple-negative breast cancer.As used herein, the expression "triple-negative breast cancer" has its general meaning in the art, and means that the breast cancer lacks or expresses low levels of the receptors for the hormones estrogen (ER negative) and progesterone (PR negative) and the protein HER2.

[0079] In some embodiments, the antibodies of the invention are specifically for the prevention of metastasis (eg, in subjects suffering from triple-negative breast cancer).

[0080] In some embodiments, the antibodies of the present invention are intended to enhance the therapeutic efficacy of cancer treatment, particularly in subjects in need thereof. In some embodiments, the methods of the present invention comprise administering to a subject a therapeutically effective amount of the antibodies of the present invention, either sequentially or simultaneously with one or more therapeutically active agents, such as chemotherapeutic or radiotherapeutic agents. Examples of chemotherapeutic agents include fludarabine, gemcitabine, capecitabine, methotrexate, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin, and oxaliplatin, mitomycin, dacarbazine, procarbazine, epipodophyllotoxins such as etoposide and teniposide, camptothecins such as irinotecan and topotecan, bleomycin, doxorubicin, idarubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epirubicin, 5- These include, but are not limited to, fluorouracil and 5-fluorouracil in combination with leucovorin, taxanes such as docetaxel and paclitaxel, levamisole, estramustine, nitrogen mustards, nitrosoureas such as carmustine and lomustine, vinca alkaloids such as vinblastine, vincristine, vindesine, and vinorelbine, imatinib mesylate, hexamethylmelamine, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, herbimycin A inhibitors, genistein, arbustatin, and lavendustin A. In some embodiments, the additional therapeutically active agent may be selected from, but is not limited to, one or a combination of the following drug classes: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, antifolates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxins, hormonal therapy agents, retinoids, photosensitizers or photodynamic therapy agents, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, anthracyclines, MDR inhibitors, and Ca2+ ATPase inhibitors.As used herein, the term "radiotherapy agent" is intended to refer to any radiotherapy agent known to those skilled in the art to be effective in treating or curing cancer, without limitation.For example, radiotherapy agent can be an agent such as that administered in brachytherapy or radionuclide therapy.Such methods can optionally further comprise the administration of one or more additional cancer therapy agents, such as but not limited to chemotherapy agents and / or other radiotherapy agents.

[0081] In some embodiments, the antibodies of the present invention are specifically for the treatment of autoimmune inflammatory diseases. In some embodiments, the autoimmune inflammatory diseases are arthritis, rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebral arthritis, and juvenile onset rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, osteoart ... deformans), chronic primary polyarthritis, reactive arthritis, and ankylosing spondylitis, inflammatory hyperproliferative skin diseases, such as psoriasis, e.g., plaque psoriasis, guttate psoriasis, pustular psoriasis, and nail psoriasis, dermatitis, including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic dermatitis, chronic allergic urticaria, including X-linked hyper-IgM syndrome, chronic autoimmune urticaria, Urticaria including measles and chronic idiopathic urticaria, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma, systemic sclerosis, sclerosis, systemic sclerosis, multiple sclerosis (MS), spino-optical MS, primary progressive MS (PPMS), and relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, and ataxic sclerosis, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, polypoid colitispolyposa), necrotizing enterocolitis, transmural colitis, autoimmune inflammatory bowel disease, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis, respiratory distress syndrome, adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune blood disorders, rheumatoid spondylitis, sudden hearing loss, e.g. anaphylaxis, and allergic and atopic nasal IgE-mediated diseases such as encephalitis, encephalitis, Rasmussen's encephalitis, limbic encephalitis and / or brainstem encephalitis, uveitis, anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, autoimmune uveitis, glomerulonephritis (GN), idiopathic membranous GN or idiopathic membranous nephropathy, membranoproliferative proliferative or membranous proliferative GN (MPGN), rapidly progressive GN, allergic conditions, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) or systemic lupus erythematodes, e.g. cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), generalized lupus erythematosusdisseminatus), lupus (including nephritis, encephalitis, pediatric, non-renal, extrarenal, discoid, alopecia), juvenile-onset (type I) diabetes mellitus including insulin-dependent diabetes mellitus of childhood (IDDM), adult-onset (type II) diabetes mellitus, autoimmune diabetes mellitus, idiopathic diabetes insipidus, immune responses related to acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, tuberculosis, sarcoidosis, granulomatosis, lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitis, large vasculitis, rheumatoid polyposis Myalgia, giant cell (Takayasu) arteritis, medium-sized vasculitis, Kawasaki disease, polyarteritis nodosa, microscopic polyarteritis, CNS vasculitis, necrotizing, cutaneous, hypersensitivity vasculitis, systemic necrotizing vasculitis, and vasculitis including ANCA-associated vasculitis, e.g., Churg-Strauss vasculitis or syndrome (CSS), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond-Blackfan anemia, hemolytic anemia, or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (perniciosa), Addison's disease, pure red cell anemia or pure red cell aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte emigration, CNS inflammatory disorders, multiple organ injury syndromes such as sepsis, trauma, or massive hemorrhage, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, antiphospholipid syndrome, allergic neuritis, Bechet's or Behcet's disease, Castleman syndrome, Goodpasture's syndrome group, Raynaud's syndrome, Sjögren's syndrome, Stevens-Johnson syndrome, pemphigoid, e.g. bullous pemphigoid and cutaneous pemphigoid, pemphigus, optionally pemphigus vulgaris, pemphigus foliaceus, mucous membrane pemphigoid, pemphigus erythematous, autoimmune polyendocrinopathy, Reiter's disease or syndrome, immune complex nephritis, antibody-mediated nephritis, neuromyelitis optica, polyneuropathy, chronic neuropathy, IgM polyneuropathy, IgM-mediated neuropathy, thrombocytopenia, thrombotic thrombocytopenic purpura (TTP), idiopathic hematologic Thrombocytopenic purpura (ITP), autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune thyroiditis, Hashimoto's thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis), subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Graves' disease, polyglandular syndromes such as autoimmune polyglandular syndrome (or polyendocrine syndrome), paraneoplastic syndromes including neurological paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff man or Stiff-person syndrome, encephalomyelitis, allergic encephalomyelitis, experimental allergic encephalomyelitis (EAE), myasthenia gravis, thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus-myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia, bronchiolitis obliterans (non-transplant) vs.NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease, Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear diseases such as autoimmune inner ear disease (AGED), autoimmune hearing loss, opsoclonus-myoclonus syndrome (OMS), polychondritides such as refractory or relapsing polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleral lymphocytosis, noncancerous lymphocytosis, monoclonal B-cell lymphocytosis, primary lymphocytosis, optionally including benign monoclonal gammopathy or monoclonal gammopathy of undetermined significance (MGUS), peripheral neuropathy, paraneoplastic syndromes, such as epilepsy, migraine, arrhythmias, myopathy, hearing loss, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveitis, chorioretinitis, autoimmune hepatopathy, fibromyalgia, polyendocrine deficiency, Schmidt's syndrome, adrenalitis, gastrotrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases, diabetic nephropathy, Dressler's syndrome, alopecia areata, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility, mixed connective tissue disease, Chagas' disease, Rheumatic fever, recurrent pregnancy loss, farmer's lung, erythema multiforme, postpericardiotomy syndrome, Cushing's syndrome, bird breeder's disease, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reactions, leprosy, malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndromesyndrome, Kaplan's syndrome, dengue fever, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, elevatum et diutinum erythema, erythroblastosis fetalis, eosinophilic fasciitis, Charmant's syndrome, Felty's syndrome, filariasis, cyclitis such as chronic cyclitis, heterochromatic cyclitis, iridocyclitis, or Fuchs' cyclitis, Henoch-Schönlein purpura, human immunodeficiency virus (HIV) infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Epstein-Barr virus infection, mumps, Evans' syndrome, autoimmune gonadal dysfunction, Sydenham's chorea, poststreptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, spinal cord fistula, choroiditis, giant cell polymyalgia, endocrine ophthalmopathy, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, retinal autoimmunity, arthritis, bronchitis, chronic obstructive airway disease, silicosis, aphthosis, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Beck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, allergic enterocolitis, erythema nodosum leprosum, idiopathic facial nerve palsy, chronic fatigue syndrome, rheumatic fever, Hammann-Rich disease, sensorineural hearing loss, paroxysmal hemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leukopenia, infectious mononucleosis infectiosa, transverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, granulomatous orchitis, pancreatitis (e.g. chronic pancreatitis), polyradiculitis acuta, pyoderma gangrenosum, Kervan thyroiditis, acquired splenic atrophy, antispermatozoan antibodiesantobody-related infertility, non-malignant thymoma, vitiligo, SCID and Epstein-Barr virus-related diseases, acquired immune deficiency syndrome (AIDS), parasitic diseases such as Leishmania, toxic shock syndrome, food poisoning, conditions involving T cell infiltration, leukocyte adhesion deficiency, immune responses related to cytokines and T lymphocyte-mediated acute and delayed hypersensitivity, diseases involving leukocyte emigration, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathy, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyglandular insufficiency, peripheral neuropathy, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis, dilated cardiomyopathy, epidermolysis bullosa acquisita (EBA), hemochromatosis, myocarditis, Nephrotic syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary or sphenoid sinusitis, eosinophil-related diseases such as eosinophilia, pulmonary infiltrative eosinophilia, eosinophilic myalgia syndrome, Löffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopulmonary aspergillosis aspergillosis, aspergilloma, or eosinophil-containing granuloma, anaphylaxis, seronegative spondyloarthritis, polyglandular autoimmune disease, sclerosing cholangitis, scleritis, episcleritis, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia-telangiectasia, autoimmune disease associated with connective tissue disease, rheumatism, neurological disease, ischemia-reperfusion injury, hyporesponsiveness to blood pressure, vascular dysfunction, antgiectasis, tissue injury, cardiovascular ischemia, hyperalgesia, brain ischemia, and diseases involving angiogenesis, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, reperfusion injury of the myocardium or other tissues, skin diseases with an acute inflammatory component, skin diseases with acute purulent meningitis or other central nervous system inflammatory disorders, inflammatory disorders of the eye and orbit, granulocyte transfusion associated syndrome, cytokine-induced toxicity, acute intense inflammation, chronic refractory inflammation, pyelitis, pneumonocirrhosis, diabetic retinopathy, diabetic aortopathy, endarterial hyperplasia, peptic ulcer, valvulitis, and endometriosis.

[0082] In some embodiments, the antibodies of the invention are particularly suitable for the treatment of systemic lupus erythematosus.

[0083] A further object of the present invention relates to a composition comprising, consisting of or consisting essentially of an antibody of the present invention.

[0084] As used herein, "consisting essentially of," in reference to a composition, means that at least one antibody of the invention, as described herein above, is the only therapeutic or biologically active agent within the composition.

[0085] In some embodiments, the composition of the invention is a pharmaceutical composition and further comprises a pharma- ceutically acceptable carrier.

[0086] The term "pharmaceutically acceptable carrier" refers to an excipient that does not produce harmful, allergic, or other untoward reactions when administered to an animal, preferably a human. This includes any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. For human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by regulatory agencies, such as the FDA or EMA.

[0087] Pharmaceutically acceptable carriers that can be used in the composition 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, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat. For use in administering to a patient, the composition can be formulated for administration to a patient. The composition of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an indwelling reservoir. As used herein, it includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. The sterile injectable form of the composition of the present invention can be an aqueous or oily suspension. These suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any bland fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharma- ceutical-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants, commonly used in the formulation of pharma- ceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween, Span, and other emulsifiers or bioavailability enhancers commonly used in the manufacture of pharma-ceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes. The compositions of the present invention may be orally administered in any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions, or solutions. In the case of tablets for oral use, commonly used carriers include lactose and cornstarch. Lubricants, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include, for example, lactose. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweeteners, flavors, or coloring agents may also be added. Alternatively, the compositions of the present invention can be administered in the form of suppositories for rectal administration. These can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, and therefore dissolves in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol. The compositions of the present invention can also be administered topically, especially when the target of treatment includes areas or organs that are easily accessible by topical application, including diseases of the eye, skin, or lower intestinal tract. Suitable topical formulations are easily prepared for each of these areas or organs. For topical application, the compositions can be formulated into a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water.Alternatively, the compositions can be formulated into a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharma- ceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. Topical application for the lower intestinal tract can be with a rectal suppository (see above) or a suitable enema formulation. Patches can also be used. The compositions of the present invention can also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and can be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents. For example, the antibody present in the pharmaceutical composition of the present invention can be provided at a concentration of 10 mg / mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. The product is formulated for intravenous administration in 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80, and sterile water for injection. The pH is adjusted to 6.5. An example of a suitable dosage range for the antibody in the pharmaceutical composition of the invention is about 1 mg / m. 2 ~500mg / m 2 However, it is understood that these plans are exemplary, and optimal plans and regimens can be adapted taking into account the affinity and tolerability of a particular antibody in the pharmaceutical composition, which needs to be determined in clinical trials. The pharmaceutical composition of the present invention for injection (e.g., intramuscular, intravenous) can be prepared to contain sterile buffer water (e.g., 1 ml for intramuscular injection) and about 1 ng to about 100 mg, for example, about 50 ng to about 30 mg or more, preferably about 5 mg to about 25 mg of the antibody of the present invention.

[0088] The present invention is further illustrated by the following figures and examples, which should not, however, be construed in any way as limiting the scope of the present invention. EXAMPLES

[0089] There is much inconsistency as to how CD95L is cleaved to release s-CD95L, and so far, various metalloprotease cleavage sites have been suggested in the sequence of CD95L. We have made various extracellular domains of CD95L fused to larger domains, including the Ig domain of gp190 (the LIF receptor). When cleaved by metalloproteases, they produce small molecular size fragments that can be easily observed by Western blotting. Using this Ig-CD95L, we have shown that MMP2 and MMP3 can cleave CD95L. These proteases show various cleavage sites within the stalk region of CD95L (Figure 1 and Figure 2A), making it difficult to address the identification of the enzyme responsible for CD95L cleavage in lupus patients. To precisely identify the cleavage site within CD95L and the metalloproteases responsible for this process, a 43-mer (ranging from residue 103 to residue 145) corresponding to the stalk region of CD95L was incubated with the same concentrations of MMPs and ADAMs. MMP2, MMP3, MMP7, MMP12, and MMP13, as well as ADAM10, efficiently degrade / cleave the 43-mer (i.e., ≥20% cleavage, Figure 1B). Subsequently, privileged cleavage sites (not seen in MMPs) were identified. 113 EL 114 , and ADAM 129 KQ 130 ) was determined from MS / MS sequencing of the resulting cleavage products.

[0090] We hypothesized that monoclonal antibodies targeting the metalloprotease cleavage site (MCSC) of CD95L might block access to these enzymes and inhibit CD95L shedding. We therefore generated antibodies corresponding to various epitopes within MCSC (Fig. 2A), and two of them (i.e., 3C7 and 9F5) effectively blocked CD95L cleavage by MMP2 and MMP3 in vitro (Fig. 2B, Table 1). In contrast, antibody 3B9, which binds to the stalk region spanning amino acid residues 130 to 136, did not block CD95L cleavage by MMP2 and MMP3 in vitro (Fig. 2B, Table 1).

[0091] In cellulo, incubation of CD95L-expressing T cells (i.e., 1A12 cells) with 9F5 did not alter the expression levels of membrane-bound CD95L, whereas 3C7 treatment led to an enhancement of m-CD95L similar to that observed with the broad-spectrum MMP inhibitor GM6001 (Figure 2C). Inhibition of CD95L cleavage reduced the soluble counterpart measured in the supernatant (Figure 2D). Using a Boyden chamber assay, incubation of 1A12 (CD95L+) cells with 3C7mAb inhibited the generation of s-CD95L, which is responsible for promoting migration of triple-negative breast cancer cells MDA-MB-231 (Figure 3A). Moreover, while 3C7mAb inhibited the cleavage of CD95L, it did not block the apoptotic function of its membrane-bound counterpart (Figure 3B). This finding indicates that these MCSC-targeted mAbs can compete with MMPs and inhibit the release of proinflammatory s-CD95L without affecting the apoptotic role of its membrane-bound counterpart, making them a unique and attractive therapeutic option.

[0092] [Table 1]

[0093] Table 1 shows the effect of the antibodies produced on CD95L cleavage by metalloproteases.

[0094] References Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are incorporated herein by reference into this disclosure.

Claims

1. An antibody that binds to the extracellular domain of CD95L, wherein the antibody prevents cleavage of CD95L by at least one metalloprotease, and the epitope of the antibody comprises at least one amino acid residue in an amino acid sequence ranging from amino acid residue 110 to amino acid residue 130 in SEQ ID NO:

1.

2. Blocking cleavage of CD95L by MMP-2 and / or MMP-3; The antibody described in claim 1.

3. binds to an epitope comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acid residues from amino acid residues 111 to 129 of SEQ ID NO:1; The antibody described in claim 1.

4. Binds to conformational epitopes, The antibody described in claim 1.

5. It is a monoclonal antibody, The antibody described in claim 1.

6. a chimeric, humanized, or human antibody; The antibody described in claim 1.

7. is an antibody fragment, The antibody described in claim 1.

8. - L-CDR1: QSLLSSR-X8-QK-X11-Y, where X8 is T or N and X11 is H or T (SEQ ID NO: 2) - L-CDR2: FTS (SEQ ID NO: 3) - L-CDR3: X1-QHYNTPWT, where X1 is K or Q (SEQ ID NO: 4); and a light chain comprising at least one or at least two of the CDRs The antibody described in claim 1.

9. - H-CDR1: GYTFT-X6-YW, where X6 is S or T (SEQ ID NO: 5) - H-CDR2: INP-X4-X5-GYT, where X4 is N or R and X5 is T or N (SEQ ID NO: 6) - H-CDR3: AVDLGY (SEQ ID NO: 7), and a heavy chain comprising at least one or at least two of the CDRs The antibody described in claim 1.

10. (i) a light chain comprising at least one CDR of VL-CDR1 shown in SEQ ID NO:8 (QSLLSSRTQKHY), (ii) a VL-CDR2 shown in SEQ ID NO:3 (FTS), and (iii) a VL-CDR3 shown in SEQ ID NO:9 (KQHYNTPWT), and / or a heavy chain comprising at least one CDR of VH-CDR1 shown in SEQ ID NO:10 (GYTFTSYW), (ii) a VH-CDR2 shown in SEQ ID NO:11 (INPNTGYT), and (iii) a VH-CDR3 shown in SEQ ID NO:7 (AVDLGY), The antibody described in claim 1.

11. (i) a light chain comprising at least one CDR having a VL-CDR1 as set forth in SEQ ID NO: 12 (QSLLSSRNQKTY), (ii) a VL-CDR2 as set forth in SEQ ID NO: 3 (FTS), and (iii) a VL-CDR3 as set forth in SEQ ID NO: 13 (QQHYNTPWT), and / or a heavy chain comprising at least one CDR having a VH-CDR1 as set forth in SEQ ID NO: 14 (GYTFTTYW), (ii) a VH-CDR2 as set forth in SEQ ID NO: 15 (INPRNGYT), and (iii) a VH-CDR3 as set forth in SEQ ID NO: 7 (AVDLGY); The antibody described in claim 1.

12. (i) a light chain comprising CDRs having a VL-CDR1 set forth in SEQ ID NO:8 (QSLLSSRTQKHY), (ii) a VL-CDR2 set forth in SEQ ID NO:3 (FTS), and (iii) a VL-CDR3 set forth in SEQ ID NO:9 (KQHYNTPWT), and a heavy chain comprising CDRs having a VH-CDR1 set forth in SEQ ID NO:10 (GYTFTSYW), (ii) a VH-CDR2 set forth in SEQ ID NO:11 (INPNTGYT), and (iii) a VH-CDR3 set forth in SEQ ID NO:7 (AVDLGY), The antibody described in claim 1.

13. (i) a light chain comprising CDRs having a VL-CDR1 set forth in SEQ ID NO: 12 (QSLLSSRNQKTY), (ii) a VL-CDR2 set forth in SEQ ID NO: 3 (FTS), and (iii) a VL-CDR3 set forth in SEQ ID NO: 13 (QQHYNTPWT), and a heavy chain comprising CDRs having a VH-CDR1 set forth in SEQ ID NO: 14 (GYTFTTYW), (ii) a VH-CDR2 set forth in SEQ ID NO: 15 (INPRNGYT), and (iii) a VH-CDR3 set forth in SEQ ID NO: 7 (AVDLGY), The antibody described in claim 1.

14. a light chain having 70% identity to SEQ ID NO: 16 or SEQ ID NO: 18, and / or a heavy chain having 70% identity to SEQ ID NO: 17 or SEQ ID NO: 19; The antibody described in claim 1.

15. cross-competes for binding to the CD95L isoform with a monoclonal antibody comprising: (i) a light chain comprising the CDRs VL-CDR1 set forth in SEQ ID NO:8 (QSLLSSRTQKHY), (ii) a VL-CDR2 set forth in SEQ ID NO:3 (FTS), and (iii) a VL-CDR3 set forth in SEQ ID NO:9 (KQHYNTPWT); and (i) a heavy chain comprising the CDRs VH-CDR1 set forth in SEQ ID NO:10 (GYTFTSYW), (ii) a VH-CDR2 set forth in SEQ ID NO:11 (INPNTGYT), and (iii) a VH-CDR3 set forth in SEQ ID NO:7 (AVDLGY); The antibody described in claim 1.

16. cross-competes for binding to the CD95L isoform with a monoclonal antibody comprising: (i) a light chain comprising CDRs which are the VL-CDR1 set forth in SEQ ID NO: 12 (QSLLSSRNQKTY), (ii) the VL-CDR2 set forth in SEQ ID NO: 3 (FTS), and (iii) the VL-CDR3 set forth in SEQ ID NO: 13 (QQHYNTPWT); and (i) a heavy chain comprising CDRs which are the VH-CDR1 set forth in SEQ ID NO: 14 (GYTFTTYW), (ii) the VH-CDR2 set forth in SEQ ID NO: 15 (INPRNGYT), and (iii) the VH-CDR3 set forth in SEQ ID NO: 7 (AVDLGY); The antibody described in claim 1.

17. 2. An antibody according to claim 1, or a nucleic acid molecule encoding the heavy and / or light chain of the antibody according to claim 1.

18. A vector comprising the nucleic acid of claim 17.

19. 20. A host cell transfected, infected or transformed with a nucleic acid according to claim 18 and / or a vector according to claim 19.

20. The antibody of claim 1 for use as a medicament.

21. A method of treatment in a patient in need of treatment, comprising administering to the patient a therapeutically effective amount of the antibody of claim 1.

22. 13. A method of treating cancer in a patient in need of treatment, comprising administering to the patient a therapeutically effective amount of the antibody of claim 1.

23. The patient is suffering from a cancer selected from the group consisting of breast cancer, colon cancer, lung cancer, prostate cancer, testicular cancer, brain cancer, skin cancer, rectal cancer, gastric cancer, esophageal cancer, sarcoma, tracheal cancer, head and neck cancer, pancreatic cancer, liver cancer, ovarian cancer, lymphatic cancer, cervical cancer, vulvar cancer, melanoma, mesothelioma, kidney cancer, bladder cancer, thyroid cancer, bone cancer, carcinoma, sarcoma, and soft tissue cancer; 23. The method of claim 22.

24. The patient is afflicted with triple-negative breast cancer.

23. The method of claim 22.

25. A method of treating an autoimmune inflammatory disease in a patient in need of treatment, comprising administering to the patient a therapeutically effective amount of the antibody of claim 1.

26. The patient is suffering from systemic lupus erythematosus.

26. The method of claim 25.

27. A pharmaceutical composition comprising the antibody of claim 1.

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