Antibodies with specificity for LTBP2 (latent transforming growth factor beta binding protein 2) and uses thereof

Abstract

The present invention relates to the treatment of cancers associated with cancer-associated fibroblasts (CAFs). In this study, we investigated a subset of LTBP2 (latent transforming growth factor beta-binding protein 2)-positive CAFs, which are responsible for the stromal fibrotic response in colorectal cancer liver metastases. We used gene nulling experiments to better understand the potential function of these cells, and we also developed a fully human antibody that enables targeting and depletion of LTBP2-positive CAFs in vitro. These novel insights, for the first time, identify the identity of CAFs within colorectal cancer liver metastases, and their targeting may be a valuable advantage in the development of novel antitumor agents. Therefore, the present invention relates to antibodies specific for LTBP2 and uses thereof.

Description

[Technical Field]

[0001] Field of the invention: The present invention relates to antibodies having specificity for LTBP2 (latent transforming growth factor beta binding protein 2) and uses thereof.

[0002] Background of the invention: Metastasis accounts for over 90% of cancer-related deaths worldwide. The liver is the epicenter of dissemination for the most lethal malignancies, including colorectal, breast, lung, and pancreatic cancers. More than 50% of patients with advanced colorectal cancer (CRC) develop liver metastases (CRC-LM) within 5 years after primary tumor resection. While only one-third of these patients are operable, the remaining patients are eligible for systemic chemotherapy with or without targeted therapy (1, 2). In the majority of cases, this leads to tumor resistance and progression. As a result, survival times for CRC-LM patients rarely exceed 5 years.

[0003] The tumor microenvironment (TME) of solid tumors offers promising opportunities for treating cancer (3), and the success of immune checkpoint inhibitors (ICIs), such as PD-L1 blockade, demonstrates that disrupting the intercellular crosstalk between cancer cells and the tumor microenvironment can result in therapeutic success. However, only a minority of patients, particularly in colorectal cancer (4), benefit from immune checkpoint inhibitors, indicating that further research is needed in this area. Cancer-associated fibroblasts (CAFs) are one of the most abundant and versatile components of the stroma. They are involved in all hallmarks of cancer (5), and growing evidence suggests that CAFs can exhibit both tumor-suppressive and tumor-promoting functions (6). Indeed, recent single-cell studies in primary breast tumors have reinforced long-held suspicions that CAFs do not adopt a unique phenotype within a single tumor (7, 8). Some CAFs may exhibit immunosuppressive properties, while others do not. In addition to their phenotypic heterogeneity, cancer-associated fibroblasts can originate from multiple sources beyond tissue-resident fibroblasts (9). There are two distinct physiological sources within the liver: resident (portal area) fibroblasts (PFs) and hepatic stellate cells (HSCs) (10). Portal area fibroblasts reside in the portal space, where they produce the connective tissue that contains bile ducts, portal veins, and arteries (three essential structures for the liver). Hepatic stellate cells are found in the space of Disse and store vitamin A and contain lipid droplets. A considerable amount of data indicates that hepatic stellate cells can be activated by liver injury, either through secreted factors or directly by immune cells, including liver-resident macrophages (Kupffer cells) (11). However, these findings are primarily based on cell fate-tracing studies, which can only be performed in mice (12).

[0004] The first cell atlas covering healthy and cirrhotic human livers (13), created using a single-cell approach, demonstrated the presence of at least four mesenchymal cell populations in the liver. Three of these populations—hepatic stellate cells expressing high levels of RGS5 (G protein signaling 5); cells expressing high levels of collagen and PDGFRA (platelet-derived growth factor receptor α) but lacking RGS5; and vascular smooth muscle cells (VSMCs) expressing strong MYH11—showed different degrees of association in the response to liver injury. Other single-cell studies of lung cancer (14) or multiple primary tumors (15) identified pericytes expressing RGS5 and VSMCs among cancer-associated fibroblasts (CAFs) in lung tumors. Two recent reports established the first single-cell atlases of the composition of colorectal cancer liver metastases (CRC-LM), including cancer-associated fibroblasts, but their focus was on changes in tumor composition upon chemotherapy ( 16 ) or on describing the entire population, with limited analysis of cancer-associated fibroblast heterogeneity ( 17 ), which is the focus of this study.

[0005] Summary of the Invention: In this study, we investigated a subset of LTBP2 (latent transforming growth factor beta-binding protein 2)-positive cancer-associated fibroblasts, which are responsible for the stromal fibrotic response in colorectal cancer liver metastases. We used gene nulling experiments to better understand the potential function of these cells, and we also developed a fully human antibody that enables targeting and depletion of LTBP2-positive cancer-associated fibroblasts in vitro. These novel insights, for the first time, identify the identity of cancer-associated fibroblasts within colorectal cancer liver metastases, and their targeting may be a valuable advantage in the development of novel antitumor agents.

[0006] The present invention relates to antibodies having specificity for LTBP2 and uses thereof. In particular, the invention is defined by the claims.

[0007] Detailed description of the invention: The present invention relates to fully human antibodies specific for LTBP-2, particularly for LTBP2-positive cancer-associated fibroblasts, and uses thereof. In particular, the present inventors have developed four fully human antibodies against LTBP2 (also referred to herein as C6, D2, F5, and F7) and demonstrated that LTBP2-positive cancer-associated fibroblasts can be depleted by targeting LTBP2.

[0008] As used herein, the term "LTBP2" for "latent transforming growth factor beta-binding protein 2" has its common meaning in the art and refers to a protein of the latent transforming growth factor (TGF) beta-binding protein (LTBP) family, which is an extracellular matrix protein with a multidomain structure. This protein is the largest member of the LTBP family, possessing unique regions and being most similar to fibrillin. The amino acid sequence of LTBP2 is Q14767 (Uniprot), and the nucleic acid sequence of LTBP2 is 4053 (Entrez).

[0009] According to the present invention, the VH region of the C6 monoclonal antibody consists of the sequence of SEQ ID NO: 1. Accordingly, the H-CDR1 of the C6 monoclonal antibody is defined by the sequence ranging from amino acid residue 31 to amino acid residue 35 of SEQ ID NO: 1. Accordingly, the H-CDR2 of the C6 monoclonal antibody is defined by the sequence ranging from amino acid residue 50 to amino acid residue 66 of SEQ ID NO: 1. Accordingly, the H-CDR3 of the C6 monoclonal antibody is defined by the sequence ranging from amino acid residue 100 to amino acid residue 113 of SEQ ID NO: 1. SEQ ID NO: 1: VH region of C6 monoclonal antibody, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. [ka]

[0010] According to the present invention, the VL region of the C6 monoclonal antibody consists of the sequence of SEQ ID NO: 2. Therefore, the L-CDR1 of the C6 monoclonal antibody is defined by the sequence ranging from amino acid residue 23 to amino acid residue 36 of SEQ ID NO: 2. Therefore, the L-CDR2 of the C6 monoclonal antibody is defined by the sequence ranging from amino acid residue 52 to amino acid residue 58 of SEQ ID NO: 2. Therefore, the L-CDR3 of the C6 monoclonal antibody is defined by the sequence ranging from amino acid residue 91 to amino acid residue 100 of SEQ ID NO: 2. SEQ ID NO: 2: VL region of the C6 monoclonal antibody, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. [ka]

[0011] According to the present invention, the VH region of the D2 monoclonal antibody consists of the sequence of SEQ ID NO: 3. Therefore, the H-CDR1 of the D2 monoclonal antibody is defined by the sequence ranging from amino acid residue 31 to amino acid residue 35 of SEQ ID NO: 3. Therefore, the H-CDR2 of the D2 monoclonal antibody is defined by the sequence ranging from amino acid residue 50 to amino acid residue 66 of SEQ ID NO: 3. Therefore, the H-CDR3 of the D2 monoclonal antibody is defined by the sequence ranging from amino acid residue 100 to amino acid residue 108 of SEQ ID NO: 3. SEQ ID NO: 3: VH region of the D2 monoclonal antibody, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. [ka]

[0012] According to the present invention, the VL region of the D2 monoclonal antibody consists of the sequence of SEQ ID NO: 4. Thus, the L-CDR1 of the D2 monoclonal antibody is defined by the sequence ranging from amino acid residue 23 to amino acid residue 36 of SEQ ID NO: 4. Thus, the L-CDR2 of the D2 monoclonal antibody is defined by the sequence ranging from amino acid residue 52 to amino acid residue 58 of SEQ ID NO: 4. Thus, the L-CDR3 of the D2 monoclonal antibody is defined by the sequence ranging from amino acid residue 91 to amino acid residue 100 of SEQ ID NO: 4. SEQ ID NO: 4: VL region of the D2 monoclonal antibody, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. [ka]

[0013] According to the present invention, the VH region of the F5 monoclonal antibody consists of the sequence of SEQ ID NO: 5. Accordingly, the H-CDR1 of the F5 monoclonal antibody is defined by the sequence ranging from amino acid residue 31 to amino acid residue 35 of SEQ ID NO: 5. Accordingly, the H-CDR2 of the F5 monoclonal antibody is defined by the sequence ranging from amino acid residue 50 to amino acid residue 66 of SEQ ID NO: 5. Accordingly, the H-CDR3 of the F5 monoclonal antibody is defined by the sequence ranging from amino acid residue 99 to amino acid residue 107 of SEQ ID NO: 5. SEQ ID NO: 5: VH region of the F5 monoclonal antibody, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. [ka]

[0014] According to the present invention, the VL region of the F5 monoclonal antibody consists of the sequence of SEQ ID NO: 6. Thus, the L-CDR1 of the F5 monoclonal antibody is defined by the sequence ranging from amino acid residue 23 to amino acid residue 36 of SEQ ID NO: 6. Thus, the L-CDR2 of the F5 monoclonal antibody is defined by the sequence ranging from amino acid residue 52 to amino acid residue 58 of SEQ ID NO: 6. Thus, the L-CDR3 of the F5 monoclonal antibody is defined by the sequence ranging from amino acid residue 91 to amino acid residue 100 of SEQ ID NO: 6. SEQ ID NO: 6: VL region of the F5 monoclonal antibody, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. [ka]

[0015] According to the present invention, the VH region of the F7 monoclonal antibody consists of the sequence of SEQ ID NO: 7. Accordingly, the H-CDR1 of the F7 monoclonal antibody is defined by the sequence ranging from amino acid residue 31 to amino acid residue 35 of SEQ ID NO: 7. Accordingly, the H-CDR2 of the F7 monoclonal antibody is defined by the sequence ranging from amino acid residue 50 to amino acid residue 66 of SEQ ID NO: 7. Accordingly, the H-CDR3 of the F7 monoclonal antibody is defined by the sequence ranging from amino acid residue 99 to amino acid residue 109 of SEQ ID NO: 7. SEQ ID NO: 7: VH region of the F7 monoclonal antibody, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. [ka]

[0016] According to the present invention, the VL region of the F7 monoclonal antibody consists of the sequence of SEQ ID NO: 8. Thus, the L-CDR1 of the F7 monoclonal antibody is defined by the sequence ranging from amino acid residue 23 to amino acid residue 36 of SEQ ID NO: 8. Thus, the L-CDR2 of the F7 monoclonal antibody is defined by the sequence ranging from amino acid residue 52 to amino acid residue 58 of SEQ ID NO: 8. Thus, the L-CDR3 of the F7 monoclonal antibody is defined by the sequence ranging from amino acid residue 91 to amino acid residue 100 of SEQ ID NO: 8. SEQ ID NO: 8: VL region of the F7 monoclonal antibody, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. [ka]

[0017] As used herein, the terms "antibody" and "immunoglobulin" have the same meaning and will be used equivalently in the present invention. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing an antigen-binding site that immunospecifically binds to an antigen. Thus, the term antibody encompasses not only complete 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 a light chain by a disulfide bond. There are two types of light chains: lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. The light chain contains two domains: a variable domain (VL) and a constant domain (CL). Heavy chains contain four domains: one variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine antigen binding recognition and specificity. The constant region domains of the light chain (CL) and the heavy chain (CH) confer important biological properties, such as antibody chain assembly, secretion, placental transport, complement binding, and Fc receptor (FcR) binding. The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of one light chain and one heavy chain variable region. Antibody specificity resides in the structural complementarity between the antibody binding site and an antigenic determinant. The antibody binding site is primarily composed of residues from hypervariable or complementarity-determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) may participate in the antibody binding site or influence the overall domain structure and thereby the binding site. Complementarity-determining regions, or CDRs, refer to amino acid sequences that together define the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively.Therefore, an antigen-binding site typically contains six CDRs, each of which comprises a set of CDRs derived from the heavy and light chain variable regions. The framework region (FR) refers to the amino acid sequence inserted between the CDRs. In one embodiment, the antibody of the present invention is a monoclonal antibody.

[0018] In the context of the present invention, the amino acid residues of the antibodies of the invention are numbered according to the KABAT numbering system. This system is set out in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, National Institutes of Health, USA (hereinafter "Kabat et al."). This numbering system is used herein. The Kabat residue designations do not always correspond directly to the linear numbering of the amino acid residues in the SEQ ID NO: sequence. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering, corresponding to a shortening of, or insertion into, a structural element of the basic variable domain structure (whether a framework region or a complementarity-determining region (CDR)). The correct Kabat numbering of residues for a given antibody can be determined by alignment of homologous residues in the antibody sequence with the "standard" Kabat numbering sequence. The CDRs of the heavy chain variable domain are located at residues 31-35 (H-CDR1), residues 50-65 (H-CDR2), and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2), and residues 89-97 (L-CDR3) according to the Kabat numbering system (http: / / www.bioinf.org.uk / abs / #cdrdef).

[0019] As used herein, the term "specificity" refers to the ability of an antibody to detectably bind to an epitope presented on an antigen, such as LTBP2, while having relatively little detectable reactivity with non-LTBP2 proteins or structures (such as other proteins presented on cancerous cells or other cell types). Specificity can be relatively determined by binding assays or competitive binding assays, such as those described elsewhere herein, using, for example, a Biacore instrument. Specificity can be demonstrated, for example, by an affinity / avidity ratio of about 10:1, about 20:1, about 50:1, about 100:1, 10,000:1, or more for binding to the specific antigen versus nonspecific binding to other unrelated molecules (in this case, the specific antigen is LTBP2).

[0020] As used herein, the term "affinity" refers to the binding strength of an antibody to an epitope. The affinity of an antibody is indicated by the dissociation constant Kd, defined as [antibody] x [antigen] / [antibody-antigen], where [antibody-antigen] is the molar concentration of the antibody-antigen complex, [antibody] is the molar concentration of unbound antibody, and [antigen] is the molar concentration of unbound antigen. The affinity constant Ka is defined by 1 / Kd. Preferred methods for determining the affinity of monoclonal antibodies 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 are incorporated herein by reference in their entireties. One preferred standard method well known in the art for determining the affinity of monoclonal antibodies is the use of a Biacore instrument.

[0021] As used herein, the terms "monoclonal antibody," "monoclonal Ab," "monoclonal antibody composition," "mAb," and the like refer to a preparation of antibody molecules of single molecular composition displaying a single binding specificity and affinity for a particular epitope.

[0022] Antibodies of the present invention can be 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, once the amino acid sequence of the desired sequence is known, one of skill in the art can readily produce the antibody using standard polypeptide production techniques. For example, they 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, CA) according to the manufacturer's instructions. Alternatively, antibodies of the present invention can be synthesized by recombinant DNA techniques well known in the art. For example, antibodies can be obtained as DNA expression products after incorporation of a DNA sequence encoding the antibody into an expression vector and introduction of such a vector into an appropriate eukaryotic or prokaryotic host that will express the desired antibody, from which they can then be isolated using well-known techniques.

[0023] In one embodiment, the antibody of the present invention is an antigen-binding fragment selected from the group consisting of Fab, F(ab')2, single-domain antibody, single-chain Fv, single-chain (Fv)2, diabody, triabody, tetrabody, unibody, minibody, maxibody, small modular immunopharmaceutical (SMIP), a minimal recognition unit consisting of amino acid residues mimicking the hypervariable region of an antibody as an isolated complementarity-determining region (CDR), and a VL chain or VH chain, and a fragment comprising or consisting of an amino acid sequence having at least 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 SEQ ID NOs: 1 to 8.

[0024] In some embodiments, the antibody of the present invention has a heavy chain comprising i) the H-CDR1 of the C6, D2, F5, or F7 monoclonal antibody, ii) the H-CDR2 of the C6, D2, F5, or F7 monoclonal antibody, and iii) the H-CDR3 of the C6, D2, F5, or F7 monoclonal antibody, and a light chain comprising i) the L-CDR1 of the C6, D2, F5, or F7 monoclonal antibody, ii) the L-CDR2 of the C6, D2, F5, or F7 monoclonal antibody, and iii) the L-CDR3 of the C6, D2, F5, or F7 monoclonal antibody.

[0025] In some embodiments, an antibody of the invention is an antibody having a heavy chain that is at least 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% identical to SEQ ID NO: 1, 3, 5 or 7, and a light chain that is at least 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% identical to SEQ ID NO: 2, 4, 6 or 8.

[0026] In particular, the antibody has a heavy chain having at least 70% identity to SEQ ID NO: 1, 3, 5 or 7 and a light chain having at least 70% identity to SEQ ID NO: 2, 4, 6 or 8.

[0027] In some embodiments, the antibody of the present invention is an antibody having a heavy chain identical to SEQ ID NO: 1, 3, 5 or 7 and a light chain identical to SEQ ID NO: 2, 4, 6 or 8.

[0028] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., [LTBP2]). The antigen-binding function of an antibody can be performed by fragments of the intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include a Fab fragment, a monovalent fragment consisting of the VL domain, VH domain, CL domain, and CH1 domain; a Fab' fragment, a monovalent fragment consisting of the VL domain, VH domain, CL domain, CH1 domain, and hinge region; a F(ab')2 fragment, a bivalent fragment containing two Fab' fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of the VH domain of a single arm of an antibody; a single-domain antibody (sdAb) fragment consisting of the VH or VL domain (Ward et al., 1989 Nature 341:544-546); and isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of an Fv fragment, i.e., VL and VH, are encoded by separate genes, they may be connected by an artificial peptide linker, allowing them to be produced using recombinant techniques as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (ScFv); see, e.g., Bird et al., 1989 Science 242:423-426; and Huston et al., 1988 proc. Natl. Acad. Sci. 85:5879-5883). A "dsFv" is a VH:VL heterodimer stabilized by a disulfide bond. Bivalent and multivalent antibody fragments may form spontaneously by association of monovalent scFvs or may be produced by linking monovalent scFvs via a peptide linker, such as a bivalent sc(Fv)2. Such single-chain antibodies comprise one or more antigen-binding portions or fragments of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Unibodies are another type of antibody fragment that lacks the hinge region of an IgG4 antibody.Deletion of the hinge region results in a molecule that is essentially half the size of traditional IgG4 antibodies and has a monovalent binding region rather than the bivalent binding region of IgG4 antibodies. Antigen-binding fragments can be incorporated into single-domain antibodies, SMIPs, maxibodies, minibodies, intrabodies, diabodies, triabodies, and tetrabodies (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The terms "diabody," "tribody," or "tetrabody" refer to small antibody fragments with multivalent antigen-binding sites (2, 3, or 4), which comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) within the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. Antigen-binding fragments can be incorporated into single-chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1), which, together with complementary light chain polypeptides, form a pair of antigen-binding regions (Zapata et al., 1995 Protein Eng. 8(10); 1057-1062 and U.S. Pat. No. 5,641,870).

[0029] The Fab of the present invention can be obtained by treating an antibody that specifically reacts with LTBP2 with the protease papain. Alternatively, the Fab can be produced by inserting DNA encoding the Fab of the antibody into a vector for a prokaryotic or eukaryotic expression system, and then introducing the vector into a prokaryotic or eukaryotic cell (as appropriate) to express the Fab.

[0030] The F(ab')2 of the present invention can be obtained by treating an antibody that specifically reacts with an antigen with the protease pepsin. Alternatively, the F(ab')2 can be produced by linking the Fab' described below via a thioether bond or a disulfide bond.

[0031] The Fab' of the present invention can be obtained by treating F(ab')2 that specifically reacts with LTBP2 with the reducing agent dithiothreitol. Alternatively, the Fab' can be produced by inserting DNA encoding the Fab' fragment of an antibody into a prokaryotic or eukaryotic expression vector, and then introducing the vector into a prokaryotic or eukaryotic cell (as appropriate) to express the fragment.

[0032] The scFvs of the present invention can be produced by obtaining cDNAs encoding the VH and VL domains as previously described, constructing DNA encoding the scFv, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then expressing the scFv by introducing the expression vector into a prokaryotic or eukaryotic cell (as appropriate). To produce humanized scFv fragments, a well-known technique called CDR grafting can be used, which involves selecting complementarity-determining regions (CDRs) from a donor scFv fragment and grafting them onto a human scFv fragment framework with a known three-dimensional structure (see, e.g., WO 98 / 45322; WO 87 / 02671; U.S. Pat. No. 5,859,205; U.S. Pat. No. 5,585,089; U.S. Pat. No. 4,816,567; EP 0173494).

[0033] Domain antibodies (dAbs) are the smallest functional binding units of antibodies (molecular weight of approximately 13 kDa) and correspond to the variable regions of either the heavy (VH) or light (VL) chains of an antibody. Further details regarding domain antibodies and methods for their production can be found in U.S. Patent Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; and 6,696,245; U.S. Patent Application No. 2004 / 0110941; European Patent Nos. 1433846, 0368684, and 0616640; WO Nos. 2005 / 035572, 2004 / 101790, 2004 / 081026, 2004 / 058821, 2004 / 003019, and 2003 / 002609, each of which is incorporated herein by reference in its entirety.

[0034] Unibodies are another antibody fragment technology based on the removal of the hinge region of an IgG4 antibody. The deletion of the hinge region results in a molecule that is essentially half the size of a traditional IgG4 antibody and has a monovalent rather than a bivalent binding region. Furthermore, because Unibodies are approximately smaller, they may exhibit better distribution on larger solid tumors, potentially with advantageous efficacy. Further details regarding Unibodies can be obtained by reference to WO2007 / 059782, which is incorporated by reference in its entirety.

[0035] In certain embodiments, the antibodies of the present invention may be used to treat cancer.

[0036] Thus, in a particular embodiment, the present invention relates to a method of treating cancer in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an antibody of the present invention.

[0037] According to the present invention, the cancer may be a liquid or solid cancer.

[0038] In one embodiment, the cancer is adrenocortical carcinoma, anal cancer, bile duct cancer (e.g., perihilar carcinoma, distal bile duct carcinoma, intrahepatic cholangiocarcinoma), bladder cancer, bone cancer (e.g., osteoblastoma, osteochondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of bone, chordoma), brain and central nervous system cancer (e.g., meningioma, astrocytoma, oligodendroglioma, ependymoma, glioma, medulloblastoma, ganglioglioma, schwannoma, germinoma, craniopharyngioma), breast cancer ( Cancers of the esophagus, gallbladder, and gallbladder (e.g., mucinous adenocarcinoma and small cell carcinoma), gastrointestinal carcinoma (e.g., choriocarcinoma and destructive villous adenoma), Hodgkin's disease, Kaposi's sarcoma, kidney cancer (e.g., renal cell carcinoma), and throat cancer (e.g., large lymph node hyperplasia and angiofollicular lymph node hyperplasia). Head and hypopharyngeal cancer, liver cancer (e.g., hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g., nasal neuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cancer, and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g., embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer The cancer may be selected from the group consisting of cancer, skin cancer (e.g., melanoma, non-melanoma skin cancer), gastric cancer, testicular cancer (e.g., seminoma, non-seminomatous germ cell carcinoma), thymic cancer, thyroid cancer (e.g., follicular adenocarcinoma, undifferentiated carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma), vaginal cancer, vulvar cancer, uterine cancer (e.g., uterine leiomyosarcoma), leukemia (such as acute myeloid leukemia, acute lymphocytic leukemia, chronic myelomonocytic leukemia (CMML), etc.), lymphoma, and myelodysplastic syndrome (MDS).

[0039] Nucleic acid sequences, vectors and host cells Therefore, a further object of the present invention relates to a nucleic acid molecule encoding any antibody according to the present invention, more particularly, said nucleic acid molecule encoding the heavy or light chain of any antibody of the present invention.

[0040] Typically, the nucleic acid is a DNA or RNA molecule, which may be contained 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 by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, thereby transforming the host and promoting expression (e.g., transcription and translation) of the introduced sequence. Thus, a further aspect of the present invention relates to a vector comprising a nucleic acid of the present invention. Such a vector may include regulatory sequences, such as a promoter, enhancer, terminator, etc., that cause or direct expression of the antibody upon administration to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include the SV (simian virus) 40 early promoter and enhancer (Mizukami T. et al. 1987), the Moloney murine leukemia virus LTR promoter and enhancer (Kuwana Y et al. 1987), and the immunoglobulin heavy chain promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983). Any expression vector for animal cells may be used as long as a gene encoding a human antibody constant region can be inserted and expressed. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), pSG1βd2-4 (Miyaji H et al. 1990), etc. Other examples of plasmids include replicative plasmids containing an origin of replication, or integrative plasmids, such as pUC, pcDNA, pBR, etc. Other examples of viral vectors include adenoviral vectors, retroviral vectors, herpesvirus vectors, and adeno-associated virus vectors.Such recombinant viruses can be produced by techniques known in the art, such as transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, glycoprotein envelope-positive 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.

[0041] A further object of the present invention relates to host cells which have been transfected, infected or transformed with the nucleic acids and / or vectors according to the invention.

[0042] The term "transformation" refers to the introduction of a "foreign" (i.e., exogenous or extracellular) gene, DNA sequence, or RNA sequence into a host cell so that the host cell will express 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 has been "transformed."

[0043] The nucleic acids of the invention can be used to produce antibodies of the invention in a suitable expression system. The term "expression system" refers to a host cell and a suitable vector under appropriate conditions for the expression of a protein encoded by foreign DNA, for example, 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, but are not limited to, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces, or Saccharomyces yeast, mammalian cell lines (e.g., Vero cells, Chinese hamster ovary (CHO) cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., generated from lymphoblast cells, fibroblast cells, embryonic cells, epithelial cells, neuronal cells, adipocytes, etc.). Examples include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dehydrofolate reductase gene (hereinafter referred to as the "DHFR gene") (Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells"), etc. The present invention also relates to a method for producing a recombinant host cell expressing an antibody according to the present invention, the method comprising the steps of (i) introducing, in vitro or ex vivo, a recombinant nucleic acid or vector as described above into a competent host cell, (ii) culturing, in vitro or ex vivo, the resulting recombinant host cell, and (iii) optionally selecting cells that express and / or secrete the antibody. Such recombinant host cells can be used for the production of the antibodies of the present invention.

[0044] 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.

[0045] Functional variants Thus, the present invention provides antibodies comprising functional variants of the VL and VH regions of the antibodies of the present invention. Functional variants of VL or VH used in the context of the monoclonal antibodies of the present invention still enable the antibody to retain at least a significant proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity / avidity and / or specificity / selectivity of the parent antibody (i.e., the C6, D2, F5, or F7 monoclonal antibody), and in some cases, such monoclonal antibodies of the present invention may have greater affinity, selectivity, and / or specificity than the parent monoclonal antibody. Such variants can be obtained by CDR mutation (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio / Technology, 10, 779-783, 1992), use of mutator strains of Escherichia coli (E. coli) (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 77-88, 1996), and sexual PCR (Crameri et al., Nature, 391, 288-291, These affinity maturation methods can be obtained by a number of affinity maturation protocols, including those described in the literature (e.g., "Affinity Maturation Protocols," ...The sequences of the CDR variants may differ from the CDR sequences of the parent antibody sequence mostly through conservative substitutions; for example, at least 10, e.g., at least 9, 8, 7, 6, 5, 4, 3, 2, or 1 of the substitutions in the variant are conservative amino acid residue substitutions. In the context of the present invention, conservative substitutions may be defined by substitutions within amino acid classes reflected as follows: aliphatic residues I, L, V, and M, Cycloalkenyl-related residues F, H, W and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W and Y Negatively charged residues D and E Polar residues C, D, E, H, K, N, Q, R, S and T Positively charged residues H, K and R Small residues A, C, D, G, N, P, S, T and V Very small residues A, G and S Residues involved in the turn: A, C, D, E, G, H, K, N, Q, R, S, P; and residue T involved in the formation of the turn. Mobile residues Q, T, K, S, G, P, D, E and R.

[0046] More conservative substitution groups include valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Conservation in terms of hydropathy / hydrophilicity and residue weight / size is also substantially maintained in the variant CDRs compared to the CDRs of [antibody name]. The importance of the hydropathic amino acid index in conferring interactive biological function to a protein is generally understood in the art. It is recognized that the relative hydropathic characteristics of amino acids contribute to the secondary structure of the resulting protein, which in turn determines the protein's interactions with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics, and these are isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). Retention of similar residues can also or alternatively be measured by a similarity score, as determined by use of a BLAST program (e.g., BLAST 2.2.8 available through NCBI (National Center for Biotechnology Information) using standard settings BLOSUM62, open gap = 11, and extended gap = 1). Suitable variants typically exhibit at least about 70% identity to the parent peptide. According to 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.According to the present invention, a first amino acid sequence having at least 90% identity to a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity to the second amino acid sequence.

[0047] In a particular embodiment, the above antibodies bind to the same antigen and have the same properties as the antibodies of the present invention, i.e., antibodies having VH and VL of SEQ ID NOs: 1 and 2, or 3 and 4, or 5 and 6, or 7 and 8.

[0048] Antibodies that compete with the antibodies of the present invention In another aspect, the present invention provides an antibody that competes with the antibody of the present invention for binding to LTBP2.

[0049] As used herein, the term "binding" in the context of antibody binding to a given antigen or epitope typically refers to binding with an affinity corresponding to a KD of about 10 M or less, e.g., about 10 M or less, e.g., about 10 M or less, about 10 M or less, or about 10 M or less, or even less, as determined by surface plasmon resonance (SPR) technology on a Biacore 3000 instrument, using, for example, a soluble form of the antigen as the ligand and the antibody as the analyte. Biacore® (GE Healthcare, Piscataway, NJ) is one of a wide variety of surface plasmon resonance assay formats routinely used for epitope binning of panels of monoclonal antibodies. Typically, an antibody binds to a given antigen with an affinity corresponding to a KD that is at least 10-fold lower, such as at least 100-fold lower, such as at least 1,000-fold lower, such as at least 10,000-fold lower, such as at least 100,000-fold lower, for example at least 100,000-fold lower, than its KD for binding to a non-specific antigen that is not identical to or closely related to the given antigen (e.g., bovine serum albumin, casein). If an antibody has a very low KD (i.e., the antibody has high affinity), the KD at which it binds to the antigen is typically at least 10,000-fold lower than its KD for the non-specific antigen. An antibody is said to not substantially bind to an antigen or epitope if such binding is not detectable (e.g., using plasmon resonance (SPR) technology on a Biacore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte) or is 100-fold, 500-fold, 1000-fold, or more than 1000-fold less than the binding detected by that antibody and an antigen or epitope having a different chemical structure or amino acid sequence.

[0050] Additional antibodies can be identified based on their ability to cross-compete with other antibodies of the present invention (e.g., competitively inhibit the binding of other antibodies of the present invention in a statistically significant manner) in standard LTBP2 binding assays. The ability of a test antibody to inhibit the binding of an antibody of the present invention to LTBP2 demonstrates that the test antibody can compete with that antibody for binding to LTBP2; such an antibody may, by non-limiting theory, bind to the same or a related (e.g., structurally similar or spatially proximal) epitope on LTBP2 as the antibody with which it competes. Thus, another aspect of the present invention provides antibodies that bind to the same antigen as the antibodies disclosed herein and compete with the antibodies disclosed herein. As used herein, an antibody "competes" for binding if the competing antibody inhibits binding of an antibody or antigen-binding fragment of the invention to LTBP2 by 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, or 99% or more in the presence of an equimolar concentration of the competing antibody.

[0051] In other embodiments, the antibodies or antigen-binding fragments of the invention bind to one or more epitopes of LTBP2. In some embodiments, the epitopes to which the antibodies or antigen-binding fragments of the invention bind are linear epitopes. In other embodiments, the epitopes to which the antibodies or antigen-binding fragments of the invention bind are non-linear, conformational epitopes.

[0052] Antibodies of the present invention can be assayed for specific binding by any method known in the art. Many different competitive binding assay formats can be used to measure epitope binding. Immunoassays that can be used include, but are not limited to, competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISAs, "sandwich" immunoassays, immunoprecipitation assays, precipitation assays, gel diffusion precipitin assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and complement fixation assays. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds., 1994 Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York).

[0053] Antibody Engineering The engineered antibodies of the present invention include antibodies in which modifications have been made to framework residues within the VH and / or VL, e.g., to improve the antibody's properties. Typically, such framework modifications are made to reduce the antibody's immunogenicity. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody's framework sequence to the germline sequence from which the antibody is derived. To return the framework region sequences to their germline structure, somatic mutations can be "backmutated" to the germline sequence, e.g., by site-directed mutagenesis or PCR-mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the present invention. Another type of framework modification involves mutating one or more residues within the framework regions, or even one or more CDR regions, to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach, also referred to as "deimmunization," is further detailed in US Patent Application Publication No. 20030153043 by Carr et al.

[0054] In some embodiments, the glycosylation of the antibody is modified. Glycosylation may be altered, for example, to increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, glycosylation sites in one or more variable framework regions can be removed, resulting in one or more amino acid substitutions that eliminate glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. Such approaches are described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0055] In some embodiments, several mutations are made to amino acids located in aggregation "hot spots" within and near the first CDR (CDR1) to reduce the antibody's susceptibility to aggregation (see Joseph M. Perchiacca et al., Proteins 2011; 79:2637-2647).

[0056] The antibodies of the present invention can be of any isotype. The choice of isotype will typically be guided by the desired effector function. IgG1 and IgG3 are isotypes that mediate such effector functions as antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity, while IgG2 or IgG4 do not or only to a lesser extent. Either kappa or lambda human light chain constant regions can be used. If desired, the class of the monoclonal antibodies of the present invention can be switched by known methods. Using typical class switching techniques, one IgG subclass can be converted to another subclass, for example, from IgG1 to IgG2. Thus, the effector function of the monoclonal antibodies of the present invention can be altered by isotype switching, for example, to an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic applications.

[0057] In some embodiments, the antibodies of the invention are full-length antibodies. In some embodiments, the full-length antibodies are IgG1 antibodies. In some embodiments, the full-length antibodies are IgG3 antibodies.

[0058] In some embodiments, the antibodies of the invention are not of the IgG2 / 4 type, e.g., IgG1 or IgG3 that have been mutated to reduce or even eliminate their ability to mediate effector functions such as antibody-dependent cellular cytotoxicity. Such mutations are described, for example, in Dall'Acqua WF et al., J Immunol. 177(2): 1129-1138(2006) and Hezareh M, J Virol. 75(24): 12161-12168(2001).

[0059] In some embodiments, the hinge region of CH1 is modified to alter, e.g., increase or decrease, the number of cysteine ​​residues in the hinge region. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine ​​residues in the hinge region of CH1 is altered, for example, to facilitate association of the light and heavy chains or to increase or decrease the stability of the antibody.

[0060] In some embodiments, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue so that the antibody has altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand for which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0061] In some embodiments, one or more selected amino acids from the amino acid residues can be substituted with a different amino acid residue, such that the antibody has altered C1q binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is further detailed in U.S. Patent No. 6,194,551 by Idusogie et al.

[0062] In some embodiments, one or more amino acid residues are altered to alter the ability of the antibody to fix complement. This approach is further described in PCT Publication No. WO 94 / 29351 by Bodmer et al.

[0063] In some embodiments, the Fc region is modified by modifying one or more amino acids to enhance the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for an Fc receptor. This approach is further described in PCT Publication No. WO 00 / 42072 by Presta. Additionally, the binding sites on human IgG1 for Fcγ receptor I, Fcγ receptor II, Fcγ receptor III, and fetal Fc receptors have been mapped, and mutants that exhibit improved binding have been described (see Shields, RL et al., 2001 J. Biol. Chen. 276:6591-6604; WO 2010106180).

[0064] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a term well understood in the art and refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcR) recognize bound antibody on target cells and subsequently cause lysis of the target cells. Nonspecific cytotoxic cells that mediate ADCC include natural killer (NK) cells, macrophages, monocytes, neutrophils, and eosinophils.

[0065] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0066] Additionally or alternatively, antibodies can be generated with an altered type of glycosylation, such as hypofucosylated or nonfucosylated antibodies with reduced or no fucosyl residues, or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the invention and thereby produce antibodies with altered glycosylation. For example, European Patent No. 1,176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in such cell lines exhibit hypofucosylation or lack fucosyl residues. Thus, in some embodiments, the monoclonal antibodies of the invention can be produced by recombinant expression in a cell line exhibiting a hypofucosylated or afucosylated pattern, e.g., a mammalian cell line deficient in expression of the FUT8 gene, which encodes fucosyltransferase. PCT Publication No. WO 03 / 035835 by Presta describes a mutant CHO cell line, Lecl3 cells, that has a reduced ability to attach fucose to the glycan linked to Asn(297), resulting in hypofucosylation of antibodies expressed in the host cells (see also Shields, RL et al., 2002 J. Biol. Chem. 277:26733-26740).PCT Publication No. WO 99 / 54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibodies (see also Umana et al., 1999 Nat. Biotech. 17: 176-180). Eureka Therapeutics, Inc. further describes genetically engineered CHO mammalian cells capable of producing antibodies with a modified mammalian glycosylation pattern lacking fucosyl residues (http: / / www.eurekainc.com / a&boutus / companyoverview.html). Alternatively, the monoclonal antibodies of the invention can be produced in yeast or filamentous fungi that can be engineered for mammalian-like glycosylation patterns and produce antibodies lacking fucose as a glycosylation pattern (see, e.g., EP 1297172 B1).

[0067] In another embodiment, the antibody is modified to extend its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 by Ward. Alternatively, to extend biological half-life, the antibody can be engineered in the CH1 or CL region to contain salvage receptor binding epitopes recruited from two loops of the CH2 domain of the IgG Fc region, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 by Presta et al. Antibodies with extended half-lives and improved binding to the neonatal Fc receptor (FcRn), which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in U.S. Patent Application Publication No. 2005 / 0014934A1 (Hinton et al.). Such antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include variants having a substitution at one or more of the Fc region residues (238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434), for example, a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0068] Another modification of the antibodies herein contemplated by the present invention is PEGylation. The antibody may be PEGylated, for example, to extend the biological (e.g., serum) half-life of the antibody. To PEGylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups become attached to the antibody or antibody fragment. PEGylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive, water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG, such as mono(C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide, that has been used to derivatize other proteins. In certain embodiments, the antibody to be PEGylated is an aglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the present invention. See, for example, European Patent No. 0154316 to Nishimura et al. and European Patent No. 0401384 to Ishikawa et al.

[0069] Another modification of the antibodies contemplated by the present invention is the conjugation or protein fusion of at least the antigen-binding region of the antibody of the invention to a serum protein, such as human serum albumin, or a fragment thereof, to extend the half-life of the resulting molecule. Such an approach is described, for example, in Balance et al., European Patent No. 0322094. Another possibility is the fusion of at least the antigen-binding region of the antibody of the invention to a protein capable of binding to a serum protein, such as human serum albumin, to extend the half-life of the resulting molecule. Such an approach is described, for example, in Nygren et al., European Patent No. 0486525.

[0070] Polysialylation is another technique that uses the natural polymer polysialic acid (PSA) to extend the shelf life and improve the stability of therapeutic peptides and proteins. PSA is a polymer (sugar) of sialic acid. When used to deliver proteins and therapeutic peptide drugs, polysialic acid provides a protective microenvironment against conjugation. This extends the shelf life of therapeutic proteins in the circulation and prevents them from being recognized by the immune system. PSA polymers are naturally found in the human body. PSA polymers have been adopted by certain bacteria, which have evolved over millions of years to coat their bacterial walls with PSA polymers. These naturally polysialylated bacteria can thus thwart the host's defense system through molecular mimicry. PSA, the ultimate natural stealth technology, can be produced in large quantities by such bacteria while retaining defined physical characteristics. Because bacterial PSA is chemically identical to human PSA, it is completely non-immunogenic, even when conjugated to proteins.

[0071] Another technique involves the use of hydroxyethyl starch ("HES") derivatives linked to antibodies. HES is a modified natural polymer derived from waxy cornstarch that can be metabolized by biological enzymes. HES solutions are typically administered to replace deficient blood volume and improve the rheological properties of blood. HES conjugation of antibodies allows for a longer circulation half-life by increasing the molecule's stability and reducing renal clearance, resulting in increased biological activity. By varying various parameters, such as the molecular weight of HES, a wide range of HES-antibody conjugates can be custom synthesized.

[0072] In another embodiment, the Fc-hinge region of an antibody is mutated to reduce the biological half-life of the antibody. More specifically, by introducing one or more amino acid mutations into the CH2-CH3 domain interface region of the Fc-hinge fragment, the antibody exhibits impaired binding to Staphylococcus aureus protein A (SpA) compared to the binding of the native Fc-hinge domain to Staphylococcus aureus protein A (SpA). This approach is further described in U.S. Patent No. 6,165,745 by Ward et al.

[0073] In a specific embodiment of the present invention, the antibody is engineered to remove deamidation sites. Deamidation is known to cause structural and functional changes in peptides or proteins. Deamidation can result in reduced biological activity and altered pharmacokinetics and antigenicity of protein pharmaceuticals (Anal Chem. 2005 Mar 1;77(5):1432-9).

[0074] In certain embodiments of the present invention, antibodies are engineered to increase their pI and improve their drug-like properties. The pI of a protein is an important determinant of the overall biophysical properties of a molecule. Antibodies with a low pI are known to be less soluble, less stable, and more prone to aggregation. Furthermore, purification of antibodies with a low pI can be challenging, especially during scale-up for clinical use. Increasing the pI of the anti-LTBP2 antibodies or fragments thereof of the present invention improves their solubility, allowing the antibodies to be formulated at higher concentrations (greater than 100 mg / ml). Formulating antibodies at high concentrations (e.g., greater than 100 mg / ml) offers the advantage of being able to administer higher doses of the antibody to a patient's eye via intravitreal injection, which can reduce dosing frequency, an important advantage for treating chronic diseases, including cardiovascular disease. A higher pI can also increase FcRn-mediated recycling of IgG-type antibodies, thereby allowing the drug to persist in the body for a longer period of time and requiring less frequent injections. Finally, the overall stability of the antibody is significantly improved due to the higher pI, resulting in a longer shelf life and bioactivity in vivo. Preferably, the pI is greater than or equal to 8.2.

[0075] Glycosylation modification can also induce enhanced anti-inflammatory properties of antibodies through the addition of sialic acid to Fc glycans. The addition of terminal sialic acid to Fc glycans reduces binding to FcγR and converts IgG antibodies into anti-inflammatory mediators through the acquisition of novel binding activity (see Robert M. Anthony et al., J Clin Immunol (2010) 30 (Suppl 1):S9-S14; Kai-Ting C et al., Antibodies 2013, 2, 392-414).

[0076] antibody mimics In some embodiments, the disclosed heavy and light chain variable region domains and CDRs can be used to prepare polypeptides containing antigen-binding regions capable of specifically binding to LTBP2. For example, immunoadhesins can be generated by incorporating the CDRs of the C6, D2, F5, or F7 monoclonal antibodies, either covalently or noncovalently, into a molecule (e.g., a polypeptide). Immunoadhesins can incorporate the CDRs as part of a larger polypeptide chain, covalently link the CDRs to another polypeptide chain, or noncovalently incorporate the CDRs. The CDRs enable the immunoadhesin to specifically bind to a particular antigen of interest (e.g., LTBP2 or an epitope thereof).

[0077] The terms "polypeptide" and "protein" are used synonymously herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise specified, a particular polypeptide sequence implicitly encompasses conservatively modified variants thereof.

[0078] In some embodiments, the antigen-binding fragments of the present invention are grafted onto non-immunoglobulin-based antibodies, also called antibody mimetics, selected from the group consisting of affibodies, affilins, affitins, adnectins, atrimers, evasins, designed ankyrin repeat proteins (DARPins), anteicalins, avimers, fynomers, and versabodies.

[0079] The term "antibody mimic" is intended to refer to a molecule that can mimic the ability of an antibody to bind to an antigen, but is not limited to a natural antibody structure. Examples of such antibody mimics include, but are not limited to, adnectins, affibodies, designed ankyrin repeat proteins (DARPins), anticalins, avimers, and versabodies, all of which utilize binding structures that mimic traditional antibody binding but arise from and function via distinct mechanisms. Antigen-binding fragments of antibodies can be grafted onto scaffolds based on polypeptides such as type III fibronectin (Fn3) (see U.S. Pat. No. 6,703,199, which describes a fibronectin polypeptide monobody). Affibodies are well known in the art and refer to affinity proteins based on a 58-amino acid residue protein domain derived from one of the IgG-binding domains of staphylococcal protein A. DARPins (designed ankyrin repeat proteins) are well known in the art and refer to antibody mimetic DRP (designed repeat protein) technology developed to exploit the binding capacity of non-antibody proteins. Anticalins are well known in the art and refer to another antibody mimetic technology, in which the binding specificity is derived from lipocalins. Anticalins may also be formatted as dual-targeting proteins, called duocalins. Avimers are well known in the art and refer to another antibody mimetic technology, derived from natural A-domain-containing proteins. Versabodies are well known in the art and refer to another antibody mimetic technology, in which small proteins of 3-5 kDa with more than 15% cysteines form a high disulfide density scaffold, replacing the hydrophobic core of typical proteins. Such antibody mimics may be contained within the scaffold. The term "scaffold" refers to a polypeptide platform for engineering novel products with tailored functions and characteristics.

[0080] In one aspect, the present invention relates to the generation of non-immunoglobulin-based antibodies, also called antibody mimetics, using non-immunoglobulin scaffolds onto which the CDRs of the present invention can be grafted. Known or future non-immunoglobulin frameworks and scaffolds may be used, as long as they contain binding regions specific for the target LTBP2 protein.

[0081] Fibronectin scaffolds are based on type III fibronectin domains (e.g., the tenth module (tenth Fn3 domain) of type III fibronectin). Type III fibronectin domains have seven or eight β-strands distributed between two β-sheets that pack together to form the core of the protein, and also contain loops (similar to CDRs) that connect the β-strands to each other and expose them to the solvent. At least three such loops are present at each end of the sandwiched β-sheets, where the edges are the boundaries of the protein perpendicular to the direction of the β-strands (see U.S. Pat. No. 6,818,418). Although these fibronectin-based scaffolds are not immunoglobulins, their overall fold is closely related to that of the variable region of the heavy chain, the smallest functional antibody fragment that contains all of the antigen recognition units in camel and llama IgG. Because of this structure, non-immunoglobulin antibodies mimic antigen-binding properties similar in nature and affinity to those of antibodies. These scaffolds can be used in in vitro loop randomization and shuffling strategies, which resemble the in vivo affinity maturation process of antibodies. These fibronectin-based molecules can be used as scaffolds in which the loop regions of the molecule can be replaced with the CDRs of the invention using standard cloning techniques.

[0082] Ankyrin technology is based on the use of proteins containing ankyrin-derived repeat modules as scaffolds to carry variable regions that can be used to bind to different targets. The ankyrin repeat module is a 33-amino acid polypeptide consisting of two antiparallel α-helices and a β-turn. The binding of the variable regions is mostly optimized using ribosome display.

[0083] Avimers are derived from naturally occurring A-domain-containing proteins, such as LRP-1. These domains are naturally used for protein-protein interactions, and over 250 proteins in humans are structurally based on "A-domain" monomers (2-10) linked via amino acid linkers. For example, methods described in U.S. Patent Application Publication Nos. 20040175756; 20050053973; 20050048512; and 20060008844 can be used to generate avimers capable of binding to target antigens.

[0084] Affibody affinity ligands are small, simple proteins composed of a three-helix bundle based on the backbone of one of the IgG-binding domains of Protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. This backbone domain consists of 58 amino acids, 13 of which are randomized to generate affibody libraries with many ligand variants (see, e.g., U.S. Pat. No. 5,831,012). Affibody molecules mimic antibodies; they have a molecular weight of 6 kDa. Despite their small size, the binding site of affibody molecules is similar to that of antibodies.

[0085] Anticalins are products developed by Pieris ProteoLab. They are derived from lipocalins, a broad group of small, robust proteins typically involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins occur in human tissues and body fluids. The protein structure is reminiscent of immunoglobulins, with hypervariable loops on a rigid framework. However, in contrast to antibodies or their recombinant fragments, lipocalins consist of a single polypeptide chain with 160–180 amino acid residues, only slightly larger than a single immunoglobulin domain. The set of four loops that make up the binding pocket exhibits remarkable structural plasticity and tolerates a wide variety of side chains. Thus, the binding site can be reshaped by a proprietary process to recognize defined target molecules of various shapes with high affinity and specificity. Anticalins were generated by mutagenesis of a set of four loops using bilin-binding protein (BBP) from Pieris brassicae, a lipocalin family protein. An example of a patent application describing anticalins is PCT Publication No. WO199916873.

[0086] Affilin molecules are small, non-immunoglobulin proteins designed for specific affinity to proteins and small molecules. Novel affilin molecules can be very rapidly selected from two libraries, each based on a scaffold protein of different human origin. Affilin molecules show no structural homology to immunoglobulin proteins. Currently, two affilin scaffolds are used: one is gamma-crystallin, a human structural intraocular lens protein, and the other is a "ubiquitin" superfamily protein. Both human scaffolds are very small, highly thermostable, and largely resistant to pH changes and denaturing agents. This high stability is primarily due to the protein's amplified beta-sheet structure. Examples of "ubiquitin-like" proteins are described in WO2004106368.

[0087] Versabodies are highly soluble and can be formulated to high concentrations. Versabodies are exceptionally heat stable, providing a long shelf life. Further information regarding Versabodies can be found in U.S. Patent Application No. 2007 / 0191272, which is incorporated herein by reference in its entirety.

[0088] The above description of antibody fragment and mimetic technologies is not intended to be exhaustive.A wide variety of additional technologies could be used in the context of the present invention, including alternative polypeptide-based technologies, such as the fusion of complementarity-determining regions as outlined in Qui et al., Nature Biotechnology, 25(8) 921-929 (2007), and nucleic acid-based technologies, such as the RNA aptamer technology described in U.S. Patent Nos. 5,789,157; ​​5,864,026; 5,712,375; 5,763,566; 6,013,443; 6,376,474; 6,613,526; 6,114,120; 6,261,774; and 6,387,620 (all of which are incorporated herein by reference).

[0089] CAR-T cells The present invention also provides a chimeric antigen receptor (CAR) comprising the antigen-binding domain of an antibody of the present invention. Typically, the chimeric antigen receptor comprises at least one VH sequence and / or VL sequence of an antibody of the present invention. The chimeric antigen receptor of the present invention also comprises an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain.

[0090] As used herein, the term "chimeric antigen receptor" or "CAR" has its common meaning in the art and refers to an artificially constructed hybrid protein or polypeptide containing an antibody antigen-binding domain (e.g., a single-chain Fv) linked to a T cell signaling domain. Characteristics of CARs include their ability to exploit the antigen-binding properties of monoclonal antibodies to redirect T cell specificity and reactivity to selected targets in an MHC (major histocompatibility complex)-unrestricted manner. MHC-unrestricted antigen recognition confers on CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thus bypassing a major tumor evasion mechanism. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) α and β chains.

[0091] In some embodiments, the present invention provides a CAR comprising an antigen-binding domain comprising, consisting of, or consisting essentially of a single-chain variable fragment (scFv) of the 14A5.2 monoclonal antibody. In some embodiments, the antigen-binding domain comprises a linker peptide. The linker peptide can be located between the light chain variable region and the heavy chain variable region.

[0092] In some embodiments, the CAR comprises an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain selected from the group consisting of CD28, 4-1BB, and CD3ζ intracellular domains. CD28 is a T cell marker important for T cell costimulation. 4-1BB delivers a potent costimulatory signal to T cells, promoting differentiation and enhancing long-term survival of T lymphocytes. CD3ζ associates with the T cell receptor to generate signals and contains an immunoreceptor tyrosine-based activation motif (ITAM).

[0093] In some embodiments, the chimeric antigen receptors of the present invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g., via disulfide bridges, or converted into acid addition salts, and / or optionally dimerized or multimerized.

[0094] The present invention also provides nucleic acids encoding the chimeric antigen receptors of the present invention. In some embodiments, the nucleic acids are incorporated into vectors as described above.

[0095] The present invention also provides host cells containing nucleic acid encoding the chimeric antigen receptor of the present invention. The host cells may be of any cell type, originate from any type of tissue, and be at any stage of development; however, the host cells are T cells, for example, isolated from peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells (PBMCs). In some embodiments, the T cells may be any T cells, such as cultured T cells, e.g., primary T cells, or T cells derived from a cultured T cell line, e.g., the Jurkat cell line, the SupT1 cell line, or the like, or T cells obtained from a mammal. If obtained from a mammal, the T cells may be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cells may also be enriched or purified. The T cells may be any type of T cell and may be at any stage of development, including, but not limited to, CD4+ / CD8+ double positive T cells, CD4+ helper T cells, e.g., Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, etc. The T cells may be CD8+ T cells or CD4+ T cells.

[0096] Such T cell populations prepared as described above can be utilized in methods and compositions for adoptive immunotherapy according to known techniques or modifications thereof that will be apparent to those skilled in the art based on the present disclosure. See, for example, U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; see also U.S. Patent No. 4,690,915 to Rosenberg. Adoptive immunotherapy of cancer refers to a therapeutic approach in which immune cells with anti-tumor reactivity are administered to a tumor-bearing host with the goal that the cells will mediate, either directly or indirectly, the regression of established tumors. Infusion of lymphocytes, particularly T lymphocytes, falls into this category. Currently, most adoptive immunotherapies are autologous lymphocyte therapies (ALTs), which are directed toward treatment using the patient's own immune cells. These therapies involve engineering the patient's own lymphocytes to either enhance immune cell-mediated responses or recognize specific antigens or foreign substances in the body, including cancer cells. Treatment is achieved by removing the patient's lymphocytes and exposing them to biologics and drugs in vitro to activate their immune function. Once the autologous cells are activated, these ex vivo activated cells are reinfused into the patient to boost the immune system and treat cancer. In some embodiments, the cells are formulated by first harvesting the cells from their culture medium, then washing and concentrating the cells to a therapeutically effective amount in a medium and container system (a "pharmaceutically acceptable" carrier) suitable for administration. Suitable infusion media can be any isotonic medium formulation, typically normal saline, Normosol® (Abbott), or Plasmalyte® (Baxter), although 5% dextrose in water or lactated Ringer's solution may also be utilized. The infusion media may be supplemented with human serum albumin. The therapeutically effective amount of cells in the composition depends on the relative proportion of T cells with the desired specificity, the age and weight of the recipient, the severity of the condition being targeted, and the immunogenicity of the antigen being targeted. The amount of these cells is approximately 10 3 / kg may be as low as 5 × 10 3 / kg; 10 7 / kg may be used, and preferably 10 8 / kg. The number of cells will depend on the intended end use of the composition, as will the type of cells contained therein. For example, if cells specific for a particular antigen are desired, the population will contain more than 70%, typically 80%, 85%, and 90-95% of such cells. For the uses provided herein, the cells will generally be in a volume of 1 liter or less, and may be 500 ml or less, or even 250 ml or 100 ml or less. A clinically relevant number of immune cells may be distributed over multiple infusions, which cumulatively equal or exceed the total amount of cells desired.

[0097] In particular, the cells of the present invention are particularly suitable for the treatment of cancer. Accordingly, a further object of the present invention relates to a method for treating cancer in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of the cell population of the present invention.

[0098] multispecific antibodies In some embodiments, the present invention provides multispecific antibodies comprising a first antigen-binding site derived from an antibody of the molecules of the invention described herein above and at least one second antigen-binding site.

[0099] In some embodiments, the second antigen-binding site is used to recruit killing mechanisms by binding to an antigen on human effector cells, such as a BiTE (bispecific T cell-engaging) antibody, which is a bispecific scFv2 directed against a target antigen and CD3 on T cells as described in U.S. Patent No. 7,235,641, or by binding to a cytotoxic agent or second therapeutic agent. As used herein, the term "effector cell" refers to an immune cell that is involved in the effector phase of an immune response, rather than the cognitive and activation phases of an immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, e.g., cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, mast cells, and granulocytes, such as neutrophils, eosinophils, and basophils. Some effector cells express specific Fc receptors (FcRs) and perform specific immune functions. In some embodiments, effector cells, such as natural killer cells, are capable of inducing ADCC. For example, monocytes and macrophages expressing Fc receptors are involved in the specific killing of target cells and They are involved in presenting antigens to other components of the immune system. In some embodiments, effector cells can phagocytose target antigens or target cells. The expression of specific Fc receptors on effector cells can be regulated by humoral factors such as cytokines. Effector cells can phagocytose target antigens or phagocytose or lyse target cells. Examples of suitable cytotoxic agents and second therapeutic agents are provided below and include toxins (such as radiolabeled peptides), chemotherapeutic agents, and prodrugs.

[0100] In some embodiments, the second antigen-binding site binds to an antigen on a human B cell, such as CD19, CD20, CD21, CD22, CD23, CD46, CD80, CD138, and HLA-DR.

[0101] In some embodiments, the second antigen-binding site binds to a tissue-specific antigen, facilitating localization of the bispecific antibody to a particular tissue.

[0102] In some embodiments, the second antigen-binding site binds to an antigen located on the same type of cell as the LTBP2-expressing cell, typically a tumor-associated antigen (TAA), but has a different binding specificity than the first antigen-binding site. Such multispecific or bispecific antibodies may have enhanced tumor cell-binding specificity and / or may engage multiple effector pathways. Exemplary TAAs include carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), RAGE (kidney antigen), alpha-fetoprotein, CAMEL (an antigen recognized by cytotoxic T lymphocytes on malignant melanoma), CT antigens (e.g., MAGE-B5, -B6, -C2, -C3, and D; Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin antigens (e.g., MUC1, mucin-CA125, etc.), ganglioside antigens, tyrosinase, gp75, c-Met, Marti, Melan-A, MUM-1, MUM-2, MUM-3, HLA-B7, Ep-CAM, or cancer-associated integrins, such as α5β3 integrin. Alternatively, the second antigen-binding site binds to a different epitope of the antigen. The second antigen-binding site may alternatively bind to an angiogenic factor or other cancer-associated growth factor, such as vascular endothelial growth factor, fibroblast growth factor, epidermal growth factor, angiogenin, or a receptor for any of these, particularly a receptor associated with cancer progression.

[0103] In some embodiments, the second antigen-binding site is derived from a second antibody or antibody-drug conjugate of the invention, such as an antibody of the invention.

[0104] Exemplary formats of the multispecific antibody molecules of the invention include: (i) two antibodies crosslinked by chemical heteroconjugation, one with specificity for [antigen] and the other with specificity for a second antigen; (ii) a single antibody comprising two different antigen-binding regions; (iii) a single-chain antibody comprising two different antigen-binding regions, e.g., two scFvs linked in tandem by an extra peptide linker; and (iv) a dual variable domain antibody (DVD-Ig), in which each light and heavy chain contains two variable domains in tandem connected through a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig)). TM) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010); (v) chemically linked bispecific (Fab') fragments; (vi) Tandabs (which are the fusion of two single-chain diabodies, resulting in tetravalent bispecific antibodies with two binding sites for each target antigen); (vii) Flexibodies (which are the combination of a single-chain Fv and a diabody, resulting in a multivalent molecule); (viii) so-called "dock and lock" molecules based on the "dimerization and docking domain" of protein kinase A (which, when applied to Fabs, can result in trivalent bispecific binding proteins consisting of two identical Fab fragments linked to different Fab fragments); (ix) so-called scorpion molecules, which contain, for example, two single-chain Fvs fused to both ends of a human Fab arm; and (x) diabodies. Another exemplary format of bispecific antibodies is an IgG-like molecule with complementary CH3 domains that force heterodimerization. Such molecules can be prepared using known techniques, such as those known as Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb (Roche), and Electrostatically Matched Antibody (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain Body (SEED Body) (EMD Serono), Biclonic (Mers), and Duobody (Genmab) technologies.

[0105] In some embodiments, bispecific antibodies are obtained or obtainable via controlled Fab-arm exchange, typically using duobody technology. Methods for in vitro production of bispecific antibodies by controlled Fab-arm exchange are described in WO2008119353 and WO2011131746 (both by Genmab, Inc.). In one exemplary method described in WO2008119353, bispecific antibodies are formed by the exchange of "Fab-arms" or "molecular halves" (swapping of heavy chains and attached light chains) between two monospecific antibodies, both of which contain an IgG4-like CH3 region, upon incubation under reducing conditions. The resulting product is a bispecific antibody with two Fab arms that may contain different sequences. In another exemplary method described in WO2011131746, a bispecific antibody of the invention is prepared by a method comprising the following steps, wherein at least one of the first antibody and the second antibody is an antibody of the invention: a) providing a first antibody comprising an Fc region of an immunoglobulin, wherein the Fc region comprises a first CH3 region; b) providing a second antibody comprising an Fc region of an immunoglobulin, wherein the Fc region comprises a second CH3 region, wherein the sequences of the first CH3 region and the second CH3 region are different such that a heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than a homodimeric interaction between each of the first CH3 region and the second CH3 region; c) incubating the first antibody with the second antibody under reducing conditions; and d) obtaining the bispecific antibody, wherein the first antibody is an antibody of the invention and the second antibody has a different binding specificity, or vice versa. Reducing conditions may be provided, for example, by adding a reducing agent, e.g., selected from 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. Step d) may further comprise restoring the conditions to non-reducing or less reducing, e.g., by removing the reducing agent, e.g., by desalting.Preferably, the sequences of the first and second CH3 regions are different and contain only a few, fairly conservative, asymmetric mutations, such that the heterodimeric interaction between the first and second CH3 regions is stronger than the homodimeric interaction between the first and second CH3 regions, respectively. Further details regarding these interactions and how they can be achieved are described in WO2011131746, which is incorporated herein by reference in its entirety. The following are exemplary embodiments of such asymmetric mutation combinations, optionally in which one or both Fc regions are of the IgG1 isotype:

[0106] In some embodiments, the first Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, 407, and 409, and the second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, 407, and 409, wherein the first and second Fc regions are not substituted at the same positions.

[0107] In some embodiments, the first Fc region has an amino acid substitution at position 405, and the second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 407, and 409, optionally at position 409.

[0108] In some embodiments, the first Fc region has an amino acid substitution at position 409, and the second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, and 407, optionally at position 405 or 368.

[0109] In some embodiments, both the first and second Fc regions are of the IgG1 isotype, the first Fc region has a Leu at position 405, and the second Fc region has an Arg at position 409.

[0110] Immunoconjugates The antibody of the present invention can be conjugated with a detectable label to form an anti-LTBP2 immunoconjugate. Suitable detectable labels include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, enzyme labels, bioluminescent labels, or colloidal gold. Methods for producing and detecting such detectably labeled immunoconjugates are well known to those skilled in the art and are described in more detail below. The detectable label may be a radioisotope that is detected by autoradiography. Particularly useful isotopes for the purposes of the present invention are 3H, 125I, 131I, 35S, and 14C.

[0111] The anti-LTBP2 immunoconjugate may also be labeled with a fluorescent compound. The presence of the fluorescently labeled antibody is determined by exposing the immunoconjugate to light of the appropriate wavelength and detecting the resulting fluorescence. Fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine.

[0112] Alternatively, the anti-LTBP2 immunoconjugate may be detectably labeled by coupling the antibody to a chemiluminescent compound. The presence of the chemiluminescent-tagged immunoconjugate is determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

[0113] Similarly, a bioluminescent compound can be used to label the anti-LTBP2 immunoconjugate of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Bioluminescent compounds useful for labeling include luciferin, luciferase, and aequorin.

[0114] Alternatively, anti-LTBP2 immunoconjugates may be detectably labeled by linking the anti-[antigen] antibody to an enzyme. When the anti-LTBP2 antibody-enzyme conjugate is incubated in the presence of an appropriate substrate, the enzyme reacts with the substrate to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorometric, or visual means. Examples of enzymes that can be used to detectably label polyspecific immunoconjugates include β-galactosidase, glucose oxidase, peroxidase, and alkaline phosphatase.

[0115] Those skilled in the art will know of other suitable labels that can be used in accordance with the present invention. Binding of marker moieties to anti-LTBP2 monoclonal antibodies can be achieved using standard techniques known in the art. Exemplary methods in this regard are described in Kennedy et al., Clin. Chim. Acta 70:1, 1976; Schurs et al., Clin. Chim. Acta 81:1, 1977; Shih et al., Int'l J. Cancer 46:1101, 1990; Stein et al., Cancer Res. 50:1330, 1990; and Coligan (supra).

[0116] Furthermore, the ease and versatility of immunochemical detection can be enhanced by using anti-LTBP2 monoclonal antibodies conjugated with avidin, streptavidin, and biotin (see, e.g., Wilchek et al. (eds.), "Avidin-Biotin Technology," Methods In Enzymology (Vol. 184) (Academic Press 1990); Bayer et al., "Immunochemical Applications of Avidin-Biotin Technology," in Methods In Molecular Biology (Vol. 10) 149-162 (Manson, ed., The Humana Press, Inc. 1992)).

[0117] Methods for performing immunoassays are well established (see, e.g., Cook and Self, "Monoclonal Antibodies in Diagnostic Immunoassays," in Monoclonal Antibodies: Production, Engineering, and Clinical Application 180-208 (Ritter and Ladyman, eds., Cambridge University Press 1995); Perry, "The Role of Monoclonal Antibodies in the Advancement of Immunoassay Technology," in Monoclonal Antibodies: Principles and Applications 107-120 (Birch and Lennox, eds., Wiley-Liss, Inc. 1995); Diamandis, Immunoassay (Academic Press, Inc. 1996)).

[0118] In some embodiments, the antibodies of the invention are conjugated to a therapeutic moiety, i.e., a drug. The therapeutic moiety can be, for example, a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulatory agent, a lytic peptide, or a radioisotope. Such conjugates are referred to herein as "antibody-drug conjugates" or "ADCs."

[0119] In some embodiments, the antibody is conjugated to a cytotoxic moiety. Cytotoxic moieties include, for example, taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicine; doxorubicin; daunorubicin; dihydroxyanthracenedione; tubulin inhibitors such as maytansine or an analogue or derivative thereof; mitotic inhibitors such as monomethyl auristatin E or F or an analogue or derivative thereof; dolastatin 10 or 15 or an analogue thereof; irinotecan or an analogue thereof; mitoxantrone; mithramycin; actinomycin D; 1-dehydrotestosterone; glucocorticoids; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or an analogue or derivative thereof; antimetabolites such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin benzodiazepine, 5-fluorouracil, dacarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine; alkylating agents such as mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C; platinum derivatives such as cisplatin or carboplatin; duocarmycin A, duocarmycin SA, rachelmycin (CC-1065), or analogs or derivatives thereof; antibiotics such as dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC); pyrrolo[2,1-c][1,4]-benzodiazepines (PDB);Diphtheria toxin and related molecules, such as diphtheria A chain and its active fragments and hybrid molecules, ricin toxin, such as ricin A or deglycosylated ricin A chain toxin, cholera toxin, Shiga-like toxins, such as Shiga-like toxin type 1, Shiga-like toxin type 2, Shiga-like toxin type IIV, heat-labile enterotoxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, Bowman-Birk soybean protease inhibitor, Pseudomonas exotoxin, allorin, saporin, modeccin, geranin, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins, such as PAPI, PAPII, and PAP-S, momordica charantia inhibitors, curcin, kurotin, saponaria officinalis inhibitors, gelonin, mitogelin, restrictocin, phenomycin, and enomycin toxins; ribonuclease (RNase); deoxyribonuclease (DNase I), Staphylococcal enterotoxin A; pokeweed antiviral protein; diphtheria toxin; and Pseudomonas endotoxin.

[0120] In some embodiments, the antibody is conjugated to a nucleic acid or nucleic acid-binding molecule. In one such embodiment, the conjugated nucleic acid is a cytotoxic ribonuclease (RNase) or deoxyribonuclease (e.g., DNase I), an antisense nucleic acid, an inhibitory RNA molecule (e.g., an siRNA molecule), or an immunostimulatory nucleic acid (e.g., an immunostimulatory CpG motif-containing DNA molecule). In some embodiments, the antibody is conjugated to an aptamer or ribozyme.

[0121] In some embodiments, the antibody is conjugated, eg, as a fusion protein, to a lytic peptide, such as CLIP (corticotropin-like intermediate lobe peptide), magainin 2, melittin, cecropin, and P18.

[0122] In some embodiments, the antibody is conjugated to a cytokine, such as IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, keratinocyte growth factor, interferon (IFN) alpha, IFN3, IFN gamma, GM (granulocyte macrophage)-CSF (colony stimulating factor), CD40L, Flt3 ligand, stem cell factor, ancestim, and tumor necrosis factor alpha.

[0123] In some embodiments, the antibody is conjugated to a radioisotope or a radioisotope-containing chelate. For example, the antibody may be conjugated to a chelator linker, such as DOTA, DTPA, or tiuxetan, which allows the antibody to be complexed with a radioisotope. The antibody may also or alternatively contain or be conjugated to one or more radiolabeled amino acids or other radiolabeled molecules. Non-limiting examples of radioisotopes include H, C, N, S, Y, Tc (technetium), I (iodine), I, Re (rhenium), Bi (bismuth), Ac (actinium), and Th (thorium). For therapeutic purposes, beta or alpha particle emitting radioisotopes can be used, such as 131I, 90Y (yttrium), 211At (astatine), 212Bi, 67Cu (copper), 186Re, 188Re, and 212Pb (lead).

[0124] In certain embodiments, the antibody-drug conjugate comprises an antitubulin agent. Examples of antitubulin agents include taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik), vinca alkaloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine), and dolastatins (e.g., auristatin E, auristatin FP, monomethyl auristatin F, monomethyl auristatin E, auristatin EB, and auristatin EVB). Other antitubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colcemid, estramustine, cryptophycin, cemadotin, maytansinoids, combretastatins, discodermolide, and ereutherobin. In some embodiments, the cytotoxic agent is a maytansinoid, another group of antitubulin agents. For example, in specific embodiments, the maytansinoid is maytansine or DM-1 (ImmunoGen, Inc.; see also Chari et al., Cancer Res. 52:127-131, 1992).

[0125] In other embodiments, the cytotoxic agent is an antimetabolite, which can be, for example, a purine antagonist (e.g., azathioprine or mycophenolate mofetil), a dihydrofolate reductase inhibitor (e.g., methotrexate), acyclovir, ganciclovir, zidovudine, vidarabine, ribavarin, azidothymidine, cytidine, arabinoside, amantadine, dideoxyuridine, iododeoxyuridine, poscarnet, or trifluridine.

[0126] In another embodiment, the anti-LTBP2 antibody is conjugated to a prodrug-converting enzyme. The prodrug-converting enzyme may be recombinantly fused to the antibody or chemically conjugated using known methods. Exemplary prodrug-converting enzymes are carboxypeptidase G2, β-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase, nitroreductase, and carboxypeptidase A.

[0127] Other molecules used as therapeutic moieties are pyrrolobenzodiazepine dimers (PBDs).

[0128] In a particular embodiment, the antibody is a chimeric antibody having a heavy chain identical to SEQ ID NO: 1 and a light chain identical to SEQ ID NO: 2, and is conjugated to monomethyl auristatin E.

[0129] In another specific embodiment, the antibody is a chimeric antibody having a heavy chain identical to SEQ ID NO: 1 and a light chain identical to SEQ ID NO: 2, and is conjugated to a pyrrolobenzodiazepine dimer (PBD).

[0130] Typically, antibody-drug conjugate compounds include a linker unit between the drug unit and the antibody unit. In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the drug unit from the antibody in the intracellular environment. In yet other embodiments, the linker unit is not cleavable, and the drug is released, for example, by degradation of the antibody.

[0131] In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., within a lysosome, endosome, or caveolae). The linker may be, for example, a peptidyl linker, which is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleavage agents may include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of the active drug within the target cell (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123).

[0132] The most typical is a peptidyl linker that is cleavable by enzymes present in 191P4D12-expressing cells. Examples of such linkers are described, for example, in U.S. Patent No. 6,214,345, which is incorporated herein by reference in its entirety for all purposes. In a specific embodiment, the peptidyl linker cleavable by intracellular proteases is a Val-Cit linker or a Phe-Lys linker (see, for example, U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin using a Val-Cit linker). One advantage of using intracellular proteolytic release of therapeutic agents is that the drug is typically attenuated upon conjugation, and the conjugate typically has high serum stability.

[0133] In other embodiments, the cleavable linker is pH sensitive, ie, sensitive to hydrolysis at certain pH values.

[0134] Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, an acid-labile linker (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic acid amide, orthoester, acetal, ketal, etc.) that is hydrolyzable in the lysosome can be used. (See, for example, U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as the pH in blood, but are unstable below pH 5.5 or 5.0, which is approximately the pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (eg, a thioether attached to the therapeutic agent via an acylhydrazone bond) (see, eg, US Pat. No. 5,622,929).

[0135] In yet other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A wide variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio)toluene), SPDB, and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel ed., Oxford U. Press, 1987). See also U.S. Pat. No. 4,880,935.

[0136] In yet other specific embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0137] In yet other embodiments, the linker unit is not cleavable and the drug is released by degradation of the antibody.

[0138] Typically, the linker is substantially insensitive to the extracellular environment. As used herein, "substantially insensitive to the extracellular environment" in the context of the linker means that when the antibody-drug conjugate compound is present in an extracellular environment (e.g., plasma), about 20% or less, typically about 15% or less, more typically about 10% or less, and even more typically about 5% or less, about 3% or less, or about 1% or less of the linker in a sample of the antibody-drug conjugate compound is cleaved. Whether a linker is substantially insensitive to the extracellular environment can be determined, for example, by incubating plasma with the antibody-drug conjugate for a predetermined time (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of free drug present in the plasma.

[0139] Techniques for conjugating molecules to antibodies are well known in the art (e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds., Alan R. Liss, Inc., 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (Robinson et al. eds., Marcel Deiker, Inc., 2nd ed. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al. eds., 1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibodies In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al. eds., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62:119-58. See also, e.g., PCT Publication No. WO 89 / 12624. Typically, the nucleic acid molecule is covalently attached to a lysine or cysteine ​​residue on the antibody through an N-hydroxysuccinimide ester or maleimide functional group, respectively.Conjugation using engineered cysteines or methods incorporating unnatural amino acids have been reported to improve conjugate homogeneity (Axup, JY, Bajjuri, KM, Ritland, M., Hutchins, BM, Kim, CH, Kazane, SA, Halder, R., Forsyth, JS, Santidrian, AF, Stafin, K., et al. (2012). Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc. Natl. Acad. Sci. USA 109, 16101-16106.; Junutula, JR, Flagella, KM, Graham, RA, Parsons, KL, Ha, E., Raab, H., Bhakta, S., Nguyen, T., Dugger, DL, Li, G., et al. (2010). Engineered Thio-trastuzumab-DM1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer. Clin. Cancer Res. 16, 4769-4778). Junutula et al. (2008) developed a cysteine-based site-specific conjugation called "thiomab" (TDC, thiomab drug conjugate), which is claimed to exhibit an improved therapeutic index compared to conventional conjugation methods. Conjugation to unnatural amino acids incorporated into antibodies has also been explored for antibody-drug conjugates; however, the generality of this approach has not yet been established (Axup et al., 2012).In particular, one skilled in the art can also envision engineered Fc-containing polypeptides with an acyl donor glutamine-containing tag (e.g., a Gin-containing peptide tag or a Q-tag) or endogenous glutamines that have been made reactive by polypeptide engineering (e.g., via deletion, insertion, substitution, or mutation of amino acids on the polypeptide). A stable and homogeneous population of engineered Fc-containing polypeptide conjugates can then be formed by covalently crosslinking the transglutaminase with an amine donor agent (e.g., a small molecule containing or bound to a reactive amine), which is site-specifically conjugated to the Fc-containing polypeptide through the acyl donor glutamine-containing tag or the accessible / exposed / reactive endogenous glutamine (WO2012059882).

[0140] therapeutic use In a further aspect, the present invention relates to an LTBP2 inhibitor for use in the treatment of cancer associated with cancer-associated fibroblasts (CAFs) in a subject in need thereof.

[0141] The term "cancer-associated fibroblasts" as used herein and according to the present invention refers to a cell type within the tumor microenvironment that promotes tumorigenic traits by initiating remodeling of the extracellular matrix or by secreting cytokines.

[0142] The terms "cancer associated with cancer-associated fibroblasts" or "tumor rich in cancer-associated fibroblasts" as used herein and described in the present invention refer to any cancer in which the effects of cancer-associated fibroblasts are particularly pronounced in their pro-angiogenic and extracellular matrix (ECM) organizing functions. Examples of cancers associated with cancer-associated fibroblasts include colorectal cancer, liver cancer, pancreatic cancer, breast cancer, and their associated liver metastases.

[0143] In certain embodiments, cancers associated with cancer-associated fibroblasts (CAFs) are cancers that have a subpopulation of cancer-associated fibroblasts designated as LTBP2-positive cancer-associated fibroblasts.

[0144] As used herein, the term "LTBP2 inhibitor" refers to a molecule or compound capable of inhibiting the interaction of LTBP2 with the microenvironment or a molecule or compound that destabilizes LTBP2. The term "LTBP2 inhibitor" also refers to an inhibitor of the expression of the gene encoding the protein. In the context of the present invention, LTBP2 inhibitors, and in particular anti-LTBP2 antibodies, can be used to deplete LTBP2-positive cancer-associated fibroblasts. More specifically, the inventors have shown that LTBP2 can also be expressed in cancer cells (see the Results section). Thus, anti-LTBP2 antibodies can be used to suppress the progression / invasion of cancer, and in particular highly malignant cancers such as hepatocellular carcinoma.

[0145] LTBP2 inhibitors are well known in the state of the art, including small interfering RNAs and short hairpin RNAs (Pang XF et al., 2019, Acta Physiol, DOI: 10.111 / alpha.13377 & Wan F et al., 2016, Oncol Res, DOI: 10.3727 / 096504016X14755368915591).

[0146] According to the present invention, the LTBP2 inhibitor may be an antibody of the present invention (C6, D2, F5 or F7 monoclonal antibody, see above).

[0147] In one embodiment, the inhibitor according to the present invention can be a low molecular weight compound, such as a small organic molecule (natural or unnatural). The term "small organic molecule" refers to a molecule (natural or unnatural) of a size comparable to those organic molecules commonly used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). The preferred size range for small organic molecules is up to about 10,000 Da, more preferably up to 5,000 Da, more preferably up to 2,000 Da, and most preferably up to about 1,000 Da.

[0148] In one embodiment, the compounds described in the present invention are aptamers. Aptamers are a class of molecules that represent an alternative to antibodies in molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences capable of recognizing virtually any class of target molecules with high affinity and specificity. Such ligands can be isolated through in vitro selection of random sequence libraries (SELEX), as described by Tuerk C. and Gold L., 1990. Random sequence libraries can be obtained by combinatorial chemical synthesis of DNA. Each member in this library is a final chemically modified linear oligomer of a unique sequence. The possible modifications, uses, and advantages of this class of molecules are reviewed in Jayasena SD, 1999. Peptide aptamers consist of conformationally constrained antibody variable regions displayed by platform proteins, such as E. coli thioredoxin A, selected from combinatorial libraries by the two-hybrid method (Colas et al., 1996). Then, for the present invention, a neutralizing aptamer of LTBP2 is selected.

[0149] In one embodiment, the compound described in the present invention is a polypeptide. In a specific embodiment, the polypeptide is an LTBP2 antagonist and can interfere with the function of LTBP2. In particular, the polypeptide can be a mutated LTBP2 protein or a similar protein that does not have the function of LTBP2. In one embodiment, the polypeptide of the present invention can be linked to a "cell-penetrating peptide" to enable the polypeptide to penetrate into cells. The term "cell-penetrating peptide" is well known in the art and refers to cell-penetrating or membrane-penetrating sequences, such as penetratin, TAT (twin-arginine permeation), mitochondrial permeability sequences, and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012). The polypeptide of the present invention can be produced by any suitable means, as will be apparent to those skilled in the art. To produce sufficient quantities of a polypeptide or functional equivalent thereof for use in accordance with the present invention, expression can be conveniently achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the present invention. Preferably, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expressing polypeptides in a wide variety of different host cells are well known. When expressed recombinantly, the polypeptide is preferably produced by expression from an encoding nucleic acid in a host cell. Any host cell can be used, depending on the individual requirements of a particular system. Suitable host cells include bacteria, mammalian cells, plant cells, yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, and many others. Bacteria are also preferred hosts for the production of recombinant proteins due to the ease with which they can be engineered and grown. A common and preferred bacterial host is Escherichia coli (E. coli).In a specific embodiment, it is believed that the polypeptides used in the therapeutic methods of the present invention can be modified to improve their therapeutic efficacy. Such modifications of therapeutic compounds can be used to reduce toxicity, extend circulation time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be significantly reduced by combining them with a variety of drug carrier vehicles that modify biodistribution. In the example of adding a dipeptide, the penetration of circulating drugs into the eye through the blood-retinal barrier can be improved by using endogenous transporters.

[0150] A strategy for improving drug viability is the use of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve cellular uptake mechanisms, alter permeability through physiological barriers, and modify clearance rates from the body. Water-soluble polymers have been synthesized containing drug moieties as terminal groups, as part of the backbone, or as pendant groups on the polymer chain to achieve either targeted or sustained-release effects. Polyethylene glycol (PEG) is widely used as a drug carrier due to its high biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and reduce toxicity. PEG can be conjugated to active agents through hydroxyl groups at the ends of the chains and through other chemical methods; however, PEG itself is limited to a maximum of two active agents per molecule. In a different approach, copolymers of PEG and amino acids have been explored as novel biomaterials, which would retain the biocompatible properties of PEG but have the added advantage of numerous attachment points per molecule (providing greater drug loading) and can be synthetically engineered to suit a wide variety of applications. Those skilled in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers consisting of alternating polymers of PEG and trifunctional monomers (such as lysine) are used by VectraMed (Plainsboro, New Jersey). PEG chains (typically less than 2000 daltons) are linked to the a- and e-amino groups of lysine through stable urethane bonds. Such copolymers retain the desirable properties of PEG while providing reactive pendant groups (carboxylic acid groups of lysine) at precisely controlled and predetermined intervals along the polymer chain. The reactive pendant groups can be used for derivatization, crosslinking, or conjugation with other molecules. These polymers are useful for generating stable, long-circulating prodrugs by varying the molecular weight of the polymer, the molecular weight of the PEG segment, and the cleavable linkage between the drug and the polymer.The molecular weight of the PEG segment affects the spacing of the drug / linking group complex and the amount of drug per molecular weight of the conjugate (smaller PEG segments provide greater drug loading). Generally, increasing the overall molecular weight of a block copolymer conjugate will extend its circulatory half-life. Nevertheless, the conjugate must be readily degradable or have a molecular weight below the glomerular filtration threshold (e.g., less than 60 kDa). In addition to the importance of the polymer backbone for maintaining circulatory half-life and biodistribution, linkers can be used to maintain the therapeutic agent in prodrug form until released from the backbone polymer by a specific trigger, typically by enzymatic activity within the target tissue. For example, this type of tissue-activated drug delivery is particularly useful when delivery to a specific site of biodistribution is required, with the therapeutic agent being released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those skilled in the art and can be based on enzyme kinetics, the distribution rate of active enzymes, and the cleavage specificity of the selected disease-specific enzyme. Such linkers can be used to modify the proteins or protein fragments described herein for delivery of therapeutic agents.

[0151] In another embodiment, the LTBP2 inhibitor according to the present invention is an inhibitor of LTBP2 gene expression.

[0152] Small inhibitory RNAs (siRNAs) can also function as LTBP2 expression inhibitors for use in the present invention. LTBP2 gene expression can be reduced by contacting a subject or cells with small double-stranded RNA (dsRNA), or a vector or construct that causes the production of small double-stranded RNA, thereby specifically suppressing LTBP2 gene expression (i.e., RNA interference, or RNAi). Methods for selecting a double-stranded RNA or a vector encoding a double-stranded RNA suitable for a gene whose sequence is known are well known in the art (see, e.g., Tuschl, T. et al. (1999); Elbashir, SM et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, ​​TR. et al. (2002); U.S. Patent Nos. 6,573,099 and 6,506,559; and International Patent Application Publication Nos. WO01 / 36646, WO99 / 32619, and WO01 / 68836).

[0153] Ribozymes can also function as LTBP2 gene expression inhibitors for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of DNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Thus, engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of the LTBP2 mRNA sequence are useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are first identified by scanning the target molecule for ribozyme cleavage sites (which typically include the following sequences: GUA, GUU, and GUC). Once identified, short RNA sequences of approximately 15-20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that may render the oligonucleotide sequence inappropriate. The suitability of a candidate target can also be evaluated by testing its availability for hybridization with complementary oligonucleotides, for example, using ribonuclease protection assays. Both antisense oligonucleotides and ribozymes useful as LTBP2 gene expression inhibitors can be prepared by known methods. These include techniques for chemical synthesis, such as solid-phase phosphoramidite chemical synthesis. Alternatively, antisense RNA molecules can be produced by in vitro or in vivo transcription of DNA sequences encoding the RNA molecules. Such DNA sequences can be incorporated into a wide variety of vectors incorporating appropriate RNA polymerase promoters, such as T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the present invention can be introduced as a means of improving intracellular stability and extending half-life.Possible modifications include, but are not limited to, the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and / or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl linkages rather than phosphodiesterase linkages within the oligonucleotide backbone.

[0154] The antisense oligonucleotide, siRNA and ribozyme of the present invention can be delivered in vivo alone or in association with a vector.In its broadest sense, "vector" refers to any vehicle that can facilitate the introduction of antisense oligonucleotide siRNA or ribozyme nucleic acid into cells, and preferably into cells that express LTBP2.Preferably, the vector transports the nucleic acid into cells with reduced degradation compared to the degradation that would occur in the absence of the vector.Generally, the vector useful in the present invention includes, but is not limited to, plasmid, phagemid, virus, or other vehicle derived from viral or bacterial origin, which has been manipulated by inserting or incorporating antisense oligonucleotide siRNA or ribozyme nucleic acid sequence. Viral vectors are a preferred type of vector, including, but not limited to, nucleic acid sequences derived from the following viruses: retroviruses, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; adenovirus, adeno-associated virus; simian virus (SV) type 40; polyomavirus; Epstein-Barr virus; papillomavirus; herpesvirus; vaccinia virus; poliovirus; and RNA viruses, such as retroviruses. Other vectors not named but known in the art can also be readily used. Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with a gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentiviruses), whose life cycle involves reverse transcription of genomic viral RNA into DNA followed by proviral integration into host cell DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins but incapable of manufacturing infectious particles). Such genetically engineered retroviral expression vectors have general utility for highly efficient transduction of genes in vivo.Standard protocols for generating replication-deficient retroviruses (including incorporating the exogenous genetic material into a plasmid, transfecting a packaging cell line with the plasmid, producing recombinant retrovirus by the packaging cell line, harvesting viral particles from tissue culture medium, and infecting target cells with the viral particles) are provided in Kriegler, 1990, and Murry, 1991. Preferred viruses for certain applications are adenoviruses and adeno-associated viruses, which are double-stranded DNA viruses already approved for use in human gene therapy. Adeno-associated viruses may be engineered to be replication-deficient and are capable of infecting a wide variety of cell types and species. They have additional advantages, such as heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages (including hematopoietic cells); and a lack of superinfection inhibition, allowing for the transduction of multiple lineages. Reportedly, adeno-associated viruses can integrate site-specifically into human cellular DNA, thereby minimizing the possibility of insertional mutagenesis and the fluctuations in inserted gene expression characteristic of retroviral infection. Furthermore, wild-type adeno-associated virus infection has been tracked in tissue culture for more than 100 passages in the absence of selective pressure, indicating that integration of adeno-associated viruses into the genome is a relatively stable event. Adeno-associated viruses can also function extrachromosomally.

[0155] Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those skilled in the art. See, e.g., Sambrook et al., 1989. Over the past several years, plasmid vectors have been used as DNA vaccines to deliver antigen-encoding genes to cells in vivo. They are particularly advantageous for this purpose because they do not have the same safety concerns as many viral vectors. However, these plasmids, which have promoters compatible with the host cell, can express peptides from genes operably encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC / cytomegalovirus, SV40, and pBlueScript. Other plasmids are well known to those skilled in the art. Furthermore, plasmids can be custom-designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids can be delivered by a variety of parenteral, mucosal, and topical routes. For example, DNA plasmids may be injected intramuscularly, intraocularly, intradermally, subcutaneously, or by other routes. They may also be administered intranasally as sprays or drops, by rectal suppositories, and orally. They may also be administered to the epidermis or mucosal surfaces using a gene gun. Plasmids may be administered in aqueous solutions, dried onto gold particles, or in conjunction with other DNA delivery systems (including, but not limited to, liposomes, dendrimers, cochleates, and microencapsulation).

[0156] In certain embodiments, the nucleic acid sequence of the antisense oligonucleotide, small interfering RNA, small hairpin RNA, or ribozyme is under the control of a heterologous regulatory region, such as a heterologous promoter, which may also be, for example, a viral promoter, such as a cytomegalovirus promoter, or any synthetic promoter.

[0157] In particular, the present invention relates to an antibody, fragment, or immunoconjugate of the present invention for use in treating cancer associated with cancer-associated fibroblasts in a subject in need thereof.

[0158] The antibodies of the present invention may be used alone or in combination with any suitable agent.

[0159] In each embodiment of the methods of treatment described herein, the anti-LTBP2 antibody or anti-LTBP2 antibody-drug conjugate is delivered in a manner consistent with conventional methods associated with the management of the disease or disorder for which treatment is sought. In accordance with the disclosure herein, an effective amount of the antibody or antibody-drug conjugate is administered to a patient in need of such treatment for a period of time and under conditions sufficient to prevent or treat the disease or disorder.

[0160] As used herein, the terms "treatment" and "treating" refer to both prophylactic or preventative treatment and curative or disease-modifying treatment (including treatment of subjects at risk of or suspected of having a disease, as well as subjects who are ill or have been diagnosed with a disease or medical condition), including the suppression of clinical recurrence. Treatment may be administered to a subject who has a medical disorder or who is likely to eventually develop a disorder to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disorder or a recurring disorder, or to extend the subject's survival beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant a pattern of disease treatment, e.g., a dosing pattern used during therapy. A therapeutic regimen can include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide high levels of drug to the subject during the initial period of the treatment regimen. An induction regimen may use (in part or in whole) a "loading regimen," which may involve administering a higher dose of drug than a physician would use during a maintenance regimen, administering a drug more frequently than a physician would administer a drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used to maintain a subject during disease treatment, e.g., so that the subject remains in remission over an extended period of time (months or years). A maintenance regimen may use continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., intermittent treatment, intermittent treatment, treatment upon relapse, or treatment upon reaching certain predetermined criteria (e.g., symptoms of disease, etc.)).

[0161] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount effective, at a dosage and for a period of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of an antibody of the present invention may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the antibody of the present invention to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the antibody or portion of the antibody are outweighed by the therapeutically beneficial effects. The effective dose and dosage regimen for an antibody of the present invention will depend on the disease or condition to be treated and can be determined by one of ordinary skill in the art. A physician of ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician can start a dose of an antibody of the present invention used in a pharmaceutical composition at a level lower than required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Generally, an appropriate dose of a composition of the present invention will be the amount of the compound that is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend on the factors described above. For example, a therapeutically effective amount for therapeutic use can be measured by its ability to stabilize the progression of a disease. Typically, the cancer-suppressing ability of a compound can be evaluated in an animal model system that is predictive of efficacy in, for example, human tumors. Alternatively, this property of a composition can be evaluated by examining the compound's ability to induce cytotoxic activity using in vitro assays known to those skilled in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise ameliorate symptoms in a subject. One skilled in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the specific composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is about 0.1-100 mg / kg, e.g., about 0.1-50 mg / kg, e.g., about 0.1-20 mg / kg, e.g., about 0.1-10 mg / kg, e.g., about 0.5, e.g., about 0.3, about 1, about 3 mg / kg, about 5 mg / kg, or about 8 mg / kg.An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg / kg, e.g., about 0.02-30 mg / kg, e.g., about 0.05-10 mg / kg, or 0.1-3 mg / kg, e.g., about 0.2-2 mg / kg. Administration can be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous, and can be administered, for example, near the target site. The dosage regimen in the above-described treatment and use methods is adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus can be administered, or several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the effectiveness of treatment is monitored during treatment, e.g., at predetermined time points. In some embodiments, effectiveness can be monitored by visualization of diseased areas or by other diagnostic methods further described herein, e.g., by performing one or more PET-CT scans, using, for example, a labeled antibody of the present invention, a fragment derived from an antibody of the present invention, or a miniantibody. If desired, the effective daily amount of the pharmaceutical composition may be administered as two, three, four, five, six, or more separate doses administered separately at appropriate intervals throughout the day, optionally in unit dosage forms. In some embodiments, the monoclonal antibodies of the invention are administered by slow continuous infusion over an extended period of time, such as more than 24 hours, to minimize any undesirable side effects. An effective dose of the antibody of the invention may also be administered using a dosing period of once a week, once every two weeks, or once every three weeks. The dosing period may also be limited, for example, to 8 weeks, 12 weeks, or until clinical progress is established.As a non-limiting example, treatment according to the present invention may be performed with a daily dose of an antibody of the present invention of about 0.1 to 100 mg / kg, for example, 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg / kg / day, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg / kg / day, after initiation of treatment. The dose may be provided using a single or divided dose every 24, 12, 8, 6, 4 or 2 hours, or any combination thereof, on at least one day of the 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40th day, or alternatively on at least one of the days of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 20th week, or any combination thereof.

[0162] Accordingly, one object of the present invention relates to a method for treating cancer associated with cancer-associated fibroblasts in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an LTBP2 inhibitor of the present invention.

[0163] In particular, the LTBP2 inhibitor is an anti-LTBP2 antibody (C6, D2, F5 or F7 monoclonal antibody, see above).

[0164] Tests are needed to test the functionality of putative LTBP2 inhibitors. To this end, cell attachment / adhesion assays can be used to identify LTBP2 inhibitors. Cancer-associated fibroblasts treated with LTBP2 inhibitors (LTBP2 blockers / suppressors) (e.g., small interfering RNAs, antibodies) will detach from the culture support and die.

[0165] Other methods for selecting suitable inhibitors may also be used. For example, LTBP2 inhibitors are identified by measuring the LTBP2 concentration in a fluid (blood, serum, plasma) before and after depleting / inhibiting this fluid with a test compound / molecule. LTBP2 is then detected using standard protocols such as ELISA or Luminex to determine the validity of the inhibition of the test compound / molecule.

[0166] In another aspect, the invention relates to an antibody of the invention, as defined in any aspect or embodiment herein, for use as a medicament.

[0167] In certain embodiments, anti-LTBP2 antibodies or LTBP2 inhibitors are used in combination with a second drug for the treatment of a disease or disorder. When used to treat cancers associated with cancer-associated fibroblasts (or tumors rich in cancer-associated fibroblasts), the anti-LTBP2 antibodies of the present invention can be used in combination with conventional cancer therapies, such as surgery, radiation therapy, chemotherapy, or a combination thereof. This is particularly true in tumors rich in cancer-associated fibroblasts, where drug penetration is hindered by the extracellular matrix (ECM) produced by cancer-associated fibroblasts. Targeting LTBP2 can reduce the extracellular matrix, thereby allowing for better drug penetration into tumors and therefore better therapeutic effects.

[0168] The present invention also provides therapeutic applications in which the antibodies of the present invention are used in combination with at least one additional therapeutic agent, e.g., for the treatment of cancer and metastatic cancer. Such administration may be simultaneous, separate, or sequential. For simultaneous administration, the agents may be administered as a single composition or as separate compositions, as appropriate. The additional therapeutic agent is typically related to the disease to be treated. Exemplary therapeutic agents include other anti-cancer antibodies, cytotoxic agents, chemotherapeutic agents, anti-angiogenic agents, anti-cancer immunogens, cell cycle / apoptosis regulators, hormone regulators, and other agents described below.

[0169] In some embodiments, the antibodies of the invention are used in combination with a chemotherapeutic agent. The term "chemotherapeutic agent" refers to a compound that is effective in inhibiting tumor growth. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylmelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatins; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (particularly cryptophycin I and cryptophycin II); syn 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novoenbiquin, fenesterine, prednimustine, trofosfamide, uracil mustard; nitroureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin (11) and calicheamicin 211, e.g., Agnew See Chem Intl. Ed. Engl. 33:183-186 (1994); dynemicins, e.g., dynemicin A; esperamicin;and neocarzinostatin chromophore and related chromoprotein-based enediyne antibiotic chromophores), aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carubicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin , esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopuricin, thiamiprine, thioguanine; pyrimidine analogues, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, e.g., calsterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal drugs, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; Bestra Bucil; Bisantrene; Edatrexate; Defofamine; Demecolcine; Diazicon; Eflornithine; Elliptinium acetate; Epothilone; Etoglucide; Gallium acetate; Hydroxyurea; Lentinan; Lonidamine; Maytansinoids such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK (registered trademark); Razoxane; Rhizoxin; Schizofuran; Spirogermanium; Tenuazonic acid;triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucarin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as paclitaxel (Taxol®, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (Taxotere®, Antony, Rhone-Poulenc, France) Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Also included within this definition are antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens (including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston)); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0170] In some embodiments, the antibodies of the present invention are used in combination with targeted cancer therapy. Targeted cancer therapy is a drug or other substance that blocks the growth and spread of cancer by interfering with specific molecules ("molecular targets") involved in cancer growth, progression, and spread. Targeted cancer therapy is sometimes referred to as a "molecularly targeted drug," "molecularly targeted therapy," "precision medicine," or similar terms. In some embodiments, the targeted therapy comprises administering a tyrosine kinase inhibitor to a subject. The term "tyrosine kinase inhibitor" refers to any of a wide variety of therapeutic agents or drugs that act as selective or non-selective inhibitors of receptor and / or non-receptor tyrosine kinases. Tyrosine kinase inhibitors and related compounds are well known in the art and are described in U.S. Patent Application Publication No. 2007 / 0254295, which is incorporated herein by reference in its entirety. It will be understood by those skilled in the art that a compound related to a tyrosine kinase inhibitor will replicate the effect of the tyrosine kinase inhibitor, for example, a related compound may act on a different member of a tyrosine kinase signaling pathway to produce the same effect as a tyrosine kinase inhibitor of that tyrosine kinase. Examples of tyrosine kinase inhibitors and related compounds suitable for use in the methods of embodiments of the present invention include dasatinib (BMS-354825), PP2, BEZ235, saracatinib, gefitinib (Iressa), sunitinib (Sutent; SU11248), erlotinib (Tarceva; OSI-1774), lapatinib (GW572016; GW2016), canertinib (CI 1033), semaxinib (SU5416), vatalanib (PTK787 / ZK222584), sorafenib (BAY43-9006), imatinib (Gleevec; STI571), leflunomide (SU101), vandetanib (Zactima; ZD6474), MK-2206 (8-[4-aminocyclobutyl]phenyl]-9-phenyl-1,2,4-triazolo[3,4-f][1,6]naphthyridin-3(2H)-one hydrochloride), derivatives thereof, analogs thereof, and combinations thereof.Additional tyrosine kinase inhibitors and related compounds suitable for use in the present invention are disclosed in, for example, U.S. Patent Application Publication No. 2007 / 0254295; U.S. Patent Nos. 5,618,829; 5,639,757; 5,728,868; 5,804,396; 6,100,254; 6,127,374; 6,245,759; 6,306,874; 6,313,138; 6,316,444; 6,329, 380, 6,344,459, 6,420,382, 6,479,512, 6,498,165, 6,544,988, 6,562,818, 6,586,423, 6,586,424, 6,740,665, 6,794,393, 6,875,767, 6,927,293, and 6,958,340, all of which are incorporated herein by reference in their entireties. In some embodiments, the tyrosine kinase inhibitor is a small molecule kinase inhibitor that is orally administered and has been the subject of at least one Phase I clinical trial, more preferably at least one Phase II clinical trial, even more preferably at least one Phase III clinical trial, and most preferably has been approved by the U.S. Food and Drug Administration for at least one hematological or oncological indication. Examples of such inhibitors include gefitinib, erlotinib, lapatinib, canertinib, BMS-599626 (AC-480), neratinib, KRN-633, CEP-11981, imatinib, nilotinib, dasatinib, AZM-475271, CP-724714, TAK-165, sunitinib, vatalanib, CP-547632, vandetanib, bosutinib, lestaurtinib, tandutinib, midostaurin, enzastaurin, AEE-788, and pazopanib. These include, but are not limited to, cediranib, axitinib, motasenib, OSI-930, cediranib, KRN-951, dovitinib, seliciclib, SNS-032, PD-0332991, MKC-I (Ro-317453; R-440), sorafenib, ABT-869, brivanib (BMS-582664), SU-14813, telatinib, SU-6668, (TSU-68), L-21649, MLN-8054, AEW-541, and PD-0325901.

[0171] In some embodiments, the antibodies of the present invention are used in combination with immunotherapeutic agents. As used herein, the term "immunotherapeutic agent" refers to a compound, composition, or treatment that indirectly or directly enhances, stimulates, or increases the body's immune response to cancer cells and / or reduces the side effects of other anti-cancer therapies. Thus, immunotherapy is a therapy that directly or indirectly stimulates or enhances the immune system's response to cancer cells and / or alleviates potential side effects caused by other anti-cancer agents. Immunotherapy is also referred to in the art as immunotherapy, biological therapy, therapy using biological response modifiers, and biologic therapy. Examples of common immunotherapeutic agents known in the art include, but are not limited to, cytokines, cancer vaccines, monoclonal antibodies, and non-cytokine adjuvants. Alternatively, immunotherapeutic treatment can consist of administering a certain amount of immune cells (such as T cells, natural killer cells, dendritic cells, or B cells) to a subject. Immunotherapeutic agents may be nonspecific, i.e., by generally enhancing the immune system, the human body becomes more effective at fighting the growth and / or spread of cancer cells, or they may be specific, i.e., targeted to cancer cells themselves. Immunotherapy regimen treatments may combine the use of nonspecific and specific immunotherapeutic agents. Nonspecific immunotherapeutic agents are substances that stimulate or indirectly improve the immune system. Nonspecific immunotherapeutic agents are used alone as the primary therapy for cancer treatment, as well as in addition to the primary therapy, in which case they function as adjuvants to enhance the effectiveness of other therapies (e.g., cancer vaccines). In this latter situation, nonspecific immunotherapeutic agents may also function to reduce side effects of other therapies, such as bone marrow suppression induced by certain chemotherapeutic agents. Nonspecific immunotherapeutic agents may act against important immune system cells and induce secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the agent itself may contain cytokines. Nonspecific immunotherapeutic agents are generally classified as cytokine or noncytokine adjuvants.Many cytokines have found application in the treatment of cancer, either as general, nonspecific immunotherapy designed to boost the immune system or as adjuvants provided with other therapies. Suitable cytokines include, but are not limited to, interferons, interleukins, and colony-stimulating factors. Interferons (IFNs) contemplated by the present invention include the general types of IFNs: IFN-alpha (IFN-α), IFN-beta (IFN-β), and IFN-gamma (IFN-γ). IFNs may act directly on cancer cells by slowing their growth, promoting the development of more normally behaving cells, and / or increasing the production of antigens by cancer cells, making them more easily recognized and destroyed by the immune system. IFNs may also act indirectly on cancer cells, for example, by slowing angiogenesis, boosting the immune system, and / or stimulating natural killer (NK) cells, T cells, and macrophages. Recombinant IFN-α is commercially available as Roferon (Roche Pharmaceuticals) and Intron A (Schering). Interleukins contemplated by the present invention include IL-2, IL-4, IL-11, and IL-12. Examples of commercially available recombinant interleukins include Proleukin® (IL-2; Chiron Pharmaceuticals) and Numega® (IL-12; Wyeth Pharmaceuticals). ZymoGenetics (Seattle, Washington) is currently testing recombinant IL-21, which is also contemplated for use in the combinations of the present invention. Colony-stimulating factors (CSFs) contemplated by the present invention include granulocyte colony-stimulating factor (G-CSF or filgrastim), granulocyte-macrophage colony-stimulating factor (GM-CSF or sargramostim), and erythropoietin (epoetin alfa, darbepoetin). Treatment with one or more growth factors may help stimulate the production of new blood cells in subjects undergoing traditional chemotherapy. Thus, treatment with colony-stimulating factors may help reduce the side effects associated with chemotherapy and may allow for the use of higher doses of chemotherapy agents.Various recombinant colony-stimulating factors are commercially available, such as Neupogen® (G-CSF; Amgen), Neulasta (perfilgrastim; Amgen), Leukine (GM-CSF; Berlex), Procrit (erythropoietin; Ortho Biotech), Epogen (erythropoietin; Amgen), and Aranesp (erythropoietin). The combination compositions and combination administration methods of the invention may also include "whole cell" and "adoptive" immunotherapy. For example, such methods can involve the use of immune system cells (e.g., tumor-infiltrating lymphocytes (TILs), e.g., CC2-positive and / or CD8-positive T cells (e.g., T cells expanded and / or genetically enhanced with tumor-specific antigens), antibody-expressing B cells or other antibody-producing or presenting cells, dendritic cells (e.g., dendritic cells cultured with dendritic cell expanders such as GM-CSF and / or Flt3-L, and / or tumor-associated antigen-loaded dendritic cells), anti-tumor natural killer cells, so-called hybrid cells, or the like. These methods and compositions may include injection or re-infusion of a combination of the above. Cell lysates may also be useful in such methods and compositions. Cellular "vaccines" in clinical trials that may be useful in this context include Canvaxin™, APC-8015 (Dendreon), HSPPC-96 (Antigenics), and Melacine® cell lysates. Antigens shed from cancer cells, and mixtures thereof (see, e.g., Bystryn et al., Clinical Cancer Research Vol. 7, 1882-1887, July 2001), optionally mixed with an adjuvant such as alum, may also be components in such methods and combination compositions.

[0172] In particular, the antibodies of the present invention may be used in combination with another antibody, such as antibody Ha22-2 (Seattle Genetics), described in patent application WO201247724.

[0173] In some embodiments, the antibodies of the invention are used in combination with radiation therapy. Radiation therapy can involve the administration of radiation or a concomitant radiopharmaceutical to the patient. The source of radiation can be either external or internal to the patient being treated (radiation treatment can be, for example, in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that can be used in the practice of such methods include, for example, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodine-123, iodine-131, and iridium-111.

[0174] In some embodiments, the antibodies of the invention are used in combination with antibodies specific for costimulatory molecules, including, but not limited to, anti-CTLA4 antibodies (e.g., ipilimumab), anti-PD1 antibodies, anti-PDL1 antibodies, anti-TIMP3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, or anti-B7H6 antibodies.

[0175] In some embodiments, the second agent is an agent that induces the death of cells expressing the antigen to which the second agent binds via ADCC. In some embodiments, the agent is an antibody (e.g., of the IgG1 or IgG3 isotype) whose mechanism of action includes inducing ADCC against cells to which the antibody binds. NK cells play an important role in inducing ADCC, and the enhanced reactivity of NK cells can be directed to target cells through the use of such a second agent. In some embodiments, the second agent is an antibody specific for a cell surface antigen, e.g., a membrane antigen. In some embodiments, the second antibody is specific for a tumor antigen (e.g., a molecule specifically expressed by tumor cells), such as those described above, e.g., CD20, CD52, ErbB2 (or HER2 / Neu), CD33, CD22, CD25, MUC-1, CEA (carcinoembryonic antigen), KDR, αVβ3, etc., particularly a lymphoma antigen (e.g., CD20). Thus, the present invention also provides a method for enhancing the antitumor effect of monoclonal antibodies directed against tumor antigen(s), in which the sequential administration of antibodies directed against one or more tumor antigens and an antibody of the present invention specifically enhances ADCC function and subsequently enhances target cell killing.

[0176] Thus, a further object relates to a method for enhancing NK cell antibody-dependent cellular cytotoxicity (ADCC) of an antibody in a subject in need thereof, comprising administering an antibody to said subject, and administering an antibody of the invention to said subject.

[0177] A further object of the present invention relates to a method for treating cancer associated with cancer-associated fibroblasts in a subject in need thereof, comprising the steps of administering to said subject a first antibody selective for a cancer cell antigen, and administering to said subject an antibody of the present invention.

[0178] Many antibodies are currently in clinical use for the treatment of cancer, and others are in various stages of clinical development. Antibodies of interest for the methods of the present invention act through ADCC and are typically selective for tumor cells; however, those skilled in the art will recognize that some clinically useful antibodies also act against non-tumor cells, such as CD20. Numerous antigens and corresponding monoclonal antibodies exist for the treatment of B-cell malignancies. One popular target antigen is CD20, which is found on B-cell malignancies. Rituximab is a chimeric, unconjugated monoclonal antibody directed against the CD20 antigen. CD20 plays an important functional role in B-cell activation, proliferation, and differentiation. The CD52 antigen is targeted by the monoclonal antibody alemtuzumab, which is applied in the treatment of chronic lymphocytic leukemia. CD22 is targeted by many antibodies, and has recently demonstrated efficacy in combination with toxins in chemotherapy-resistant hairy cell leukemia. Monoclonal antibodies targeting CD20 also include tositumomab and ibritumomab. Monoclonal antibodies used in solid tumors that are useful in the methods of the present invention include, but are not limited to, edrecolomab and trastuzumab (Herceptin). Edrecolomab targets the 17-1A antigen found in colon and rectal cancer and is approved for use in Europe for these indications. Its antitumor effects are mediated through induction of ADCC, CDC, and anti-idiotypic networks. Trastuzumab targets the HER-2 / neu antigen, which is found in 25% to 35% of breast cancers. Trastuzumab is thought to work in a variety of ways: downregulating HER-2 receptor expression, inhibiting the growth of human tumor cells overexpressing the HER-2 protein, enhancing immune recruitment and ADCC against tumor cells overexpressing the HER-2 protein, and downregulating angiogenic factors. Alemtuzumab (Campus) is used to treat chronic lymphocytic leukemia; colon cancer and lung cancer; gemtuzumab (Mylotarg) finds use in the treatment of acute myeloid leukemia; ibritumomab (Zevalin) finds use in the treatment of non-Hodgkin's lymphoma; panitumumab (Vectibix) finds use in the treatment of colon cancer.Cetuximab (Erbitux) is also of interest for use in the methods of the invention. The antibody binds to the epidermal growth factor receptor (EGFR) and has been used in the treatment of solid tumors, including colon cancer and squamous cell carcinoma of the head and neck (SCCHN).

[0179] kit parts A fifth aspect of the present invention relates to i) an LTBP2 inhibitor, and ii) at least one anti-cancer agent, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer associated with cancer-associated fibroblasts.

[0180] In some embodiments, the present invention relates to i) an antibody of the present invention directed against LTBP2 (C6, D2, F5 or F7 monoclonal antibody, see above), and ii) at least one anti-cancer agent, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancers with predominant stroma.

[0181] In some embodiments, the at least one anti-cancer agent is gemcitabine and / or follifirinox.

[0182] As used herein, the term "concurrent use" refers to the use of an LTBP2 inhibitor and at least one anti-cancer agent simultaneously.

[0183] As used herein, the term "separate use" refers to the use of an LTBP2 inhibitor and at least one anti-cancer agent that do not exist simultaneously.

[0184] As used herein, the term "sequential use" refers to the use of an LTBP2 inhibitor and at least one anti-cancer agent present in sequence.

[0185] Pharmaceutical Composition: Typically, the antibody or inhibitor of the present invention is administered to a subject in the form of a pharmaceutical composition containing a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that can be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based 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 will be formulated for administration to a patient. The compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. The term "use" herein includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intracapsular, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. Sterile injectable forms of the compositions of the present invention may be aqueous or oily suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile, fixed oils are conveniently used as solvents or suspending media. For this purpose, any non-irritating, fixed oil may be used, including synthetic monoglycerides or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.These oily solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants, commonly used in formulating pharmaceutically 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 pharmaceutically 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. For tablets for oral use, commonly used carriers include lactose and cornstarch. Lubricants, such as magnesium stearate, are also typically added. Diluents useful for oral administration in capsule form 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 may be administered in the form of rectal suppositories. 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 will melt in the rectum to release the drug. Such excipients include cocoa butter, beeswax, and polyethylene glycol. The compositions of the present invention may also be administered topically, especially when the target of treatment includes areas or organs easily accessible by topical application, including diseases of the eyes, skin, or lower gastrointestinal tract. Suitable topical formulations for each of these areas or organs are easily prepared. The compositions for topical application may 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 in a suitable lotion or cream containing the active compounds suspended or dissolved in one or more pharmaceutically 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 to the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches may also be used. The compositions of the present invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation, and may be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, hydrogen fluoride, and / or other convenient solubilizing or dispersing agents. For example, the antibody present in the pharmaceutical composition of the present invention may be supplied 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 exemplary suitable dose range for the antibody in the pharmaceutical composition of the invention is about 1 mg / mL. 2 to 500 mg / m 2 However, it will be understood that these schedules are exemplary, and that optimal schedules and formulation regimens can be adapted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition, which must be determined in clinical trials. Pharmaceutical compositions of the present invention for injection (e.g., intramuscular, intravenous) can be prepared to contain sterile buffered water (e.g., 1 ml for intramuscular administration) 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 anti-myosin 18A antibody of the present invention.

[0186] In certain embodiments, the use of liposomes and / or nanoparticles is contemplated for the introduction of antibodies into host cells. The formation and use of liposomes and / or nanoparticles is known to those of skill in the art.

[0187] Nanocapsules are generally capable of encapsulating compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymer overloading, such ultrafine particles (size approximately 0.1 μm) are generally designed using polymers that are degradable in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles can be easily fabricated.

[0188] Liposomes are formed from phospholipids dispersed in an aqueous medium, spontaneously forming multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs)). MLVs typically have diameters between 25 nm and 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters ranging from 200 to 500 Å, which contain aqueous solutions in their cores. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0189] The present invention will be further illustrated by the following figures and examples, which, however, should not be construed as limiting the scope of the present invention in any way. [Brief explanation of the drawings]

[0190] [Figure 1] LTBP2 is essential for the viability of cancer-associated fibroblasts, but less or not at all essential for cancer cells. A) MTT assay showing proliferation of CCD18Co cells exposed to HT29 conditioned medium (CM) and treated for 96 hours with various antibodies against LTBP2 (F5, C6, F7, D2) or irrelevant antibodies (rituximab, anti-CD20 antibody). B) Same as A for HT29 cancer cells. Mean values ​​are normalized to the non-treated condition (NT). Error bars indicate standard deviation. P values ​​were obtained by performing a t-test. [Figure 2]Expression of LTBP2 in hepatocellular carcinoma. Immunohistochemical analysis of LTBP2 expression in human hepatocellular carcinoma, its stromal and cancer cell components. Two magnifications (A) and (B), 100x and 400x magnifications, respectively, are shown. Dark colors indicate positive LTBP2 expression. Image (B) mainly shows selective LTBP2 positivity in specific cancer cells. [Figure 3] LTBP2 enhances hepatocellular carcinoma invasion in vitro. Examination of the invasive potential of hepatocellular carcinoma cells engineered to express LTBP2, specifically human hepatoma (HLE) cells (A) and Alexander (ALEX) cells (B). [Figure 4] Comparison of anti-LTBP-D2 antibody with rituximab and cetuximab. Test of the LTBP inhibitors rituximab (negative control) and cetuximab (positive control) on the invasion of LTBP2-expressing HLE cells (A) and LTBP2-expressing ALEX cells (B).

[0191] Working Example: Materials and Methods Patient materials The Sample Analysis and Research Committee of the Montpellier Regional Cancer Hospital (ICM, Montpellier Cancer Institute) approved this study. In accordance with French law, patients provided written consent authorizing the use of their material for research purposes. Liver metastases derived from 25 colorectal cancer patients (CRC-LM) were used in this study. All patients were treated with neoadjuvant chemotherapy before surgery and sample collection. Six different tumors from five patients were analyzed using single-cell RNA sequencing. Four patients presented with a single liver metastasis, while one had two metastases. The remaining cohort of CRC-LM patients was involved in a validation study.

[0192] Tumor collection and dissociation Fresh primary tumors and liver metastases were cut into multiple fragments (10–15 mm), taking care to avoid surrounding non-tumor tissue and areas of apparent necrosis. 2Tumor samples were further dissociated into small pieces using surgical scissors and washed with cold Hank's Balanced Salt Solution (product number 14025092, Gibco, Thermo Fisher Scientific, Waltham, MA, USA). To 200 mg of sample, 8 mL of enzyme digestion mixture was added. The latter consisted of 1 mL of collagenase (20 mg / mL, product number 0130, Sigma-Aldrich, St. Louis, MO, USA), 1 mL of hyaluronidase (20 mg / mL, product number H3506, Sigma-Aldrich), and 2.5 μL of deoxyribonuclease (100 μg / μL, product number D5025, Sigma-Aldrich) in 8 mL of RPMI medium (product number 21875042, Gibco). For further details, please see the supplemental material.

[0193] Cell sorting Three to ten million cells were transferred to a 15 mL clear conical tube, and the volume was adjusted to 1 mL using 0.5% bovine serum albumin in PBS. Five tubes (each containing 100,000 cells in 100 μL of 0.5% bovine serum albumin in PBS) were then prepared for individual staining / negative controls. Cells were stained (according to the manufacturer's instructions) using the following antibodies / dyes: EPCAM-PE (product number 347198, Becton Dickinson (BD), Franklin Lakes, NJ, USA); CD31-Alexa488 (product number 558068, BD); CD45-APC (product number 560973, BD); and Live-Dead-NearIR (product number L34961, Life Technologies, Thermo Fisher Scientific). The antibody-sample mixture was incubated for 30 minutes at room temperature, then topped up with 0.5% bovine serum albumin in PBS to a volume of 10 mL and centrifuged at 300 × g for 5 minutes at 4 °C. Cells were suspended in 1 mL of 0.5% bovine serum albumin in PBS and sorted using a FACS Aria2 (BD). For further details, see the supplemental material.

[0194] Single-cell RNA sequencing As outlined above, samples were processed using the 10x Genomics Single Cell 3' Reagent Kit Version 3 (10x Genomics, Pleasanton, CA, USA) user guide. The process can be briefly described as follows: starting with a cell suspension, gel beads-in-emulsion (GEM) was prepared, barcodes were generated, and reverse transcription was performed. The purified cDNA was then amplified for 12 cycles, and the resulting cDNA was analyzed using a fragment analyzer (High Sensitivity Kit Next-Generation Sequencer) (Agilent Technologies, Santa Clara, CA, USA) to determine its quantity. Subsequently, cDNA libraries were prepared, adjusting the PCR cycles based on the calculated cDNA concentration. For this purpose, the Chromium Single Cell 3' Library and Gel Beads Kit Version 3, the Chromium Single Cell 3' Chip Kit Version 3, and the Chromium i7 Multiplex were used. The ratios of each library were calculated based on a previous shallow sequencing run using the MiniSeq (Illumina, San Diego, CA, USA) and Mid Output Reagent Cartridge (Illumina). After assessing cell number, reads, and sequencing saturation, libraries were then pooled into two or three samples per run and normalized to the final loading concentration. Each run was sequenced on a NovaSeq using v1 chemistry. A sequencing depth of 50,000 reads per cell was targeted for each sample. Sequencing fastq files that passed Illumina's quality control standards were further analyzed using 10x Genomics' CellRanger pipeline versions 3.0.2 and 3.1.0.

[0195] Data preparation and initial filtering Raw data were processed using 10x Genomics' CellRanger software (version 3.0.2). For each sample, cells with the top 0.05% or total UMIs (unique molecular identifiers) were considered doublets and therefore removed. Cells with fewer than 1,000 distinct genes measured were also discarded. Unless otherwise noted, each cell trascriptome was normalized by total UMI counts (divided by the sum and multiplied by 10) and log-transformed (log2(1 + standard UMI counts)). For further details, see the supplemental material.

[0196] 2D Projection and Clustering Two-dimensional projections of EpCAM (epithelial cell adhesion molecule)-positive cells and naive T (NT) cells were obtained separately using the 1,500 most variable genes (variation coefficients) among the 5,000 most highly expressed genes in each cell population. We considered only genes expressed in at least 1% of the cells. Principal component analysis (PCA) was computed, and the first 30 principal components were then subjected to t-SNE (perplexity = 30). Clustering of naive T cells was achieved by computing Euclidean distances between transcriptomes and constructing a dendrogram using Ward's method. This computation used the same 1,500 genes as the projections.

[0197] Differential gene expression analysis and gene signatures Differentially expressed genes were identified using edgeR.41. To this end, TMM normalization was applied using the calcNormFactors function, and the glmFit and glmLRT functions, with default parameters, were used to identify differentially expressed genes. Corrected P values ​​were obtained according to the Benjamini-Hochberg procedure. Gene signatures for each of the four naive T cell clusters (CLU (clusterin)-positive, C3-positive, RGS5 (G protein signaling 5)-positive, and POSTN (periostin)-positive) were selected by requiring expression in at least 20% of the cluster cells, a fold change (FC) of 2 or greater in one comparison to three other clusters pooled together or three comparisons to each cluster separately, and a corrected P value of 1% or less. The top 30 such genes were retained and sorted according to fold change. Signature genes for the two major naive T cell clusters (MCAM (melanoma cell adhesion molecule)-positive and LTBP2-positive) were obtained by requiring expression in at least 20% of the main cluster cells and at least 10% of each subcluster cell (e.g., for MCAM-positive, in CLU-positive and RGS5-positive), a fold change of 2 or more compared with other major clusters (MCAM-positive vs. LTBP2-positive) or its subclusters (e.g., MCAM-positive vs. C3-positive or POSTN-positive), and a corrected P value of 1% or less. We retained the top 30 such genes sorted according to fold change.

[0198] Non-cancerous mesenchymal hepatocytes Liver mesenchymal single-cell transcriptomes were obtained from a publicly available atlas covering four healthy and three cirrhotic human livers. 13 Among these cells, the authors identified four clusters: Mes(1), Mes(2), Mes(3), and Mes(4). Mes(4) was discarded in our study because it was identified as a mesothelial cell. We constructed a Mes(1-3) gene signature following the same procedure as above: expression was required in at least 20% of cells in a given cluster, a fold change of 2 or greater compared to each other or the union of two other clusters, and a corrected P value of 1% or less. Because fewer genes met these criteria in this dataset, we limited the size of the signature to the top 16 genes according to fold change.

[0199] Machine Learning We constructed three different models to classify novel cells as belonging to one of Ramachandran et al.'s Mes(1), Mes(2), or Mes(3) mesenchymal cell subtypes. 13 We used random forests (R package Random Forest, default parameters), k-nearest neighbors (R package DMwR, function kNN, k = 100, standard = false), and support vector machines (R package caret; trainControl with method = "repeatedcv", number = 10, repeats = 3; train with method = "svmLinear", preprocess = c("center", "scale"), tuneLength = 10). The performance of the algorithms was evaluated by 20-fold repeated cross-validation, using 90% of the cells to train the models and 10% to test them.

[0200] cell culture HT29, LOVO, and CCD18Co cells were obtained from the American Type Culture Collection (ATCC, Virginia, USA). SW1222 cells were a kind gift from Dr. W. Bodmer, Department of Medical Oncology, Weatherall Institute, Oxford, UK. A cancer-associated fibroblast cell line was isolated from CRC-LM. LX2, CCD18Co cells, were immortalized. All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (all Gibco, Thermo Fisher Scientific, Waltham, MA, USA) at 37°C in 5% carbon dioxide.

[0201] Conditioned medium (CM) from colorectal cancer cell lines was obtained after incubating 80% confluent cells in serum-free DMEM for 48 hours. The conditioned medium was collected, centrifuged at 150 × g for 5 minutes at room temperature, and then added to CCD18Co, LX2, and cancer-associated fibroblast cell monolayers (cells pre-starved for 6 hours in serum-free medium) for 48 hours. A control consisted of the addition of serum-free DMEM. Conditioned medium was then collected for Western blot analysis and processed as before. Cell monolayers were washed twice with PBS and lysed for RNA extraction.

[0202] Human anti-LTBP2 siRNA (on-target and human LTBP2 (4053), catalog number L-011078-00-0005) and control siRNA (on-target and non-targeting pool, catalog number D-001810-10-05) were purchased from Dharmacon (Lafayette, CA, USA). CCD18Co cells were transfected with 40 nM siRNA using Lipofectamine (Lipofectamine 2000 Reagent, catalog number 11668-019, Life Technologies, Carlsbad, CA, USA). After 48 h, cell monolayers were washed twice with PBS and lysed for RNA extraction.

[0203] Selection of LTBP2 antibodies by phage display Histidine-tagged LTBP2 was produced by cloning the LTBP2 open reading frame (product number OHu107637, GeneScript, Piscataway, NJ, USA) into the pCMV vector (product number 212220, Agilent, Santa Clara, CA, USA) and transiently transfecting HEK293 cells. After culturing HEK293 cells under standard conditions, the medium was collected and centrifuged, and recombinant LTBP2 was purified using nickel chromatography (product number A50585, Thermo Fisher Scientific, Waltham, MA, USA). Phage display selection of anti-LTBP2 antibodies was performed by the academic platform GenAc at Montpellier Biocampus.

[0204] Treatment of cell culture medium with antibodies and MTT assay CCD18Co cells were cultured in DMEM under standard conditions, while cancer cell-conditioned medium was derived as described above. For the experiment, the conditioned medium was diluted 1:1 with fresh DMEM, and 1% fetal bovine serum was added to the mixture. The mixture was then transferred to CCD18Co cells, while antibodies were also added at a concentration of 5 μg / mL. The cells were then incubated for 120 hours under standard cell culture conditions. Cell viability was then assessed using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) staining (catalog number M5655, Sigma-Aldrich). Absorbance at 540 nm was measured.

[0205] Western blot analysis The conditioned medium was concentrated 10-fold using a Vivaspin column and a 10 kDa filter (catalog number VS0102, Sartorius Stedim Biotech, Stonehouse, UK). The cell culture medium was exchanged for RIPA buffer (150 mM NaCl, 0.5% sodium deoxycholate, 1% Triton X-100, 0.5% SDS, 50 mM Tris-HCl (pH 7.5)). Laemmli buffer (0.1% 2-mercaptoethanol, 0.0005% bromophenol blue, 10% glycerol, 2% SDS in 63 mM Tris-HCl (pH 6.8)) was added to 20 μl of the concentrated conditioned medium. The conditioned medium was then boiled for 5 minutes and loaded onto a 6% polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane at 100 V for 2 hours. After blocking in 5% skim milk for 1 h, the membrane was incubated (4°C overnight) with an antibody against LTBP2 (1:500; catalogue no. AF3850, R&D Systems, Minneapolis, USA).

[0206] Silver staining Laemmli buffer was added to 5 μl of 10-fold concentrated conditioned medium. After boiling the samples for 5 minutes, they were loaded onto a 10% polyacrylamide gel. The gel was stained using PlusOne Silver Staining Kit, Protein (Cat. No. 17-1150-01, GE Healthcare, Uppsala, USA).

[0207] Gene expression analysis by real-time quantitative PCR (RT-qPCR) Total RNA was isolated using the Monarch Total RNA Miniprep Kit (Cat. No. T2010S, New England Biolabs). For RT-qPCR analysis, RNA was reverse transcribed using SuperScript III Reverse Transcriptase (Cat. No. 18080; Invitrogen, Carlsbad, CA, USA). 20 ng of cDNA was used for each PCR reaction.

[0208] Comparisons of basal gene expression were performed in three biological replicates for cancer-associated fibroblasts and LX2, and four replicates for CCD18Co. Cp values ​​were compared using an unpaired Welch's test using the t-test R function (var.equal=false).

[0209] Evaluation of the effect of conditioned medium from colorectal cancer cells on hTert (telomerase reverse transcriptase) in cancer-associated fibroblasts, LX2, and CCD18Co, respectively, was performed with two, three, and four biological replicates. For each cell line, the ΔCp values ​​between the two conditions were compared using a paired Student's test (var.equal=false). Fold changes compared to the control condition are reported.

[0210] Bulk RNA sequencing Total RNA was isolated as previously described. For each sample, 1 μg of total RNA was used to construct a sequencing library. Libraries were prepared using the RNA Stranded Total RNA prep Ligation with Ribo-Zero Plus Kit (Illumina, San Diego, USA) to remove ribosomal RNA. They were then sequenced on a NovaSeq6000 with SP-200 cycles (Illumina), generating 66 million reads per direction per sample. Base calling and demultiplexing steps were performed using Illumina's Dragen 3.8.4 software. Fastq files were aligned to the human genome (Ensemble GRCh38) using our pipeline (STAR ​​using default parameters and two passes, read counts extracted using HTSeq-count).

[0211] TMM normalization was applied with the calcNormFactors function (edgeR package), and differentially expressed genes were identified using the glmFit and glmLRT functions with default parameters. P-value correction was obtained according to the Benjamini-Hochberg procedure (multitest package). Normalized transcriptomes were then log-transformed (×log2(x+1)), and z-scores were computed.

[0212] Functional analysis of differentially expressed genes (corrected p-value < 0.01, absolute fold change > 2) was achieved by performing a hypergeometric distribution test on Gene Ontology of Biological Processes (GOBPs) containing at least three differentially expressed genes.

[0213] Data access and sample identification numbers The single-cell transcriptome is available from Gene Expression Omnibus (GEO) under reference number GSE158692. In these data, patient 1 metastasis (P1_MP) is referenced as SC_196081, P2_MP as 19G00619, P3_MP as 19G00635, patient 4 metastasis a (P4_MP) as 19G02977_Big, b (P4_MPb) as 19G02977_Small, and P5_MP as 20G00953. RNA-seq data for cell culture media is available from Gene Expression Omnibus (GEO) under reference number GSE191323.

[0214] result Cancer-associated fibroblasts in CRC-LM consist of distinct subpopulations In this study, we isolated cancer-associated fibroblasts from metastases based on a triple-negative selection strategy (EPCAM-negative / CD45-negative / CD31-negative / LiveDead-negative). This strategy was chosen because of the lack of universal cancer-associated fibroblast cell surface markers and the potential heterogeneity of this cell population. Other recent single-cell cancer-associated fibroblast studies followed a similar procedure (7, 8). After data quality filtering and excluding several contaminating cells (n = 215, primarily hepatocytes; data not shown), we obtained a total of 4,397 individual transcriptomes of cancer-associated fibroblasts (data not shown). These cancer-associated fibroblasts clustered into two major groups, indicating the existence of two main cancer-associated fibroblast populations (data not shown). A second level of clustering resolved the two major populations into four more specialized cancer-associated fibroblast clusters (data not shown). Differential gene expression analysis identified population-specific genes at two levels of resolution (data not shown). Normalized expression levels of six representative genes were performed alongside a dendrogram of the complete cancer-associated fibroblast transcriptome, identifying four cancer-associated fibroblast populations. Each metastasis contained all cancer-associated fibroblast populations except for P3_MP, which lacked CLU positivity (data not shown).

[0215] All cancer-associated fibroblasts contained genes underlying typical cancer-associated fibroblast functions, such as the essential production of collagen and extracellular matrix-related components (data not shown). Nevertheless, there were significantly different activity levels in several gene ontology-based biological processes between these populations (data not shown). LTBP2-positive cancer-associated fibroblasts were involved in extracellular matrix remodeling (data not shown) and collagen production (data not shown). FAP (fibroblast activation protein), a marker of fibroblast activation and proliferation, and PDGFRA (platelet-derived growth factor receptor α), a marker of connective tissue remodeling, were also specifically expressed by LTBP2-positive cancer-associated fibroblasts (data not shown). LTBP2-positive / POSTN-positive cancer-associated fibroblasts, which account for the majority of LTBP2-positive cancer-associated fibroblasts, were even more active in many regions. These included collagen production (data not shown), transforming growth factor-β responses as exemplified by the expression of POSTN and INHBA (data not shown), angiogenesis (vascular endothelial growth factor C and two known angiogenesis-related genes, UNC5B and SRPX2 (19, 20), data not shown), and Wnt signaling. LTBP2-positive / C3-positive cancer-associated fibroblasts expressed complement genes (e.g., C7 and complement factor D, data not shown) but also expressed intermediate levels of clusterin (data not shown). Complement genes are known to have immunosuppressive effects in certain tumors, including colorectal cancer, especially when coexpressed with regulators of the complement cascade, such as clusterin (21). MCAM-positive cancer-associated fibroblasts expressed markers of the blood vessel wall, such as RGS5, a known pericyte gene. MCAM itself is a known pericyte and vascular smooth muscle cell (VSMC) gene. MYH11 expression was also strong in MCAM-positive cancer-associated fibroblasts; it is a marker of contractile force.Consistent with their higher muscle contractile properties, MCAM-positive / CLU (clusterin)-positive cancer-associated fibroblasts expressed additional markers of contractile force, such as PLN and ACTG2 (data not shown), but not exclusively. Furthermore, MCAM-positive / RGS5-positive cancer-associated fibroblasts exhibited an average cancer-associated fibroblast phenotype (data not shown) and were not specifically enriched (data not shown). Recent reports examining the heterogeneity of the tumor microenvironment across multiple cancers15 identified five commonly recognized cancer-associated fibroblast populations. We aligned our cancer-associated fibroblast gene signature with these data and found highly significant overlap (data not shown). We also found significant overlap of the LTBP2-positive gene signature with a cancer-associated fibroblast (CAF-S1) enriched in triple-negative breast tumors7 (data not shown).

[0216] To explain the phenotypic heterogeneity observed in the identified cancer-associated fibroblast populations in CRC-LM, we compared our data with an atlas depicting hepatic cell types in healthy and cirrhotic livers (13). As explained in the introduction, we identified three relevant mesenchymal cell populations in the liver: vascular smooth muscle cells (VSMCs), hepatic stellate cells (HSCs), and cells strongly associated with fibrosis that expressed PDGFRA (platelet-derived growth factor receptor α) but lacked RGS5 expression. These latter cells have been designated SAMs (scar-associated mesenchymal cells). Using the same procedures as above, we computed gene signatures for these three populations (data not shown). Notably, significant crossover of our four cancer-associated fibroblasts with these signatures indicated phenotypic proximity between vascular smooth muscle cells and MCAM-positive / CLU-positive cancer-associated fibroblasts, hepatic stellate cells and MCAM-positive / RGS5-positive cancer-associated fibroblasts, and scar-associated mesenchymal cells and LTBP2-positive cancer-associated fibroblasts (data not shown). Using a machine learning (ML) approach, we first constructed and evaluated various classifiers (random forest, support vector machine, and k-nearest neighbor) based on single-cell data from healthy and cirrhotic livers (13). Standard cross-validation (training models using 90% of the data and testing them using 10%) yielded good performance estimates (data not shown). Therefore, we applied these classifiers to the transcriptomes of our cancer-associated fibroblasts from CRC-LM and indeed confirmed the association patterns (data not shown). Notably, comparison of cancer-associated fibroblasts with their matched noncancerous mesenchymal cells showed systematically close and significantly increased process reporting compared to the noncancerous situation (data not shown).Finally, we investigated the single-cell transcriptomes of cancer-associated fibroblasts from hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) to assess the presence of cancer-associated fibroblasts similar to the LTBP2- and MCAM-positive cancer-associated fibroblasts in our CRC-LM (22). Strikingly, these two cancer-associated fibroblast populations featured intrahepatic cholangiocarcinomas of comparable size to our data in CRC-LM, but LTBP2-positive cancer-associated fibroblasts were strongly depleted in HCC (data not shown).

[0217] We confirmed the expression of three reporter genes (LTBP2, C3, and POSTN) associated with LTBP2-positive cancer-associated fibroblasts in related mesenchymal cells upon exposure to conditioned medium from colorectal cancer cells by RT-qPCR. Specifically, we used an astrocyte cell line (LX2), fibroblasts (CCD18Co), and cancer-associated fibroblasts from a patient's CRC-LM. Basal expression levels were obtained in cells incubated in DMEM (data not shown). We found that LTBP2 and POSTN were overexpressed in cancer-associated fibroblasts compared with LX2 and CCD18Co cells, whereas the reverse pattern was observed for C3. This suggested that the cancer-associated fibroblasts obtained from the patient at hand were of the LTBP2-positive / POSTN-positive subtype. Furthermore, LX2 stellate cells tended to express fewer LTBP2-positive gene markers (LTBP2, C3, and POSTN), consistent with the RGS5-positive cancer-associated fibroblasts originating from stellate cells in our single-cell data. To compare this with the situation in which mesenchymal cells are exposed to nearby cancer cells, we incubated LX2 and CCD18Co cells, as well as patient-derived cancer-associated fibroblasts, with conditioned medium from three different colorectal cancer cell lines. We observed that only fibroblast CCD18Co cells increased the expression of C3 and POSTN (data not shown). The lack of induction in cancer-associated fibroblasts may be due to very high basal levels, while LX2 stellate cells failed to respond to challenge with these two genes. Regarding LTBP2, we found a modest trend, suggesting that the primary regulation may operate at the protein level. Indeed, Western blots on conditioned medium of such cells used for RT-qPCR confirmed this hypothesis (data not shown).

[0218] LTBP2 is a secreted protein with the potential for systemic accessibility and targetability in vivo. Guided by this, we used phage display technology to select a series of four fully human IgG anti-LTBP2 antibodies. Treatment of fibroblasts (CCD18Co cells) with the series of anti-LTBP2 antibodies significantly reduced their viability (as judged by MTT assay) (Fig. 1A), but not HT29 colorectal cancer cells (Fig. 1B), indicating specificity for fibroblasts. Closer examination showed that 96 hours after treatment with the anti-LTBP2 antibodies, fibroblasts began to round up and detach from the wells. Staining of detached cells with propidium iodide / Hoechst did not reveal any obvious apoptosis or necrosis (data not shown), suggesting a more subtle LTBP2-mediated mechanism of action. To obtain information about the functional importance of LTBP2 for the biological function of fibroblasts, we silenced its gene expression by siRNA and performed RNA sequencing. We found 496 significantly deregulated genes, suggesting an important role (data not shown). Approximately half of the deregulated genes showed increased expression upon LTBP2 silencing, while the other half showed decreased expression. Gene ontology biological process enrichment analysis identified several deregulated pathways. Representative genes of several pathways were identified, including integrins, collagens, LOX (involved in extracellular matrix collagen cross-linking and stiffness), and CD151 (involved in cell adhesion).

[0219] LTBP2-positive fibroblasts are found in the portal space of normal human liver Scar-associated mesenchymal cells (SAMs) globally expressed portal fibroblast markers, and a subpopulation of scar-associated mesenchymal cells was located in the periportal space. 13 Our analysis above demonstrated the association of LTBP2-positive cancer-associated fibroblasts with scar-associated mesenchymal cells. Therefore, we sought to verify their origin in adjacent normal human liver. Triple immunofluorescence staining for pancytokeratin (epithelial compartment; data not shown), α-smooth muscle actin (hepatic stellate cells / fibroblasts), and LTBP2 revealed a clear enrichment of LTBP2-positive cells in the portal area of ​​normal liver (data not shown). Further investigation revealed that LTBP2 staining was consistent with the typical appearance of collagen-containing connective tissue found in the portal space. LTBP2-expressing cells were also positive for α-smooth muscle actin, although the two proteins did not colocalize. This was not surprising. This is because α-smooth muscle actin is known to be a cytoskeletal protein, whereas LTBP2 is primarily secreted. α-Smooth muscle actin is particularly positive in the Disse space and labels stellate cells. However, no significant LTBP2 staining was observed in the Disse space or hepatocytes. Within tumor tissue, double-stained LTBP2-positive / α-smooth muscle actin-positive cancer-associated fibroblasts (data not shown) were clearly distinguishable from cancer-associated fibroblasts expressing only α-smooth muscle actin (data not shown). For simplicity reasons, we chose to further identify only LTBP2-positive and α-smooth muscle actin-positive cancer-associated fibroblasts. We believe that LTBP2-positive cancer-associated fibroblasts are also positive for α-smooth muscle actin.

[0220] LTBP2-positive cancer-associated fibroblasts accumulate in tumor sites, accompanied by intense interstitial fibrosis. The above bioinformatics analysis demonstrated that LTBP2-positive cancer-associated fibroblasts are significantly involved in extracellular matrix remodeling. Further analysis revealed that LTBP2-positive cancer-associated fibroblasts tend to accumulate in areas of intense interstitial fibrosis. To confirm this initial observation, we utilized the invasion fronts of a cohort of 20 liver metastases, classified based on their so-called histopathological growth patterns (HGPs) (23-25). This classification evaluates the transition zone, where cancer cells proliferate toward normal liver parenchyma, surrounding stromal cells, and the extracellular matrix (ECM). Three patterns have been defined: the interstitial fibrotic (or encapsulated) histopathological growth pattern, characterized by extensive collagen deposition, prominent angiogenesis, and a complete lack of contact between tumor cells and hepatocytes; The extrusion (or proliferative) histopathological pattern lacks interstitial fibrosis; tumor cells are separated from hepatocytes by a thin layer of reticulin fibers, which are pushed aside by the metastases. A mild immune infiltrate may be present at the border. In replacement HGP, unlike the other two types of HGP, cancer cells infiltrate the liver parenchyma without disrupting its architecture. There is no fibrosis and almost no inflammation. Some metastases may exhibit distinctly different HGPs depending on their location. Analysis of immunofluorescence images clearly demonstrated that the proportion of LTBP2-positive cancer-associated fibroblasts was significantly higher at the invasive front of tumors exhibiting interstitial fibrosis HGP compared with those exhibiting replacement or extrusion patterns (data not shown). Cases with mixed growth patterns (two distinct areas exhibiting different HGPs) were also included and counted separately at the regional level.

[0221] Independently of the invasive front and HGP, metastases with a strong stromal fibrotic reaction in their center also contained an increased proportion of LTBP2-positive cancer-associated fibroblasts (data not shown).

[0222] LTBP2-positive cancer-associated fibroblasts and angiogenesis In addition to extracellular matrix remodeling, bioinformatics revealed elevated activity of angiogenesis-related pathways in LTBP2-positive cancer-associated fibroblasts. Therefore, we tested whether angiogenesis also correlates with the presence of LTBP2-positive cancer-associated fibroblasts. To this end, we evaluated CD31 positivity in 20 CRC-LMs, along with LTBP2 staining. The normal / cancer interface with a displacement / extrusion pattern yielded areas with few LTBP2-positive cancer-associated fibroblasts, and immunofluorescence analysis here revealed small capillary-type vessels (data not shown), similar to those observed in normal liver parenchyma. This contrasts sharply with areas of severe interstitial fibrosis, where numerous large vessels (not capillaries) could be readily observed (data not shown). The latter is consistent with previous reports highlighting the importance of neovascularization in interstitial fibrotic HGP (26). Similar to extracellular matrix remodeling, angiogenesis, when accompanied by a strong interstitial fibrotic response, was associated with a high density of LTBP2-positive cancer-associated fibroblasts in the center of metastases (data not shown). LTBP2 expression was positively correlated with vessel size across all cases (data not shown).

[0223] LTBP2-positive cancer-associated fibroblasts and cancer cell proliferation Areas with abundant LTBP2-positive cancer-associated fibroblasts exhibited significant interstitial fibrosis. Although we did not specifically stain for collagen, hematoxylin / eosin images and single-cell RNA-sequencing data clearly demonstrated significant collagen deposition / production by LTBP2-positive cancer-associated fibroblasts. Collagen leads to a more abundant (and certainly stiffer) extracellular matrix. Therefore, we hypothesized that this mechanical constraint would reduce the proliferative potential of cancer cells. To confirm this, we examined CRC-LM cases for Ki67 expression, which showed that Ki67 positivity in cancer cells was negatively correlated with the abundance of LTBP2-positive cancer-associated fibroblasts (data not shown).

[0224] Mapping cancer-associated fibroblast / cancer cell interactions EPCAM-positive cells (5,331 cells in total) obtained from six CRC-LMs formed well-defined clusters that correlated with the original patients (data not shown). Notably, two metastases from patient 4 grouped together, indicating less transcriptional diversity. To infer ligand-receptor (LR) interactions between cancer cells and the two main cancer-associated fibroblast populations (MCAM-positive and LTBP2-positive), we used our recently published Bioconductor package, SingleCellSignalR (27). Inference relies on a curated database of known in vivo and in vitro ligand-receptor interactions and the calculation of a score for each interaction, the so-called LR score. An LR score of 0.5 or greater is sufficient to confirm the interaction (27). For LR interactions between cancer cells and cancer-associated fibroblasts, we calculated six LR scores, one per metastasis, and imposed a threshold of 0.5 on the median LR score (generally, the LR scores of separate metastases were close to each other; data not shown). We found that the greatest number of paracrine LR interactions occurred between cancer-associated fibroblasts, followed by interactions between cancer-associated fibroblasts and cancer cells, and between cancer cells and cancer-associated fibroblasts (data not shown). Most of the molecules involved in these interactions were growth factors or related to the extracellular matrix, cell-cell interactions, or chemotaxis.

[0225] Next, we focused on the differences between interactions linking LTBP2- or MCAM-positive cancer-associated fibroblasts to cancer cells. Based on the median LR score difference (|MCAM.median LR score - LTBP2.median LR score| > 0.1) and differential gene expression of CAF-secreted ligands between MCAM-positive and LTBP2-positive cancer-associated fibroblasts (FDR < 1%, fold change > 2), we were able to determine LR interactions with a significant strength bias (data not shown). We identified 178 significantly stronger interactions between cancer cells and LTBP2-positive cancer-associated fibroblasts (data not shown) and 14 significantly stronger interactions with MCAM-positive cancer-associated fibroblasts (data not shown). Stronger interactions originating from LTBP2-positive cancer-associated fibroblasts included interactions with growth factors, Wnt signaling, and angiogenesis. Furthermore, MCAM- and LTBP2-positive cancer-associated fibroblasts appeared to differentially regulate laminin trimers. Selection of significantly biased LR interactions associated with cancer cells for the two cancer-associated fibroblast populations identified 81 stronger interactions toward LTBP2-positive cancer-associated fibroblasts and 13 stronger interactions toward MCAM-positive cancer-associated fibroblasts (data not shown). The greater number of significantly stronger interactions with LTBP2-positive cancer-associated fibroblasts in both directions reflects a greater propensity to act with more specific phenotypes, as previously suggested (data not shown).

[0226] Canonical Wnt signaling is upregulated in cancer cells found in the vicinity of LTBP2-positive cancer-associated fibroblasts Speculation of cell-cell interactions suggested that LTBP2-positive cancer-associated fibroblasts induce Wnt / β-catenin signaling in cancer cells (data not shown). Because the importance of Wnt in colorectal cancer is known, we sought to verify this observation at the protein level. We co-stained LTBP2 and β-catenin in CRC-LM and assessed the degree of nuclear β-catenin staining near LTBP2-positive cancer-associated fibroblasts. Nuclear accumulation of β-catenin could be observed in cancer cells located in areas rich in LTBP2-positive cancer-associated fibroblasts. Correlation analysis confirmed a significant association between the two proteins (data not shown).

[0227] Comparison of original cancer-associated fibroblasts in CRC-LM and other liver malignancies Published single-cell data on intrahepatic cholangiocarcinoma and hepatocellular carcinoma22 already allowed us to demonstrate the presence of a population of cancer-associated fibroblasts with LTBP2+ and MCAM-positive gene signatures (data not shown). Application of our machine learning model showed that both intrahepatic cholangiocarcinoma and CRC-LM have a comparable and significant proportion of cancer-associated fibroblasts derived from portal vein fibroblasts (data not shown). Conversely, hepatocellular carcinoma has a majority of cancer-associated fibroblasts derived from hepatic stellate cells (data not shown).

[0228] Cancer cells can adopt features of cancer-associated fibroblasts Surprisingly, the present inventors have shown that LTBP2 may be expressed in some cancer cells, particularly in highly malignant cancer cells (FIG. 2).

[0229] LTBP2 enhances hepatocellular carcinoma invasion in vitro The rate of hepatocellular carcinoma invasion (human hepatocellular carcinoma cells and Alexander cells) was higher in hepatocellular carcinoma cells engineered to express LTBP2 than in other groups (Figure 3). Thus, LTBP2-expressing cells are strongly involved in cancer progression and metastasis. Furthermore, LTBP2 is a functional target that promotes tumor development in human hepatocellular carcinoma.

[0230] LTBP2 inhibitors inhibit the invasion of LTBP2-expressing hepatocellular carcinoma cells Anti-LTBP2 inhibitors, particularly the antibodies of the present invention, reduce the invasion of LTBP2-expressing hepatocellular carcinoma cells. The rate of hepatocellular carcinoma invasion is reduced more effectively with D2 (FIG. 4), particularly compared to rituximab and cetuximab.

[0231] References: Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are hereby incorporated by reference into the present disclosure.

[0232] [Table 1] TIFF2025531832000010.tif242165 TIFF2025531832000011.tif241165 TIFF2025531832000012.tif46165

Claims

1. An antibody having a heavy chain comprising i) H-CDR1 of the C6 monoclonal antibody, ii) H-CDR2 of the C6 monoclonal antibody, and iii) H-CDR3 of the C6 monoclonal antibody, and a light chain comprising i) L-CDR1 of the C6 monoclonal antibody, ii) L-CDR2 of the C6 monoclonal antibody, and iii) L-CDR3 of the C6 monoclonal antibody. (wherein H-CDR1 of the C6 monoclonal antibody is defined by the sequence ranging from amino acid residue 31 to amino acid residue 35 of SEQ ID NO: 1; H-CDR2 of the C6 monoclonal antibody is defined by the sequence ranging from amino acid residue 50 to amino acid residue 66 of SEQ ID NO:1; The H-CDR3 of the C6 monoclonal antibody is defined by the sequence ranging from amino acid residue 100 to amino acid residue 113 of SEQ ID NO:1; L-CDR1 of the C6 monoclonal antibody is defined by the sequence ranging from amino acid residue 23 to amino acid residue 36 of SEQ ID NO:2; L-CDR2 of the C6 monoclonal antibody is defined by the sequence ranging from amino acid residue 52 to amino acid residue 58 of SEQ ID NO:2; The L-CDR3 of the C6 monoclonal antibody is defined by the sequence ranging from amino acid residue 91 to amino acid residue 100 of SEQ ID NO:2); Alternatively, an antibody having a heavy chain comprising i) the H-CDR1 of the D2 monoclonal antibody, ii) the H-CDR2 of the D2 monoclonal antibody, and iii) the H-CDR3 of the D2 monoclonal antibody, and a light chain comprising i) the L-CDR1 of the D2 monoclonal antibody, ii) the L-CDR2 of the D2 monoclonal antibody, and iii) the L-CDR3 of the D2 monoclonal antibody. (wherein H-CDR1 of the D2 monoclonal antibody is defined by the sequence ranging from amino acid residue 31 to amino acid residue 35 of SEQ ID NO: 1; H-CDR2 of the D2 monoclonal antibody is defined by the sequence ranging from amino acid residue 50 to amino acid residue 66 of SEQ ID NO:1; The H-CDR3 of the D2 monoclonal antibody is defined by the sequence ranging from amino acid residue 100 to amino acid residue 108 of SEQ ID NO:1; L-CDR1 of the D2 monoclonal antibody is defined by the sequence ranging from amino acid residue 23 to amino acid residue 36 of SEQ ID NO:2; L-CDR2 of the D2 monoclonal antibody is defined by the sequence ranging from amino acid residue 52 to amino acid residue 58 of SEQ ID NO:2; L-CDR3 of the D2 monoclonal antibody is defined by the sequence ranging from amino acid residue 91 to amino acid residue 100 of SEQ ID NO:2), Alternatively, an antibody having a heavy chain comprising i) the H-CDR1 of the F5 monoclonal antibody, ii) the H-CDR2 of the F5 monoclonal antibody, and iii) the H-CDR3 of the F5 monoclonal antibody, and a light chain comprising i) the L-CDR1 of the F5 monoclonal antibody, ii) the L-CDR2 of the F5 monoclonal antibody, and iii) the L-CDR3 of the F5 monoclonal antibody. (wherein H-CDR1 of the F5 monoclonal antibody is defined by the sequence ranging from amino acid residue 31 to amino acid residue 35 of SEQ ID NO: 1; H-CDR2 of the F5 monoclonal antibody is defined by the sequence ranging from amino acid residue 50 to amino acid residue 66 of SEQ ID NO:1; The H-CDR3 of the F5 monoclonal antibody is defined by the sequence ranging from amino acid residue 99 to amino acid residue 107 of SEQ ID NO:1; L-CDR1 of the F5 monoclonal antibody is defined by the sequence ranging from amino acid residue 23 to amino acid residue 36 of SEQ ID NO:2; L-CDR2 of the F5 monoclonal antibody is defined by the sequence ranging from amino acid residue 52 to amino acid residue 58 of SEQ ID NO:2; The L-CDR3 of the F5 monoclonal antibody is defined by the sequence ranging from amino acid residue 91 to amino acid residue 100 of SEQ ID NO:

2. Alternatively, an antibody having a heavy chain comprising i) the H-CDR1 of the F7 monoclonal antibody, ii) the H-CDR2 of the F7 monoclonal antibody, and iii) the H-CDR3 of the F7 monoclonal antibody, and a light chain comprising i) the L-CDR1 of the F7 monoclonal antibody, ii) the L-CDR2 of the F7 monoclonal antibody, and iii) the L-CDR3 of the F7 monoclonal antibody. (wherein H-CDR1 of the F7 monoclonal antibody is defined by the sequence ranging from amino acid residue 31 to amino acid residue 35 of SEQ ID NO: 1; H-CDR2 of the F7 monoclonal antibody is defined by the sequence ranging from amino acid residue 50 to amino acid residue 66 of SEQ ID NO:1; The H-CDR3 of the F7 monoclonal antibody is defined by the sequence ranging from amino acid residue 99 to amino acid residue 109 of SEQ ID NO:1; L-CDR1 of the F7 monoclonal antibody is defined by the sequence ranging from amino acid residue 23 to amino acid residue 36 of SEQ ID NO:2; L-CDR2 of the F7 monoclonal antibody is defined by the sequence ranging from amino acid residue 52 to amino acid residue 58 of SEQ ID NO:2; The L-CDR3 of the F7 monoclonal antibody is defined by the sequence ranging from amino acid residue 91 to amino acid residue 100 of SEQ ID NO:2).

2. The antibody of claim 1, having a heavy chain that is at least 70% identical to SEQ ID NO: 1, 3, 5 or 7 and a light chain that is at least 70% identical to SEQ ID NO: 2, 4, 6 or 8.

3. 3. The antibody of claim 2, having a heavy chain identical to SEQ ID NO: 1, 3, 4 or 7 and a light chain identical to SEQ ID NO: 2, 4, 6 or 7.

4. The antibody of claim 1, which is a chimeric antibody.

5. The antibody of claim 1, which is a humanized antibody comprising the CDRs of the C6, D2, F5 or F7 monoclonal antibody.

6. A nucleic acid molecule encoding the antibody of claim 1.

7. The antibody of claim 1 conjugated to a cytotoxic moiety.

8. The antibody of claim 1 for use as a pharmaceutical.

9. 10. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody of claim 1.

10. 10. The method of claim 9, wherein the cancer is colorectal cancer, liver cancer, pancreatic cancer, breast cancer, and liver metastases associated therewith.

11. 1. An LTBP2 (latent transforming growth factor beta 2 binding protein) inhibitor for use in a subject in need thereof for the treatment of cancer associated with cancer associated with cancer-associated fibroblasts (CAF).

12. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and at least one antibody of claim 1 or an inhibitor of claim 11.

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