Anti-SLC1A4 monoclonal antibody and its use

Abstract

The present disclosure relates to monoclonal antibodies directed against an external loop of SLC1A4, preferably human monoclonal antibodies. The present disclosure also relates to therapeutic uses of the monoclonal antibodies, particularly methods of use for treating serine-dependent cancers, particularly cancers that express SLC1A4. The present disclosure also relates to in vitro and in vivo methods of using the anti-SLC1A4 monoclonal antibodies disclosed herein for diagnostic purposes.

Description

[Technical Field]

[0001] The present disclosure relates to the field of diagnosis and treatment of serine-dependent cancers, particularly SLC1A4-expressing cancers. The present disclosure relates to monoclonal antibodies directed against human SLC1A4 and their use in diagnostic and therapeutic methods. [Background technology]

[0002] Cancer refers to a group of diseases characterized by the development of abnormal cells that divide uncontrollably and invade and destroy normal body tissues. Cancer is the second leading cause of death in the world. Numerous therapies have been developed to treat various cancer diseases. However, sometimes cancer cells can overcome the effectiveness of anti-cancer treatments. Therefore, it is important to specifically investigate and target the mechanisms of cancer and provide novel and more effective therapies.

[0003] As is known in the art, metabolic pathways leading to the synthesis, uptake, and utilization of the non-essential amino acid serine are frequently amplified in cancer. Many cancer types exhibit enhanced serine biosynthesis and uptake from the extracellular medium, as evidenced by the amplified expression and utilization of the de novo serine synthases phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT1), phosphoserine phosphatase (PSPH), and solute carrier family 1 members 4 and 5 (i.e., SLC1A4 and SLC1A5). Much of this serine is delivered to mitochondria for catabolism to glycine and 1C units. A meta-analysis study revealed that enzymes involved in the mitochondrial 1C pathway are among the most frequently overexpressed genes in cancer (Nilsson et al., 2014, Nat. Commun., Vol. 5:3128).

[0004] Many scientific groups have reported on the efficacy and mechanism of pharmacological inhibitors of serine glycine one-carbon (SGOC). In particular, the following pharmacological inhibitors can be cited: (i) SHINI targeting SHMT1 / 2 (Ducker et al., 2017, Proc Natl Acad Sci USA, Vol. 114:11404:11409), (ii) AGF347 targeting SHMT1 / 2, GART and ATIC (Dekhme et al., 2019, Mol Cancer Ther, Vol. 18:1787-1799), (iii) 2.12 targeting SHMT1 / 2 (Marani et al., 2016, Oncotarget, Vol. 7:4570-4583), (iv) LY345899 targeting MTHFD1 / 2 (Gustafsson et al., 2017, Cancer Res, Vol. 77:937-948), (v) Carolacton targeting MTHFD1 / 2 (Fu et al., 2017, Cancer Res, Vol. 77:937-948). al.,2017,Nat Commun.,Vol.8:1529),(vi) LY231514 / MTA / Pemetrexed targeting TYMS,DHFR,GART and ATIC (Chattopadhyay et al.,2007,Mol Cancer Ther,Vol.6:404~417),(vii) Amethopterin / MTX / Methothrexate targeting TYMS and DHFR (Kremer, 2004, Arthritis Rheum, Vol. 50:1370~1382), and (viii) 5-FU targeting TYMS (Danenberg, 1977, Biochim Biophys Acta, Vol. 473: 73~92).

[0005] In liposarcomas, increased recruitment of MDM2 to chromatin and a high dependency of these cancers on serine and glycine metabolism have been reported. In this context, genetic or pharmacological targeting of chromatin-bound MDM2 or the 3-phosphoglycerate dehydrogenase (PHGDH) enzyme, which catalyzes the first limiting step in de novo serine synthesis, has been reported to represent a highly effective therapeutic strategy for liposarcomas (PCT Application WO 2019 / 106126). In this literature, targeting key genes involved in serine metabolism or transport (including PGHDH, PSAT1, PSPH, and SLC1A4) and therapeutic strategies that limit serine availability or uptake were proposed for the treatment of liposarcomas with chromatin-bound MDM2. Although antibodies are listed among the various MDM2 inhibitor candidates that have been investigated, in reality, no antibodies have been prepared and, by definition, no antibodies have been tested. Summary of the Invention [Problem to be solved by the invention]

[0006] There remains a need to identify additional therapeutic strategies aimed at preventing or treating cancers (including their progression) associated with dysregulation of the synthesis, uptake, and utilization of the non-essential amino acid serine.

[0007] There remains a need to provide methods that make it possible to determine cancers associated with dysregulation of the synthesis, uptake and use of the non-essential amino acid serine, including its amplification.

[0008] The present disclosure aims to meet all or part of these needs. [Means for solving the problem]

[0009] The present disclosure provides a monoclonal antibody directed against an extracellular loop of the human SLC1A4 protein, comprising: a) a monoclonal antibody or antigen-binding fragment thereof, (i) a heavy chain, the variable domain of which is VH-CDR1 having the sequence set forth as SEQ ID NO: 1; VH-CDR2 having the sequence set forth as SEQ ID NO: 2; and VH-CDR3 having the sequence set forth as SEQ ID NO: 3 Including, and / or (ii) a light chain, the variable domain of which comprises: VL-CDR1 having the sequence set forth as SEQ ID NO: 5; VL-CDR2 having the sequence set forth as SEQ ID NO: 6; and VL-CDR3 having the sequence set forth as SEQ ID NO: 7 Including, the monoclonal antibody or antigen-binding fragment thereof, b) a monoclonal antibody or an antigen-binding fragment thereof, (i) a heavy chain, the variable domain of which is VH-CDR1 having the sequence set forth as SEQ ID NO: 9; VH-CDR2 having the sequence set forth as SEQ ID NO: 10; and VH-CDR9 having the sequence set forth as SEQ ID NO: 11 Including, and / or (ii) a light chain, the variable domain of which comprises: VL-CDR1 having the sequence set forth as SEQ ID NO: 13; VL-CDR2 having the sequence set forth as SEQ ID NO: 14; and VL-CDR3 having the sequence set forth as SEQ ID NO: 15 Including, the monoclonal antibody or antigen-binding fragment thereof, c) a monoclonal antibody or an antigen-binding fragment thereof, (i) a heavy chain, the variable domain of which is VH-CDR1 having the sequence set forth as SEQ ID NO: 17; VH-CDR2 having the sequence set forth as SEQ ID NO: 18; and VH-CDR9 having the sequence set forth as SEQ ID NO: 19 Including, and / or (ii) a light chain, the variable domain of which comprises: VL-CDR1 having the sequence set forth as SEQ ID NO: 21; VL-CDR2 having the sequence set forth as SEQ ID NO: 22; and VL-CDR3 having the sequence set forth as SEQ ID NO: 23 Including, the monoclonal antibody or antigen-binding fragment thereof, d) a monoclonal antibody or an antigen-binding fragment thereof, (i) a heavy chain, the variable domain of which is VH-CDR1 having the sequence set forth as SEQ ID NO: 25; VH-CDR2 having the sequence set forth as SEQ ID NO: 26; and VH-CDR9 having the sequence set forth as SEQ ID NO: 27 Including, and / or (ii) a light chain, the variable domain of which comprises: VL-CDR1 having the sequence set forth as SEQ ID NO: 29; VL-CDR2 having the sequence set forth as SEQ ID NO: 30; and VL-CDR3 having the sequence set forth as SEQ ID NO: 31 Including, the monoclonal antibody or antigen-binding fragment thereof, e) a monoclonal antibody or antigen-binding fragment thereof, (i) a heavy chain, the variable domain of which is VH-CDR1 having the sequence set forth as SEQ ID NO: 33; VH-CDR2 having the sequence set forth as SEQ ID NO: 34; and VH-CDR9 having the sequence set forth as SEQ ID NO: 35 Including, and / or (ii) a light chain, the variable domain of which comprises: VL-CDR1 having the sequence set forth as SEQ ID NO: 37; VL-CDR2 having the sequence set forth as SEQ ID NO: 38; and VL-CDR3 having the sequence set forth as SEQ ID NO: 39 Including, the monoclonal antibody or antigen-binding fragment thereof, f) a monoclonal antibody or an antigen-binding fragment thereof, (i) a heavy chain, the variable domain of which is VH-CDR1 having the sequence set forth as SEQ ID NO: 41; VH-CDR2 having the sequence set forth as SEQ ID NO: 42; and VH-CDR9 having the sequence set forth as SEQ ID NO: 43 Including, and / or (ii) a light chain, the variable domain of which comprises: VL-CDR1 having the sequence set forth as SEQ ID NO: 45; VL-CDR2 having the sequence set forth as SEQ ID NO: 46; and VL-CDR3 having the sequence set forth as SEQ ID NO: 47 Including, The monoclonal antibody or antigen-binding fragment thereof comprising The present invention relates to the above monoclonal antibody selected from the group consisting of:

[0010] In some embodiments, the monoclonal antibody is (i) a heavy chain variable domain having at least 85% amino acid identity with the heavy chain variable domain of SEQ ID NO: 4 and comprising a VH-CDR1 of SEQ ID NO: 1, a VH-CDR2 of SEQ ID NO: 2, and a VH-CDR3 of SEQ ID NO: 3; and / or (ii) a light chain variable domain having at least 85% amino acid identity with the light chain variable domain of SEQ ID NO: 8 and comprising a VL-CDR1 of SEQ ID NO: 5, a VL-CDR2 of SEQ ID NO: 6, and a VL-CDR3 of SEQ ID NO: 7. Includes:

[0011] In some embodiments, the monoclonal antibody is (i) a heavy chain variable domain having at least 85% amino acid identity with the heavy chain variable domain of SEQ ID NO: 12 and comprising a VH-CDR1 of SEQ ID NO: 9, a VH-CDR2 of SEQ ID NO: 10, and a VH-CDR3 of SEQ ID NO: 11; and / or (ii) a light chain variable domain having at least 85% amino acid identity with the light chain variable domain of SEQ ID NO: 16 and comprising a VL-CDR1 of SEQ ID NO: 13, a VL-CDR2 of SEQ ID NO: 14, and a VL-CDR3 of SEQ ID NO: 15. Includes:

[0012] In some embodiments, the monoclonal antibody is (i) a heavy chain variable domain having at least 85% amino acid identity with the heavy chain variable domain of SEQ ID NO: 20 and comprising a VH-CDR1 of SEQ ID NO: 17, a VH-CDR2 of SEQ ID NO: 18, and a VH-CDR3 of SEQ ID NO: 19; and / or (ii) a light chain variable domain having at least 85% amino acid identity with the light chain variable domain of SEQ ID NO: 24 and comprising a VL-CDR1 of SEQ ID NO: 21, a VL-CDR2 of SEQ ID NO: 22, and a VL-CDR3 of SEQ ID NO: 23. Includes:

[0013] In some embodiments, the monoclonal antibody is (i) a heavy chain variable domain having at least 85% amino acid identity with the heavy chain variable domain of SEQ ID NO: 28 and comprising a VH-CDR1 of SEQ ID NO: 25, a VH-CDR2 of SEQ ID NO: 26, and a VH-CDR3 of SEQ ID NO: 27; and / or (ii) a light chain variable domain having at least 85% amino acid identity with the light chain variable domain of SEQ ID NO: 32 and comprising a VL-CDR1 of SEQ ID NO: 29, a VL-CDR2 of SEQ ID NO: 30, and a VL-CDR3 of SEQ ID NO: 31. Includes:

[0014] In some embodiments, the monoclonal antibody is (i) a heavy chain variable domain having at least 85% amino acid identity with the heavy chain variable domain of SEQ ID NO: 36 and comprising a VH-CDR1 of SEQ ID NO: 33, a VH-CDR2 of SEQ ID NO: 34, and a VH-CDR3 of SEQ ID NO: 35; and / or (ii) a light chain variable domain having at least 85% amino acid identity with the light chain variable domain of SEQ ID NO: 40 and comprising a VL-CDR1 of SEQ ID NO: 37, a VL-CDR2 of SEQ ID NO: 38, and a VL-CDR3 of SEQ ID NO: 39. Includes:

[0015] In some embodiments, the monoclonal antibody is (i) a heavy chain variable domain having at least 85% amino acid identity with the heavy chain variable domain of SEQ ID NO: 44 and comprising a VH-CDR1 of SEQ ID NO: 41, a VH-CDR2 of SEQ ID NO: 42, and a VH-CDR3 of SEQ ID NO: 43; and / or (ii) a light chain variable domain having at least 85% amino acid identity with the light chain variable domain of SEQ ID NO: 48 and comprising a VL-CDR1 of SEQ ID NO: 45, a VL-CDR2 of SEQ ID NO: 46, and a VL-CDR3 of SEQ ID NO: 47. Includes:

[0016] In some embodiments, the monoclonal antibody is a human antibody.

[0017] The present disclosure further relates to an antigen-binding fragment of the antibody, which can be selected from the group consisting of fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabody.

[0018] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof can be linked to a therapeutic or diagnostic agent to form an immunoconjugate.

[0019] The present disclosure also relates to nucleic acid sequences encoding the monoclonal antibodies or antigen-binding fragments thereof described herein, as well as to vectors containing the nucleic acids, and also to host cells, particularly prokaryotic or eukaryotic host cells, containing the nucleic acid sequences or vectors described herein.

[0020] The present disclosure further relates to the in vitro use of the monoclonal antibodies or antigen-binding fragments thereof described herein for detecting SLC1A4 in a sample, preferably in a cancer tissue sample.

[0021] The present disclosure further relates to in vitro uses of the monoclonal antibodies, or antigen-binding fragments thereof, or immunoconjugates described herein for detecting SLC1A4-expressing tumors in cancer patients, for example, by immuno-imaging techniques.

[0022] The present disclosure also relates to pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof described herein, or an immunoconjugate described herein, in combination with a pharmaceutically acceptable carrier.

[0023] The present disclosure also relates to the monoclonal antibodies described herein, or antigen-binding fragments thereof, or immunoconjugates thereof, for use as drugs.

[0024] The present disclosure also relates to the monoclonal antibodies, or antigen-binding fragments thereof, or immunoconjugates thereof described herein for use in treating cancers that express SLC1A4. [Brief explanation of the drawings]

[0025] [Figure 1A] Figure 1 shows SLC1A4 protein expression in multiple human cell lines. Figure 1A is a photograph from a Western blot assay. Top line: Detection with a commercially available anti-SLC1A4 antibody. Bottom line: Detection with a commercially available anti-beta-actin antibody. Lanes, from left to right: (i) SK-MEL-5 melanoma cell line; (ii) SK-MEL-28 melanoma cell line; (iii) HPAC pancreatic cancer cell line; (iv) A2058 melanoma cell line; (v) MW2664 melanoma cell line; and (vi) IB115 sarcoma cell line. [Figure 1B] Figure 1 shows SLC1A4 protein expression in multiple human cell lines. Figure 1B is a graph showing the mRNA expression levels of the SLC1A4 gene in multiple cell lines. From left to right: (i) IB115 sarcoma cell line; (ii) SK-MEL-5 melanoma cell line; (iii) HPAC pancreatic cancer cell line. Vertical axis: SLC1A4 mRNA expression level, expressed in arbitrary units (AU). Cell lines were selected after gene expression analysis in CCLE (Cancer Cell Line Encyclopedia). [Figure 1C]Figure 1 shows SLC1A4 protein expression in multiple human cell lines. Figure 1C is a graph showing the mRNA expression levels of the SLC1A4 gene in multiple cell lines. From left to right: (i) IB111 sarcoma cell line; (ii) IB115 sarcoma cell line; (iii) SK-MEL-5 melanoma cell line; (iv) A375 melanoma reporter cell line; and (v) HPAC pancreatic cancer cell line. Vertical axis: SLC1A4 mRNA expression level, expressed in arbitrary units (AU). Cell lines were selected after gene expression analysis in the Cancer Cell Line Encyclopedia (CCLE). [Figure 2A] Figure 2 shows the binding of anti-SLC1A4 monoclonal antibodies of the present disclosure to the target extracellular loop of human SLC1A4. Figure 2A: Top frame: Amino acid sequence of human SLC1A4 extracellular loop (SEQ ID NO: 51). Main frame: Scheme of membrane-embedded SLC1A4 protein including intra-, extra-, and trans-membrane domains and the extracellular loop. Left portion: SLC1A4. Right portion: Illustrative scheme of binding of anti-SLC1A4 monoclonal antibodies to the SLC1A4 extracellular loop. [Figure 2A-2] Figure 2 shows the binding of an anti-SLC1A4 monoclonal antibody of the present disclosure to the target extracellular loop of human SLC1A4. Figure 2A-A, like Figure 2A, shows a scheme of the membrane-embedded SLC1A4 protein, including intramembrane, extramembrane, and transmembrane domains, as well as the extracellular loop. Left part: Receptor typology of SLC1A4. Right: Illustrative scheme of the binding of an anti-SLC1A4 monoclonal antibody to the SLC1A4 extracellular loop receptor. [Figure 2B]Figure 2 shows the binding of anti-SLC1A4 monoclonal antibodies of the present disclosure to the target extracellular loops of human SLC1A4. Figure 2B is a photograph of a Western blot assay of six anti-SLC1A4 human monoclonal antibodies of the present disclosure, shown from left to right as follows: (i) B5.1, (ii) E10.2, (iii) D6.2, (iv) G1.4, (v) B8.3, and (vii) A10.1. The left panel shows non-reducing conditions; the right panel shows reducing conditions. The human antibodies used in this assay were engineered to replace their Fc fragments with rabbit Fc fragments to facilitate their detection with a labeled anti-rabbit IgG commercially available antibody. Such engineering is known not to alter the specificity of the engineered antibodies present in the variable VH and VL fragments. [Figure 3] Figure 3 shows the binding of several human anti-SLC1A4 monoclonal antibodies of the present disclosure to the target SLC1A4 in a flow cytometry assay. Horizontal axis: fluorescence intensity, expressed in arbitrary units. Vertical axis: cumulative number of fluorescence-positive cellular events detected by the instrument. From left to right: (i) SK-MEL-5 melanoma cell line; (ii) IB115 sarcoma cell line; (iii) HPAC pancreatic cancer cell line. Fluorescent signal peaks labeled with the following antibodies: (i) 13R4 control anti-beta-galactosidase antibody; (ii) E10 anti-SLC1A4; (iii) A10 anti-SLC1A4; (iv) G11 anti-SLC1A4; (v) D6 anti-SLC1A4; (vi) B5 anti-SLC1A4; and (vii) B8 anti-SLC1A4. The human antibodies used in this assay have been engineered to have their Fc fragments replaced by rabbit Fc fragments to facilitate their detection by labeled anti-rabbit IgG commercial antibodies as well. [Figure 4A]Figure 4 shows the binding of several human IgG1 monoclonal antibodies to SLC1A4 expressed on the membranes of (i) SK-MEL-5 melanoma cell line (Figures 4A and 4B), (ii) HPAC pancreatic cell line (Figures 4C and 4D), (iii) IB111 sarcoma cell line (Figure 4E), (iv) IB115 sarcoma cell line (Figure 4E), (v) SKMEL-5 melanoma cell line (Figure 4F), (vi) SKBR3 breast cancer cell line (Figure 4F), (vii) A375 melanoma cell line (Figure 4G), (viii) A431 melanoma cell line (Figure 4G), (ix) SKOV3 ovarian cancer cell line (Figure 4H), or (x) U937 leukemia cell line (Figure 4H). Horizontal axis: fluorescence intensity, expressed in arbitrary units. Vertical axis: cumulative number of fluorescent-positive cell events detected by the instrument. Figure 4A, left to right: (i) 13R4 control anti-beta-galactosidase antibody; (ii) E10 anti-SLC1A4 antibody; (iii) D6 anti-SLC1A4 antibody. [Figure 4B] Figure 4 shows the binding of several human IgG1 monoclonal antibodies to SLC1A4 expressed on the membranes of (i) SK-MEL-5 melanoma cell line (Figures 4A and 4B), (ii) HPAC pancreatic cell line (Figures 4C and 4D), (iii) IB111 sarcoma cell line (Figure 4E), (iv) IB115 sarcoma cell line (Figure 4E), (v) SKMEL-5 melanoma cell line (Figure 4F), (vi) SKBR3 breast cancer cell line (Figure 4F), (vii) A375 melanoma cell line (Figure 4G), (viii) A431 melanoma cell line (Figure 4G), (ix) SKOV3 ovarian cancer cell line (Figure 4H), or (x) U937 leukemia cell line (Figure 4H). Horizontal axis: fluorescence intensity, expressed in arbitrary units. Vertical axis: cumulative number of fluorescent-positive cell events detected by the instrument. Figure 4B, left to right: (i) B8 anti-SLC1A4, (ii) G11 anti-SLC1A4. [Figure 4C]Figure 4 shows the binding of several human IgG1 monoclonal antibodies to SLC1A4 expressed on the membranes of (i) SK-MEL-5 melanoma cell line (Figures 4A and 4B), (ii) HPAC pancreatic cell line (Figures 4C and 4D), (iii) IB111 sarcoma cell line (Figure 4E), (iv) IB115 sarcoma cell line (Figure 4E), (v) SKMEL-5 melanoma cell line (Figure 4F), (vi) SKBR3 breast cancer cell line (Figure 4F), (vii) A375 melanoma cell line (Figure 4G), (viii) A431 melanoma cell line (Figure 4G), (ix) SKOV3 ovarian cancer cell line (Figure 4H), or (x) U937 leukemia cell line (Figure 4H). Horizontal axis: fluorescence intensity, expressed in arbitrary units. Vertical axis: cumulative number of fluorescent-positive cell events detected by the instrument. Figure 4C, left to right: (i) 13R4 control anti-beta-galactosidase antibody; (ii) E10 anti-SLC1A4; (iii) D6 anti-SLC1A4. [Figure 4D] Figure 4 shows the binding of several human IgG1 monoclonal antibodies to SLC1A4 expressed on the membranes of (i) SK-MEL-5 melanoma cell line (Figures 4A and 4B), (ii) HPAC pancreatic cell line (Figures 4C and 4D), (iii) IB111 sarcoma cell line (Figure 4E), (iv) IB115 sarcoma cell line (Figure 4E), (v) SKMEL-5 melanoma cell line (Figure 4F), (vi) SKBR3 breast cancer cell line (Figure 4F), (vii) A375 melanoma cell line (Figure 4G), (viii) A431 melanoma cell line (Figure 4G), (ix) SKOV3 ovarian cancer cell line (Figure 4H), or (x) U937 leukemia cell line (Figure 4H). Horizontal axis: fluorescence intensity, expressed in arbitrary units. Vertical axis: cumulative number of fluorescence-positive cell events detected by the instrument. Figure 4D, left to right: (i) B8 anti-SLC1A4, (ii) G11 anti-SLC1A4. Horizontal axis: fluorescence intensity, expressed in arbitrary units. [Figure 4E]Figure 4 shows the binding of several human IgG1 monoclonal antibodies to SLC1A4 expressed on the membranes of (i) SK-MEL-5 melanoma cell line (Figures 4A and 4B), (ii) HPAC pancreatic cell line (Figures 4C and 4D), (iii) IB111 sarcoma cell line (Figure 4E), (iv) IB115 sarcoma cell line (Figure 4E), (v) SKMEL-5 melanoma cell line (Figure 4F), (vi) SKBR3 breast cancer cell line (Figure 4F), (vii) A375 melanoma cell line (Figure 4G), (viii) A431 melanoma cell line (Figure 4G), (ix) SKOV3 ovarian cancer cell line (Figure 4H), or (x) U937 leukemia cell line (Figure 4H). Horizontal axis: fluorescence intensity, expressed in arbitrary units. Vertical axis: cumulative number of fluorescent-positive cell events detected by the instrument. Figure 4E, boxed graphs, from left to right: (a) IB111 sarcoma cell line and (b) IB115 sarcoma cell line. Fluorescent signal peaks labeled with the following antibodies: (i) IgG1 antibody, (ii) E10 anti-SLC1A4, (iii) D6 anti-SLC1A4, and (iv) B8 anti-SLC1A4. [Figure 4F] Figure 4 shows the binding of several human IgG1 monoclonal antibodies to SLC1A4 expressed on the membranes of (i) SK-MEL-5 melanoma cell line (Figures 4A and 4B), (ii) HPAC pancreatic cell line (Figures 4C and 4D), (iii) IB111 sarcoma cell line (Figure 4E), (iv) IB115 sarcoma cell line (Figure 4E), (v) SKMEL-5 melanoma cell line (Figure 4F), (vi) SKBR3 breast cancer cell line (Figure 4F), (vii) A375 melanoma cell line (Figure 4G), (viii) A431 melanoma cell line (Figure 4G), (ix) SKOV3 ovarian cancer cell line (Figure 4H), or (x) U937 leukemia cell line (Figure 4H). Horizontal axis: fluorescence intensity, expressed in arbitrary units. Vertical axis: cumulative number of fluorescent-positive cell events detected by the instrument. Figure 4F, boxed graphs, from left to right: (a) SKMEL-5 melanoma cell line and (b) SKBR3 breast cancer cell line. Fluorescent signal peaks labeled with the following antibodies: (i) IgG1 antibody, (ii) E10 anti-SLC1A4, (iii) D6 anti-SLC1A4, and (iv) B8 anti-SLC1A4. [Figure 4G]Figure 4 shows the binding of several human IgG1 monoclonal antibodies to SLC1A4 expressed on the membranes of (i) SK-MEL-5 melanoma cell line (Figures 4A and 4B), (ii) HPAC pancreatic cell line (Figures 4C and 4D), (iii) IB111 sarcoma cell line (Figure 4E), (iv) IB115 sarcoma cell line (Figure 4E), (v) SKMEL-5 melanoma cell line (Figure 4F), (vi) SKBR3 breast cancer cell line (Figure 4F), (vii) A375 melanoma cell line (Figure 4G), (viii) A431 melanoma cell line (Figure 4G), (ix) SKOV3 ovarian cancer cell line (Figure 4H), or (x) U937 leukemia cell line (Figure 4H). Horizontal axis: fluorescence intensity, expressed in arbitrary units. Vertical axis: cumulative number of fluorescent-positive cell events detected by the instrument. Figure 4G, boxed graphs, from left to right: (a) A375 melanoma cell line, and (b) A431 melanoma cell line. Fluorescent signal peaks labeled with the following antibodies: (i) IgG1 antibody and (ii) B8 anti-SLC1A4. [Figure 4H] Figure 4 shows the binding of several human IgG1 monoclonal antibodies to SLC1A4 expressed on the membranes of (i) SK-MEL-5 melanoma cell line (Figures 4A and 4B), (ii) HPAC pancreatic cell line (Figures 4C and 4D), (iii) IB111 sarcoma cell line (Figure 4E), (iv) IB115 sarcoma cell line (Figure 4E), (v) SKMEL-5 melanoma cell line (Figure 4F), (vi) SKBR3 breast cancer cell line (Figure 4F), (vii) A375 melanoma cell line (Figure 4G), (viii) A431 melanoma cell line (Figure 4G), (ix) SKOV3 ovarian cancer cell line (Figure 4H), or (x) U937 leukemia cell line (Figure 4H). Horizontal axis: fluorescence intensity, expressed in arbitrary units. Vertical axis: cumulative number of fluorescent-positive cell events detected by the instrument. Figure 4H, boxed graphs, from left to right: (a) SKOV3 ovarian cancer cell line and (b) U937 leukemia cell line. Fluorescent signal peaks labeled with the following antibodies: (i) IgG1 antibody and (ii) B8 anti-SLC1A4. [Figure 5]Figure 5 shows the in vitro effects of several human IgG1 monoclonal anti-SLC1A4 antibodies of the present disclosure on the IB115 liposarcoma cell line. Horizontal axis, left to right: (i) control culture in the absence of antibody; (ii) B8 anti-SLC1A4 antibody; (iii) D6 anti-SLC1A4 antibody; (iv) E10 anti-SLC1A4 antibody; (v) G11 anti-SLC1A4 antibody. Vertical axis: % living cells compared to reference (untreated) control cells. [Figure 6A] Figure 6 shows the antiproliferative activity of the following antibodies in the IB111 sarcoma cell line (Figure 6A): (i) B8 anti-SLC1A4 (dark line on each graph), (ii) E10 anti-SLC1A4 (dashed line on each graph), and (iii) D6 anti-SLC1A4 (gray line on each graph). Horizontal axis: antibody drug concentration (μg / mL). Vertical axis: percentage of living cells compared to reference (untreated) control cells. Growth curves showing growth inhibition of cell lines treated for 7 days with a range of increasing antibody concentrations. The IC50 (half-maximal inhibitory concentration) for each antibody is presented in μg / mL below each graph in Figures 6A-6D. [Figure 6B] Figure 6 shows the antiproliferative activity of the following antibodies in the following cancer cell lines: IB115 sarcoma cell line (Figure 6B): (i) B8 anti-SLC1A4 (dark line on each graph), (ii) E10 anti-SLC1A4 (dashed line on each graph), and (iii) D6 anti-SLC1A4 (gray line on each graph). Horizontal axis: antibody drug concentration (μg / mL). Vertical axis: percentage of living cells (%) compared to reference (untreated) control cells. Growth curves showing growth inhibition of cell lines treated for 7 days with a range of increasing antibody concentrations. The IC50 (half-maximal inhibitory concentration) for each antibody is presented in μg / mL below each graph in Figures 6A-6D. [Figure 6C]Figure 6 shows the antiproliferative activity of the following antibodies in the following cancer cell lines: SKMEL-5 melanoma cell line (Figure 6C): (i) B8 anti-SLC1A4 (dark line on each graph), (ii) E10 anti-SLC1A4 (dashed line on each graph), and (iii) D6 anti-SLC1A4 (gray line on each graph). Horizontal axis: antibody drug concentration (μg / mL). Vertical axis: percentage of living cells (%) compared to reference (untreated) control cells. Growth curves showing growth inhibition of cell lines treated for 7 days with a range of increasing antibody concentrations. The IC50 (half-maximal inhibitory concentration) for each antibody is presented in μg / mL below each graph in Figures 6A-6D. [Figure 6D] Figure 6 shows the antiproliferative activity of the following antibodies in the following cancer cell lines: HPAC pancreatic cancer cell line (Figure 6D): (i) B8 anti-SLC1A4 (dark line on each graph), (ii) E10 anti-SLC1A4 (dashed line on each graph), and (iii) D6 anti-SLC1A4 (gray line on each graph). Horizontal axis: antibody drug concentration (μg / mL). Vertical axis: percentage of living cells (%) compared to reference (untreated) control cells. Growth curves showing growth inhibition of cell lines treated for 7 days with a range of increasing antibody concentrations. The IC50 (half-maximal inhibitory concentration) for each antibody is presented in μg / mL below each graph in Figures 6A-6D. [Figure 7A]Figure 7 shows individual tumor growth and survival curves for vehicle (NaCl) and anti-SLC1A4 antibody-treated mice inoculated with the IB111 liposarcoma cell line. Each group consisted of 10 mice. Vertical axis: mouse tumor volume, expressed in mm3 (cubic millimeters). Horizontal axis: days of treatment for mice. Figure 7A is a graph showing individual tumor growth curves in mice inoculated with the IB111 sarcoma cell line treated with vehicle (dark line) or different concentrations of B8 anti-SLC1A4 antibody (dashed line): left panel: B8 anti-SLC1A4 at 20 mg / kg, and right panel: B8 anti-SLC1A4 at 2 mg / kg. [Figure 7B] Figure 7 shows individual tumor growth and survival curves for vehicle (NaCl) and anti-SLC1A4 antibody-treated mice inoculated with the IB111 liposarcoma cell line. Each group consisted of 10 mice. Vertical axis: mouse tumor volume, expressed in mm3 (cubic millimeters). Horizontal axis: days of treatment for mice. Figure 7B is a graph showing individual tumor growth curves in mice inoculated with the IB111 sarcoma cell line treated with vehicle (dark line) or different concentrations of D6 anti-SLC1A4 antibody (dashed line): left panel: D6 anti-SLC1A4 at 20 mg / kg, and right panel: D6 anti-SLC1A4 at 2 mg / kg. [Figure 7C] Figure 7 shows individual tumor growth and survival curves for vehicle (NaCl) and anti-SLC1A4 antibody-treated mice inoculated with the IB111 liposarcoma cell line. Each group consisted of 10 mice. Vertical axis: mouse tumor volume, expressed in mm3 (cubic millimeters). Horizontal axis: days of treatment for mice. Figure 7C is a graph showing individual tumor growth curves in mice inoculated with the IB111 sarcoma cell line treated with vehicle (dark line) or different concentrations of E10 anti-SLC1A4 antibody (dashed line): left panel: E10 anti-SLC1A4 at 20 mg / kg, and right panel: E10 anti-SLC1A4 at 2 mg / kg. [Figure 8]Figure 8 shows Kaplan-Meier curves illustrating the survival of mice treated with the IB111 liposarcoma cell line treated with vehicle (thick line) or different anti-SLC1A4 antibodies. From left to right, the boxes show: (i) mice treated with vehicle or B8 anti-SLC1A4 antibody at 20 mg / kg or 2 mg / kg, (ii) mice treated with vehicle or D6 anti-SLC1A4 antibody at 20 mg / kg or 2 mg / kg, and mice treated with liver cancer vehicle or E10 anti-SLC1A4 antibody at 20 mg / kg or 2 mg / kg. Vertical axis: percentage of mouse survival. Horizontal axis: number of days of treatment. [Figure 9A] Figure 9 shows individual tumor growth and survival curves for vehicle (NaCl) and anti-SLC1A4 antibody-treated mice inoculated with the IB115 liposarcoma cell line. Each group consisted of 10 mice. Horizontal axis: mouse tumor volume, expressed in mm (cubic millimeters). Figure 9A is a graph showing individual tumor growth curves in mice inoculated with the IB115 sarcoma cell line treated with vehicle (dark line) or different concentrations of B8 anti-SLC1A4 antibody (dashed line): left panel: B8 anti-SLC1A4 at 20 mg / kg, and right panel: B8 anti-SLC1A4 at 2 mg / kg. [Figure 9B] Figure 9 shows individual tumor growth and survival curves for vehicle (NaCl) and anti-SLC1A4 antibody-treated mice inoculated with the IB115 liposarcoma cell line. Each group consisted of 10 mice. Horizontal axis: mouse tumor volume, expressed in mm3 (cubic millimeters). Horizontal axis: days of treatment for mice. Figure 9B is a graph showing individual tumor growth curves in mice inoculated with the IB115 sarcoma cell line treated with vehicle (dark line) or different concentrations of D6 anti-SLC1A4 antibody (dashed line): left panel: D6 anti-SLC1A4 at 20 mg / kg, and right panel: D6 anti-SLC1A4 at 2 mg / kg. [Figure 9C]Figure 9 shows individual tumor growth and survival curves for vehicle (NaCl) and anti-SLC1A4 antibody-treated mice inoculated with the IB115 liposarcoma cell line. Each group consisted of 10 mice. Horizontal axis: mouse tumor volume, expressed in mm3 (cubic millimeters). Horizontal axis: days of treatment for mice. Figure 9C is a graph showing individual tumor growth curves in mice inoculated with the IB115 sarcoma cell line treated with vehicle (dark line) or different concentrations of E10 anti-SLC1A4 antibody (dashed line): left panel: E10 anti-SLC1A4 at 20 mg / kg, and right panel: E10 anti-SLC1A4 at 2 mg / kg. [Figure 10A] Figure 10 shows Kaplan-Meier curves showing survival time in mice inoculated with the IB115 liposarcoma cell line and treated with vehicle or different anti-SLC1A4 antibodies. Vertical axis: percentage of mouse survival. Horizontal axis: days of treatment. Figure 10A: Mice treated with vehicle (dark line) or B8 anti-SLC1A4 antibody at 20 mg / kg (dark dashed line) or 2 mg / kg (gray dashed line). [Figure 10B] Figure 10 shows Kaplan-Meier curves showing survival time in mice inoculated with the IB115 liposarcoma cell line treated with vehicle or different anti-SLC1A4 antibodies. Vertical axis: percentage of mouse survival. Horizontal axis: days of treatment. Figure 10B: Mice treated with vehicle (dark line) or D6 anti-SLC1A4 antibody at 20 mg / kg (dark dashed line) or 2 mg / kg (gray dashed line). [Figure 10C] Figure 10 shows Kaplan-Meier curves showing survival time in mice inoculated with the IB115 liposarcoma cell line and treated with vehicle or different anti-SLC1A4 antibodies. Vertical axis: percentage of mouse survival. Horizontal axis: days of treatment. Figure 10C: Mice treated with vehicle (dark line) or E10 anti-SLC1A4 antibody at 20 mg / kg (dark dashed line) or 2 mg / kg (gray dashed line). [Figure 11]Figure 11 shows the distribution of membrane SLC1A4 H-scores in a tissue microarray containing various cancer lines. Each dot is associated with the quantitative expression of cell surface SLC1A4 in one human cancer line sample. The H-score is calculated by the following formula: 3 x percentage of strong staining + 2 x percentage of moderate staining + percentage of weak staining, giving a range of 0 to 300. Vertical axis: SLC1A4 protein expression on the cell surface, expressed as H-score. Horizontal axis: Cancer cell lines, from left to right: (i) Breast cancer, (ii) Endometrium cancer, (iii) Melanoma cancer, (iv) Ovary cancer, (v) Prostate cancer, (vi) Kidney cancer, (vii) Esophagus cancer, (viii) Stomach cancer, (ix) Liver cancer, (x) Pancreas cancer, (xi) Colon cancer, and (xii) Gastrointestinal Stromal Tumor (GIST). [Figure 12A] Figure 12 shows the internalization of several human IgG1 monoclonal anti-SLC1A4 antibodies, including IgG antibodies (●), B8 antibodies (■), D6 antibodies (▲), and E10 antibodies (▼), in various cancer cell lines, including the IB111 sarcoma cell line (Figure 12A). Cells were exposed to an unlimited amount of anti-SLC1A4 antibodies labeled with Fabfluor, a dye that fluoresces in response to lowering pH. The dye serves as a localization indicator within the acidified lysosomal compartment, where the antibody is degraded after internalization. Cells were imaged over time, and antibody-Fabfluor internalization was quantified by fluorescence microscopy. Vertical axis: ratio of red area per well, normalized to t0. Horizontal axis: time, expressed in hours. [Figure 12B]Figure 12 shows the internalization of several human IgG1 monoclonal anti-SLC1A4 antibodies, including IgG antibodies (●), B8 antibodies (■), D6 antibodies (▲), and E10 antibodies (▼), in various cancer cell lines, including the IB115 sarcoma cell line (Figure 12B). Cells were exposed to an unlimited amount of anti-SLC1A4 antibodies labeled with Fabfluor, a dye that fluoresces in response to lowering pH. The dye serves as a localization indicator within the acidified compartment of the lysosome, where the antibody is degraded after internalization. Cells were imaged over time, and antibody-Fabfluor internalization was quantified by fluorescence microscopy. Vertical axis: ratio of red area per well, normalized to t0. Horizontal axis: time, expressed in hours. [Figure 12C] Figure 12 shows the internalization of several human IgG1 monoclonal anti-SLC1A4 antibodies, including IgG (●), B8 (■), D6 (▲), and E10 (▼), in various cancer cell lines: the SKMEL-5 melanoma cell line (Figure 12C). Cells were exposed to an unlimited amount of anti-SLC1A4 antibodies labeled with Fabfluor, a dye that fluoresces in response to lowering pH. The dye serves as a localization indicator within the acidified lysosomal compartment, where the antibody is degraded after internalization. Cells were imaged over time, and antibody-Fabfluor internalization was quantified by fluorescence microscopy. Vertical axis: ratio of red area per well, normalized to t0. Horizontal axis: time, expressed in hours. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present inventors aimed to devise an agent that binds to SLC1A4, particularly an agent that binds to cells that express SLC1A4, to target SLC1A4-expressing tumor cells and kill these tumor cells. More precisely, the present inventors aimed to devise a novel anti-SLC1A4 monoclonal antibody that can be used to detect or kill SLC1A4-expressing tumor cells.

[0027] In some embodiments, these SLC1A4 monoclonal antibodies are intended to block serine transport by SLC1A4 and / or exert a cytotoxic anti-tumor effect via activation of the antibody-dependent cellular cytotoxicity natural process (ADCC), and / or, after conjugation with cytotoxic drugs (to form antibody drug conjugates, or ADCs), these SLC1A4 monoclonal antibodies can be used as shuttles to enable specific entry of cytotoxic drugs into cancer cells, resulting in their death. The inventors' goal was to devise in vitro and / or in vivo (e.g., by immuno-imaging techniques) diagnostic and therapeutic tools useful in the prevention and / or treatment of SLC1A4-expressing cancers, which are serine-dependent cancers. As used herein, treating a SLC1A4-expressing cancer includes preventing the development of a SLC1A4-expressing cancer, which includes preventing the development of a SLC1A4-expressing cancer in patients who have already been treated with other anti-cancer agents, which includes patients whose SLC1A4-expressing cancer has become resistant to one or more of the other anti-cancer agents.

[0028] To this end, the inventors have designed a family of monoclonal antibodies directed against the external loops of the SLC1A4 protein, more particularly against the external loops of the human SLC1A4 protein. These monoclonal antibodies are also referred to herein as "anti-xlSLC1A4" monoclonal antibodies ("xl" is used to mean "external loop").

[0029] The antibodies disclosed herein are comprised of monoclonal antibodies, including human monoclonal antibodies, and are therefore directed against a determinable target polypeptide sequence contained in the human SLC1A4 polypeptide sequence, in contrast to polyclonal antibodies, which include antibodies directed against multiple different antigenic peptides contained in the target protein. Furthermore, because the antibodies disclosed herein are comprised of structurally defined monoclonal antibodies, including human monoclonal antibodies, they are readily reproducible by multiple well-known techniques, in contrast to polyclonal antibodies.

[0030] The monoclonal antibodies disclosed herein are readily available for binding to SLC1A4 external loops exposed on the cell membrane. Binding of the monoclonal antibodies disclosed herein to the cell membrane of tumor cells expressing SLC1A4 (i) optionally induces tumor cell killing through ADCC (antibody-dependent cell cytotoxicity), (ii) optionally induces tumor cell killing through CDC (complement-dependent cell cytotoxicity), and (iii) optionally, when associated with a cytotoxic moiety (typically in the form of an ADC), induces tumor cell killing by internalization of the monoclonal antibody associated with the cytotoxic moiety.

[0031] As shown in the examples herein, the anti-SLC1A4 monoclonal antibodies of the present disclosure can be internalized in tumor cells expressing the target SLC1A4, and therefore can be used in an ADC format.

[0032] Because the monoclonal antibodies disclosed herein bind to the extracellular loops of the SLC1A4 protein, these monoclonal antibodies may block serine transport by SLC1A4, thereby contributing to the killing of tumor cells expressing SLC1A4.

[0033] To the inventors' knowledge, this is the first time that a monoclonal antibody directed against an external loop of human SLC1A4 has been disclosed.

[0034] Monoclonal antibodies directed against the external loops of human SLC1A4 may also be referred to herein as "anti-xlSLC1A4."

[0035] The present inventors have demonstrated that the anti-xlSLC1A4 monoclonal antibodies disclosed herein can specifically bind to cancer cells expressing SLC1A4. Consequently, the anti-xlSLC1A4 monoclonal antibodies disclosed herein can be advantageously used to detect cancer cells expressing SLC1A4 for diagnostic purposes.

[0036] The present inventors have also demonstrated that the anti-xlSLC1A4 monoclonal antibody disclosed herein has anti-proliferative activity against human cancer cells expressing SLC1A4.

[0037] Importantly, the inventors also demonstrated that the in vitro anti-proliferative effects are transcribed in vitro by the anti-tumor properties of the anti-xlSLC1A4 monoclonal antibodies disclosed herein.

[0038] Consequently, the anti-xlSLC1A4 monoclonal antibodies of the present disclosure are useful as novel therapeutic agents, either alone or in combination with one or more other anti-cancer therapies, for preventing the progression of cancer and / or treating cancer, particularly for preventing and / or treating SLC1A4-expressing cancers, including serine-dependent cancers.

[0039] definition

[0040] The term "SLC1A4" has its common meaning in the art and refers to solute carrier family 1 member 4, a neutral amino acid transporter of serine, having UniProtKB accession number P43007. The term "SLC1A4" may be used interchangeably with the term "ASCT1" (Alanine / Serine / Cysteine / Threonine Transporter 1).

[0041] As used herein, "xlSLC1A4" refers to the extracellular loop of the SLC1A4 protein.

[0042] As used herein, the outer loop of the human SLC1A4 protein (also referred to herein as human "xlSLC1A4") consists of the amino acid sequence of SEQ ID NO:51.

[0043] According to the present disclosure, "antibody" and "immunoglobulin" have the same meaning and are used interchangeably. 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 specifically binds to an antigen. Thus, the term "antibody" encompasses not only whole antibody molecules but also antibody variants (including derivatives). In a native antibody, 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. The light and heavy chains of an immunoglobulin each have three CDRs, designated VL-CDR1, VL-CDR2, VL-CDR3, and VH-CDR1, VH-CDR2, VH-CDR3, respectively, and therefore contain at least three CDRs contained within either the heavy or light chain. An antigen-binding site may contain six CDRs, including a set of CDRs from each of the heavy and light chain V regions. The framework region (FR) refers to an amino acid sequence located between CDRs.

[0044] As used herein, the term "antibody" or "immunoglobulin" is used in the broadest sense and includes fully assembled antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments capable of binding to human SLC1A4 external loops, and recombinant peptides as described above, so long as the "antibody" or "immunoglobulin" exhibits the desired biological activity as defined herein.

[0045] As used herein, the terms "monoclonal antibody" or "monoclonal antibody composition" refer to a preparation of antibody molecules of single molecular composition that display a single binding specificity and affinity for a particular epitope of a target protein, including an external loop of human SLC1A4.

[0046] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from the human immunoglobulin sequence repertoire. The human antibodies of the present disclosure may include amino acid residues not directly encoded by the human immunoglobulin sequence repertoire (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0047] 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., a human SLC1A4 extracellular loop). Antigen-binding fragments include Fv, Fab, F(ab)'2, single-domain antibodies, dsFv, ScFv, Sc(Fv)2, and diabodies. Typically, an antigen-binding fragment includes at least (i) a set of VH-CDR1, VH-CDR2, and VH-CDR3, or (ii) a set of VL-CDR1, VL-CDR2, and VL-CDR3, or both sets from the parent antibody.

[0048] The terms "treatment" or "therapy" refer to the administration of an active agent with the intent to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect a condition (e.g., a disease) or a symptom of a condition, or to prevent or delay the onset of a symptom, complication, or biochemical sign of a condition, or to arrest or inhibit the further development of a disease, condition, or disorder in a statistically significant manner.

[0049] The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of the drug may reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow to some extent, and preferably stop) cancer cell invasion into peripheral organs; inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with the disorder. To the extent the drug may prevent growth and / or kill existing cancer cells, the drug may be cytostatic and / or cytotoxic. In the case of cancer treatment, in vivo efficacy can be measured, for example, by assessing survival time, progression-free survival (PFS), response rates (RR), duration of response, and / or quality of life. In the context of this specification, this term encompasses an amount of anti-xlSLC1A4 monoclonal antibody that has an anti-tumor effect when administered to a subject suffering from a cancer that expresses SLC1A4.

[0050] In accordance with the present disclosure, the terms "subject," "patient," "individual," "subject in need thereof," "patient in need thereof," or "individual in need thereof" are equivalent and refer to a human or non-human mammal suffering from or likely to suffer from a cancer associated with expression of human SLC1A4, also referred to herein as an SLC1A4-expressing cancer. The individual is preferably a human.

[0051] As used herein, a "SLC1A4-expressing" cancer is a cancer in which the tumor cells contained therein or derived therefrom express the SLC1A4 protein on their surface. SLC1A4-expressing cells can be identified by binding of the anti-xlSLC1A4 monoclonal antibodies disclosed herein.

[0052] As used herein, a "serine-dependent" cancer is a cancer whose viability and proliferation depend on the availability of serine in its environment. A serine-dependent cancer is a cancer comprising cancer cells whose growth or proliferation is reduced or inhibited in the absence of serine.

[0053] For each amino acid sequence of interest, and particularly for each antibody amino acid sequence of interest, a reference sequence is set forth herein. The present specification also encompasses amino acid sequences that have a specified percentage of amino acid identity with the reference amino acid sequence.

[0054] As used herein, the "percentage of identity" between two amino acid sequences or two nucleic acid sequences is determined by comparing optimally aligned sequences over a comparison window.

[0055] Thus, the portion of the amino acid or nucleic acid sequence within the comparison window may contain additions or deletions (e.g., "gaps") compared to the reference sequence (which does not contain these additions or these deletions) in order to obtain optimal alignment between the two sequences.

[0056] The terms "sequence identity" and "identity" are used interchangeably herein. For purposes of this disclosure, determining the percentage of sequence identity between two amino acid sequences or two nucleic acid sequences is defined as aligning the sequences for optimal comparison purposes. Gaps may be introduced into either of the two sequences being compared to optimize the alignment between the two sequences. Such alignments can be performed over the entire length of the sequences being compared. Alternatively, the alignments can be performed over a shorter length, for example, about 20, about 50, about 100, or more amino acids or nucleotides. Sequence identity is the percentage of identical matches between the two sequences over the reported aligned region. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using a mathematical algorithm. Those skilled in the art will be aware of the fact that several different computer programs are available for aligning two sequences and determining the identity between two sequences (Kruskal, JB (1983) An overview of sequence comparison In D. Sankoff and JB Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley).

[0057] The percent sequence identity between two amino acid sequences or two nucleic acid sequences is most preferably determined using the Needleman and Wunsch algorithm for aligning two sequences (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned using this algorithm. The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE. The NEEDLE program from the EMBOSS package can be used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden J. and Bleasby, A. Trends in Genetics 16, (6) pp. 276-277, http: / / emboss.bioinformatics.nl / ). For protein sequences, EBLOSUM62 can be used for the substitution matrix. For nucleotide sequences, EDNAFULL can be used. Optional parameters are preferably a gap opening penalty of 10 and a gap extension penalty of 0.5. No end gap penalty is added. In the Output section, "Yes" is displayed in response to the question "Brief identity and similarity", and "SRS pairwise" is displayed as the Output alignment format. As described above, after alignment by the program NEEDLE, the percentage of sequence identity between the query sequence and the sequences of the disclosure is calculated as follows: the number of corresponding positions in the alignment that show identical amino acids or identical nucleotides in both sequences, divided by the total length of the alignment after subtracting the total number of gaps in the alignment.Identity as defined herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled as "longest-identity" in the program's output.

[0058] Anti-xlSLC1A4 monoclonal antibody

[0059] As previously mentioned herein, the present disclosure provides a family of monoclonal antibodies ("anti-xlSLC1A4" monoclonal antibodies) directed against the external loops of the SLC1A4 protein, more particularly against the external loops of the human SLC1A4 protein.

[0060] The present disclosure provides a monoclonal antibody directed against an extracellular loop of the human SLC1A4 protein, comprising: a) a monoclonal antibody or antigen-binding fragment thereof, (i) a heavy chain, the variable domain of which is VH-CDR1 having the sequence set forth as SEQ ID NO: 1; VH-CDR2 having the sequence set forth as SEQ ID NO: 2; and VH-CDR9 having the sequence set forth as SEQ ID NO: 3 Including, and / or (ii) a light chain, the variable domain of which comprises: VL-CDR1 having the sequence set forth as SEQ ID NO: 5; VL-CDR2 having the sequence set forth as SEQ ID NO: 6; and VL-CDR3 having the sequence set forth as SEQ ID NO: 7 Including, the monoclonal antibody or antigen-binding fragment thereof, b) a monoclonal antibody or an antigen-binding fragment thereof, (i) a heavy chain, the variable domain of which is VH-CDR1 having the sequence set forth as SEQ ID NO: 9; VH-CDR2 having the sequence set forth as SEQ ID NO: 10; and VH-CDR9 having the sequence set forth as SEQ ID NO: 11 Including, and / or (ii) a light chain, the variable domain of which comprises: VL-CDR1 having the sequence set forth as SEQ ID NO: 13; VL-CDR2 having the sequence set forth as SEQ ID NO: 14; and VL-CDR3 having the sequence set forth as SEQ ID NO: 15 Including, the monoclonal antibody or antigen-binding fragment thereof, c) a monoclonal antibody or an antigen-binding fragment thereof, (i) a heavy chain, the variable domain of which is VH-CDR1 having the sequence set forth as SEQ ID NO: 17; VH-CDR2 having the sequence set forth as SEQ ID NO: 18; and VH-CDR9 having the sequence set forth as SEQ ID NO: 19 Including, and / or (ii) a light chain, the variable domain of which comprises: VL-CDR1 having the sequence set forth as SEQ ID NO: 21; VL-CDR2 having the sequence set forth as SEQ ID NO: 22; and VL-CDR3 having the sequence set forth as SEQ ID NO: 23 Including, the monoclonal antibody or antigen-binding fragment thereof, d) a monoclonal antibody or an antigen-binding fragment thereof, (i) a heavy chain, the variable domain of which is VH-CDR1 having the sequence set forth as SEQ ID NO: 25; VH-CDR2 having the sequence set forth as SEQ ID NO: 26; and VH-CDR9 having the sequence set forth as SEQ ID NO: 27 Including, and / or (ii) a light chain, the variable domain of which comprises: VL-CDR1 having the sequence set forth as SEQ ID NO: 29; VL-CDR2 having the sequence set forth as SEQ ID NO: 30; and VL-CDR3 having the sequence set forth as SEQ ID NO: 31 Including, the monoclonal antibody or antigen-binding fragment thereof, e) a monoclonal antibody or antigen-binding fragment thereof, (i) a heavy chain, the variable domain of which is VH-CDR1 having the sequence set forth as SEQ ID NO: 33; VH-CDR2 having the sequence set forth as SEQ ID NO: 34; and VH-CDR9 having the sequence set forth as SEQ ID NO: 35 Including, and / or (ii) a light chain, the variable domain of which comprises: VL-CDR1 having the sequence set forth as SEQ ID NO: 37; VL-CDR2 having the sequence set forth as SEQ ID NO: 38; and VL-CDR3 having the sequence set forth as SEQ ID NO: 39 Including, the monoclonal antibody or antigen-binding fragment thereof, f) a monoclonal antibody or an antigen-binding fragment thereof, (i) a heavy chain, the variable domain of which is VH-CDR1 having the sequence set forth as SEQ ID NO: 41; VH-CDR2 having the sequence set forth as SEQ ID NO: 42; and VH-CDR9 having the sequence set forth as SEQ ID NO: 43 Including, and / or (ii) a light chain, the variable domain of which comprises: VL-CDR1 having the sequence set forth as SEQ ID NO: 45; VL-CDR2 having the sequence set forth as SEQ ID NO: 46; and VL-CDR3 having the sequence set forth as SEQ ID NO: 47 Including, The monoclonal antibody or antigen-binding fragment thereof comprising The present invention relates to the above monoclonal antibody selected from the group consisting of:

[0061] In some embodiments, monoclonal antibodies directed against the extracellular loops of the human SLC1A4 protein according to the present disclosure comprise either specific CDRs of the heavy chain or specific CDRs of the light chain.

[0062] In some other embodiments, monoclonal antibodies directed against the extracellular loops of the human SLC1A4 protein according to the present disclosure comprise both a particular CDR of the heavy chain and a particular CDR of the light chain.

[0063] The family of monoclonal antibodies described herein includes: (i) a heavy chain variable domain having at least 85% amino acid identity with the heavy chain variable domain of SEQ ID NO: 4 and comprising a VH-CDR1 of SEQ ID NO: 1, a VH-CDR2 of SEQ ID NO: 2, and a VH-CDR3 of SEQ ID NO: 3; and / or (ii) a light chain variable domain having at least 85% amino acid identity with the light chain variable domain of SEQ ID NO: 8 and comprising a VL-CDR1 of SEQ ID NO: 5, a VL-CDR2 of SEQ ID NO: 6, and a VL-CDR3 of SEQ ID NO: 7. The present invention encompasses anti-xlSLC1A4 monoclonal antibodies, including:

[0064] The family of monoclonal antibodies described herein includes: (i) a heavy chain variable domain having at least 85% amino acid identity with the heavy chain variable domain of SEQ ID NO: 12 and comprising a VH-CDR1 of SEQ ID NO: 9, a VH-CDR2 of SEQ ID NO: 10, and a VH-CDR3 of SEQ ID NO: 11; and / or (ii) a light chain variable domain having at least 85% amino acid identity with the light chain variable domain of SEQ ID NO: 16 and comprising a VL-CDR1 of SEQ ID NO: 13, a VL-CDR2 of SEQ ID NO: 14, and a VL-CDR3 of SEQ ID NO: 15. The present invention encompasses anti-xlSLC1A4 monoclonal antibodies, including:

[0065] The family of monoclonal antibodies described herein includes: (i) a heavy chain variable domain having at least 85% amino acid identity with the heavy chain variable domain of SEQ ID NO: 20 and comprising a VH-CDR1 of SEQ ID NO: 17, a VH-CDR2 of SEQ ID NO: 18, and a VH-CDR3 of SEQ ID NO: 19; and / or (ii) a light chain variable domain having at least 85% amino acid identity with the light chain variable domain of SEQ ID NO: 24 and comprising a VL-CDR1 of SEQ ID NO: 21, a VL-CDR2 of SEQ ID NO: 22, and a VL-CDR3 of SEQ ID NO: 23. The present invention encompasses anti-xlSLC1A4 monoclonal antibodies, including:

[0066] The family of monoclonal antibodies described herein includes: (i) a heavy chain variable domain having at least 85% amino acid identity with the heavy chain variable domain of SEQ ID NO: 28 and comprising a VH-CDR1 of SEQ ID NO: 25, a VH-CDR2 of SEQ ID NO: 26, and a VH-CDR3 of SEQ ID NO: 27; and / or (ii) a light chain variable domain having at least 85% amino acid identity with the light chain variable domain of SEQ ID NO: 32 and comprising a VL-CDR1 of SEQ ID NO: 29, a VL-CDR2 of SEQ ID NO: 30, and a VL-CDR3 of SEQ ID NO: 31. The present invention encompasses anti-xlSLC1A4 monoclonal antibodies, including:

[0067] The family of monoclonal antibodies described herein includes: (i) a heavy chain variable domain having at least 85% amino acid identity with the heavy chain variable domain of SEQ ID NO: 36 and comprising a VH-CDR1 of SEQ ID NO: 33, a VH-CDR2 of SEQ ID NO: 34, and a VH-CDR3 of SEQ ID NO: 35; and / or (ii) a light chain variable domain having at least 85% amino acid identity with the light chain variable domain of SEQ ID NO: 40 and comprising a VL-CDR1 of SEQ ID NO: 37, a VL-CDR2 of SEQ ID NO: 38, and a VL-CDR3 of SEQ ID NO: 39. The present invention encompasses anti-xlSLC1A4 monoclonal antibodies, including:

[0068] The family of monoclonal antibodies described herein includes: (i) a heavy chain variable domain having at least 85% amino acid identity with the heavy chain variable domain of SEQ ID NO: 44 and comprising a VH-CDR1 of SEQ ID NO: 41, a VH-CDR2 of SEQ ID NO: 42, and a VH-CDR3 of SEQ ID NO: 43; and / or (ii) a light chain variable domain having at least 85% amino acid identity with the light chain variable domain of SEQ ID NO: 48 and comprising a VL-CDR1 of SEQ ID NO: 45, a VL-CDR2 of SEQ ID NO: 46, and a VL-CDR3 of SEQ ID NO: 47. The present invention encompasses anti-xlSLC1A4 monoclonal antibodies, including:

[0069] As used herein, an amino acid sequence having at least 85% amino acid identity with a given heavy chain variable domain means an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity with the given heavy chain variable domain. It also encompasses an amino acid sequence consisting of the given heavy chain variable domain, i.e., an amino acid sequence having 100% amino acid identity with the given heavy chain variable domain.

[0070] As used herein, an amino acid sequence having at least 85% amino acid identity with a given light chain variable domain has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity with the given light chain variable domain. It also encompasses an amino acid sequence consisting of the given light chain variable domain, i.e., an amino acid sequence having 100% amino acid identity with the given light chain variable domain.

[0071] The present disclosure further provides a monoclonal antibody directed against an extracellular loop of human SLC1A4 protein, comprising: a) the monoclonal antibody sometimes referred to herein as "B5", (i) a heavy chain variable domain having a heavy chain variable domain of SEQ ID NO: 4; and / or (ii) a light chain variable domain having the light chain variable domain of SEQ ID NO: 8 The monoclonal antibody comprising b) the monoclonal antibody sometimes referred to herein as "D6", (i) a heavy chain variable domain having a heavy chain variable domain of SEQ ID NO: 12; and / or (ii) a light chain variable domain having the light chain variable domain of SEQ ID NO: 16 The monoclonal antibody comprising c) the monoclonal antibody sometimes referred to herein as "A10", (i) a heavy chain variable domain having a heavy chain variable domain of SEQ ID NO: 20; and / or (ii) a light chain variable domain having the light chain variable domain of SEQ ID NO: 24 The monoclonal antibody comprising d) the monoclonal antibody sometimes referred to herein as "E10", (i) a heavy chain variable domain having a heavy chain variable domain of SEQ ID NO: 28; and / or (ii) a light chain variable domain having the light chain variable domain of SEQ ID NO: 32 The monoclonal antibody comprising e) the monoclonal antibody sometimes referred to herein as "G11", (i) a heavy chain variable domain having a heavy chain variable domain of SEQ ID NO: 36; and / or (ii) a light chain variable domain having the light chain variable domain of SEQ ID NO: 40 The monoclonal antibody comprising f) the monoclonal antibody sometimes referred to herein as "B8", (i) a heavy chain variable domain having a heavy chain variable domain of SEQ ID NO: 44; and / or (ii) a light chain variable domain having the light chain variable domain of SEQ ID NO: 48 The monoclonal antibody comprising The present invention relates to the above monoclonal antibody, which is selected from the group consisting of:

[0072] In some embodiments, the directed monoclonal antibody directed against an extracellular loop of the human SLC1A4 protein selected from B5, D6, A10, E10, G11 and B8 described above comprises either a designated heavy chain or a designated light chain.

[0073] In some other embodiments, the directed monoclonal antibody directed against an extracellular loop of the human SLC1A4 protein selected from B5, D6, A10, E10, G11 and B8 described above comprises both the designated heavy chain and the designated light chain.

[0074] In some embodiments, the anti-xlSLC1A4 monoclonal antibody of the present disclosure can be a chimeric monoclonal antibody, i.e., an antibody having a human heavy chain variable domain, a human light chain variable domain, and a non-human Fc domain. The non-human Fc domain can be, for example, a mouse Fc domain, a guinea pig Fc domain, or a rabbit Fc domain. For example, the rabbit Fc domain can consist of the Fc domain of SEQ ID NO: 49 disclosed herein, which Fc domain is exemplified in the Examples herein.

[0075] In some embodiments, the anti-xlSLC1A4 monoclonal antibody of the present disclosure can be a human monoclonal antibody, i.e., an antibody having a human heavy chain variable domain, a human light chain variable domain, and a human Fc domain. The human Fc domain can be a human IgG Fc domain, such as a human IgG1 Fc domain. For example, the human Fc domain can consist of the human IgG1 Fc domain of SEQ ID NO: 50 disclosed herein, which is exemplified in the Examples herein.

[0076] The present disclosure also relates to antigen-binding fragments of the monoclonal antibodies directed against the extracellular loops of the human SLC1A4 protein described herein.

[0077] Most preferably, the antigen-binding fragment of a monoclonal antibody directed against an extracellular loop of the human SLC1A4 protein described herein comprises at least (i) a set of VH-CDR1, VH-CDR2, and VH-CDR3, or (ii) a set of VL-CDR1, VL-CDR2, and VL-CDR3, or both sets, from the parent antibody to which the binding fragment refers. Exemplary antigen-binding fragments derived from any of the B5, D6, A10, E10, G11, and B8 monoclonal antibodies are encompassed herein.

[0078] In some embodiments, the antigen-binding fragment of a monoclonal antibody directed against an extracellular loop of the human SLC1A4 protein comprises the variable domain of the heavy chain of the antibody or the variable domain of the light chain of the antibody.

[0079] In some other embodiments, the antigen-binding fragment of a monoclonal antibody directed against an extracellular loop of the human SLC1A4 protein comprises the variable domain of the heavy chain of the antibody and the variable domain of the light chain of the antibody.

[0080] Antigen-binding fragments of the monoclonal antibodies described herein can in particular be selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2 and diabodies. They can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies according to methods well known to those skilled in the art.

[0081] "Single-chain Fv" (or "scFv") antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see "The Pharmacology of Monoclonal Antibodies," Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0082] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH and 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 and form two antigen-binding sites. Diabodies are described more fully in, for example, European Patent No. EP 404,097; International Publication No. WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0083] Diabodies or bispecific antibodies can be broadly divided into two types: immunoglobulin G (IgG)-like molecules and non-IgG-like molecules. IgG-like bsAbs retain Fc-mediated effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP) (Spiess et al., 2015, Mol Immunol., Vol. 67(2):95-106). The Fc region of Abs facilitates purification and improves solubility and stability. Bispecific antibodies in IgG-like formats usually have longer serum half-lives due to their larger size and FcRn-mediated recycling (Kontermann et al., 2015, Bispecific antibodies. Drug Discovery Today Vol. 20(7):838-47). Non-IgG-like bsAbs have good size and offer enhanced tissue penetration (Kontermann et al., 2015, Bispecific antibodies. Drug Discovery Today Vol. 20(7):838-47).

[0084] Methods for producing antibodies of the present disclosure

[0085] The anti-xlSLC1A4 monoclonal antibodies of the present disclosure are selected as novel antibody entities exhibiting innovative functional properties, and after they have been cloned and sequenced, they can be produced by any technique known in the art, including, but not limited to, chemical, biological, genetic or enzymatic techniques, either alone or in combination.

[0086] Knowing the amino acid sequence of a desired polypeptide, e.g., the amino acid sequence of the heavy or light chain of a monoclonal antibody, one of skill in the art can readily produce any of the monoclonal antibodies or antigen-binding fragments thereof disclosed herein by standard techniques for producing polypeptides. For example, they can be synthesized using the well-known solid phase method, preferably using a commercially available peptide synthesis instrument (e.g., one manufactured by Applied Biosystems, Foster City, California) and following the manufacturer's instructions. Alternatively, antibodies of the present disclosure can be synthesized by recombinant DNA techniques well known in the art. For example, after incorporating a DNA sequence encoding the antibody into an expression vector and introducing such a vector into an appropriate eukaryotic or prokaryotic host that will express the desired antibody, the antibody can be obtained as a DNA expression product, from which it can then be isolated using well-known techniques.

[0087] Nucleic acid sequence

[0088] Thus, a further object of the present disclosure relates to nucleic acid sequences encoding the antibodies according to the present disclosure. Based on the known amino acid sequence, one skilled in the art can easily design a suitable polypeptide-encoding nucleic acid sequence, taking into account, for example, the preferred codon usage used by the organism in which the antibody synthesis is intended (e.g., bacteria, yeast, animal cells, human cells).

[0089] Typically, the nucleic acid is a DNA or RNA molecule, which may be comprised in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector, as are well known in the art.

[0090] vector

[0091] 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 so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence.

[0092] Another object of the present disclosure relates to a vector comprising the nucleic acid of the present disclosure.

[0093] Any expression vector for animal cells can be used as long as it can insert and express a gene encoding a human antibody C region. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), and pSG1βd2-4- (Miyaji H et al. 1990).

[0094] host cell

[0095] A further object of the present disclosure relates to host cells transfected, infected or transformed with the nucleic acids and / or vectors disclosed herein.

[0096] The term "transformation" means the introduction of a "foreign" (i.e., extrinsic or extracellular) gene, DNA, or RNA sequence into a host cell, such that the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA has been "transformed."

[0097] The nucleic acids of the present disclosure can be used to produce the anti-SLC1A4 monoclonal antibodies of the present disclosure in a suitable expression system. The term "expression system" refers to a host cell and a compatible vector under suitable conditions for expression of a protein encoded by, for example, foreign DNA carried by the vector and introduced into the host cell.

[0098] 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.). Hosts according to the present disclosure can be specifically prokaryotic or eukaryotic cells. Specific examples include E. coli, Kluyveromyces or Saccharomyces yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neural cells, adipocytes, etc.). For example, mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") (Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells"), and the like are also included.

[0099] Immunoconjugate

[0100] In the present disclosure, the term "immunoconjugate" is used to define an anti-xlSLC1A4 associated with another moiety, wherein the other moiety is linked to the anti-xlSLC1A4 monoclonal antibody either covalently or non-covalently.

[0101] The immunoconjugates of the present disclosure include anti-xlSLC1A4 monoclonal antibodies linked, most preferably covalently bound, to a detectable molecule, which may also be referred to herein as a "detectable label." Such immunoconjugates can be used in vitro or in vivo to detect the presence of tumor cells expressing SLC1A4.

[0102] The immunoconjugates of the present disclosure also include anti-xlSLC1A4 monoclonal antibodies linked, most preferably covalently bound, to a cytotoxic moiety such that the immunoconjugates can exert a cytotoxic effect upon internalization in tumor cells expressing SLC1A4.

[0103] Detectable label

[0104] The antibodies of the present disclosure can be conjugated with a detectable label to form an anti-xlSLC1A4 immunoconjugate, which can be used for in vitro diagnosis or in vivo immunoimaging of tumors in cancer patients. Suitable detectable labels include, for example, a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label, or colloidal gold. Methods for making and detecting such detectably labeled immunoconjugates are well known to those of skill in the art and are described in more detail below.

[0105] In some embodiments, the detectable label is selected to be suitable for determining the presence of cells expressing SLC1A4 in vitro. For example, the detectable label can be selected from radioactive compounds, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds, and enzyme compounds.

[0106] In some other embodiments, the detectable label is selected to be suitable for determining the presence of cells expressing SLC1A4 in vivo, e.g., the detectable label can be selected from radioactive compounds, including positron emitter compounds.

[0107] The detectable label can be a radioisotope that is detected by autoradiography. Particularly useful isotopes for the purposes of the present disclosure are: 3 H, 125 I, 131 I, 35 S and 14 It is C.

[0108] The anti-xlSLC1A4 immunoconjugates of the present disclosure can 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, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine, among others.

[0109] Alternatively, the anti-xlSLC1A4 immunoconjugate of the present disclosure can 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 esters, imidazole, acridinium salts, and oxalate esters.

[0110] Similarly, a bioluminescent compound can be used to label the anti-xlSLC1A4 immunoconjugate of the present disclosure. 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.

[0111] Alternatively, anti-xlSLC1A4 immunoconjugates can be detectably labeled by linking the anti-human-xlSLC1A4 monoclonal antibody to an enzyme. When the anti-xlSLC1A4 enzyme conjugate is incubated in the presence of a suitable substrate, the enzyme reacts with the substrate to produce a chemical moiety that can be detected, for example, by spectrophotometry, fluorometry, or visual means. Examples of enzymes that can be used to detectably label polyspecific immunoconjugates include β-galactosidase, glucose oxidase, peroxidase, and alkaline phosphatase.

[0112] Those skilled in the art will know of other suitable labels that can be used in accordance with the present disclosure. Binding of marker moieties to anti-human-xlSLC1A4 monoclonal antibodies can be achieved using standard techniques known in the art. Exemplary methodologies in this regard are described by 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.

[0113] Furthermore, the use of anti-human-xlSLC1A4 monoclonal antibodies conjugated with avidin, streptavidin, and biotin can enhance the convenience and versatility of in vitro immunochemical detection (see, for example, 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) pp. 149-162 (Manson, ed., The Humana Press, Inc. 1992)).

[0114] 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).

[0115] The use of immunoconjugates according to the present disclosure in in vivo immunoimaging methods includes diagnostic imaging using radioimmunoscintigraphy or positron-emission tomography (PET) techniques. Immunopositron emission tomography (PET) is a diagnostic imaging tool that utilizes monoclonal antibodies labeled with positron-emitting substances, combining the targeting properties of antibodies with the sensitivity of positron emission tomography cameras (see, e.g., *The Oncologist*, 12:1379 (2007); *Journal of Nuclear Medicine*, 52(8).1171 (2011)). ImmunoPET allows for the visualization and quantification of antigen and antibody accumulation in vivo, serving as an important tool for complementary diagnostics and therapeutics. For example, immunoPET is useful in selecting potential patient candidates for specific therapies and in monitoring treatment. In the context of the present disclosure, the in vivo use of immunoconjugates of anti-xlSLC1A4 monoclonal antibodies linked to a detectable label, e.g., linked to a positron-emitting agent, makes it possible to determine whether a subject is suffering from a cancer that expresses SLC1A4, and thus also makes it possible to localize SLC1A4-expressing tumor cells within the subject's body, and therefore also makes it possible to monitor the status of the cancer within the subject's body, for example, responsiveness to a selected anti-cancer treatment (which includes responsiveness to anti-cancer treatment using the anti-xlSLC1A4 monoclonal antibodies and their cytotoxic immunoconjugates disclosed herein).

[0116] Antibody-drug conjugates (ADCs)

[0117] In another aspect, the present disclosure provides an anti-xlSLC1A4 monoclonal antibody-drug conjugate. As used herein, "anti-xlSLC1A4 monoclonal antibody-drug conjugate" refers to an anti-xlSLC1A4 monoclonal antibody according to the present disclosure conjugated to a therapeutic agent.

[0118] The present disclosure also relates to immunoconjugates comprising the anti-xlSLC1A4 monoclonal antibodies or antigen-binding fragments thereof described herein, linked to a therapeutic agent.

[0119] Such anti-xlSLC1A4 monoclonal antibody-drug conjugates (ADCs) are expected to provide clinically beneficial effects in the prevention and / or treatment of SLC1A4-expressing cancers when the ADCs are administered to subjects suffering from SLC1A4-expressing cancers, typically when administered alone but also when administered in combination with one or more other therapeutic agents.

[0120] In typical embodiments, an anti-xlSLC1A4 monoclonal antibody of the present disclosure is conjugated to a cytotoxic agent such that the resulting antibody-drug conjugate (ADC) exerts a cytotoxic or cytostatic effect when taken up or internalized by a cell expressing SLC1A4 (e.g., a cancer cell expressing SLC1A4). Particularly suitable moieties for conjugation to an antibody are chemotherapeutic agents, prodrug-converting enzymes, radioisotopes or compounds, or toxins. For example, an anti-xlSLC1A4 monoclonal antibody can be conjugated to a cytotoxic agent, e.g., a chemotherapeutic agent or toxin (e.g., a cytostatic or cytocidal agent, abrin, ricin A, pseudomonas exotoxin, diphtheria toxin).

[0121] Useful classes of cytotoxic agents include, for example, antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes, e.g., cisplatin, mono-, di-, and tri-platinum complexes, carboplatin), anthracyclines, antibiotics, antifluoride agents, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinol, preforming compounds, purine antimetabolites, puromycin, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, and the like.

[0122] Individual cytotoxic agents include, for example, androgens, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoximine, camptothecin, carboplatin, carmustine (BSNU), CC-1065 (Li et al, Cancer Res.42:999-1004, 1982), chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (formerly actinomycin), daunorubicin, decarbazine, docetaxel, doxorubicin, estrogen, 5-fluorodeoxyuridine, etoposide phosphate (VP-16), 5-fluorouracil, gramicidin D D), hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C C), mitoxantrone, nitroimidazole (VM-26), paclitaxel (VM-26), plicamycin, procarbizine, streptozotocin, tenoposide (VM-26), 6-thioguanine, thioTEPA, topotecan (VM-26), vinblastine (VM-26), vincristine (VM-26), and vinorelbine (VM-26).

[0123] Particularly preferred cytotoxic agents to be grafted onto antibodies to form ADCs include, for example, dolastatins (e.g., auristatin E, AFP, MMAF, MMAE), DNA minor groove binders (e.g., ribonucleotides ... binders (e.g., enediynes and lexitropsins), duocarmycins, taxanes (e.g., paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38 (7-ethyl-10-hydroxy-camptothecin), topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin, epothilone A and B B), estramustine, cryptophysins, cemadotin, maytansinoids, discodermolide, eleutherobin, benzodiazepine derivatives, mitoxantrone.

[0124] In some embodiments, the cytotoxic agent is a conventional chemotherapeutic agent, such as doxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate, mitomycin C, or etoposide. Additionally, potent drugs such as CC-1065 analogs, calicheamicin, maytansine, analogs of dolastatin 10, rhizoxin, palytoxin, etc., can be linked to anti-xlSLC1A4-expressing antibodies.

[0125] In another variation, the cytotoxic agent is a DNA minor groove binding agent (see, e.g., U.S. Pat. No. 6,130,237). For example, in some embodiments, the minor groove binding agent is a CBI compound. In other embodiments, the minor groove binding agent is an enediyne (e.g., calicheamicin).

[0126] In other embodiments, the anti-xlSLC1A4 monoclonal antibody of the present disclosure is conjugated to a prodrug-converting enzyme. The prodrug-converting enzyme can 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] Techniques for conjugating therapeutic agents to proteins, particularly antibodies, are well known (e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds., Alan R. Liss, Inc., 1985); Hellstrom et al., "Antibodies For Drug Delivery," Controlled Drug Delivery (Robinson et al. eds., Marcel Deiker, Inc., 2nd ed. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al. eds., 1985); "Analysis, Results, and Future Prospect of the Therapeutic Use of Radiolabeled Antibodies In Cancer Therapy," 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).

[0128] Linkers in antibody-drug conjugates

[0129] 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 non-cleavable, and the drug is released, for example, by degradation of the antibody.

[0130] In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., in a lysosome, endosome, or caveolae). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal protease or an endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleavage agents can 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 target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123).

[0131] The most typical one is a peptidyl linker that can be cleaved by enzymes present in cells that express 191P4D12. 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 certain embodiments, the peptidyl linker that can be cleaved 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 with a Val-Cit linker). One advantage of utilizing intracellular proteolytic release of therapeutic drugs is that the drug is typically weakened when conjugated, and the serum stability of the conjugate is typically high.

[0132] In other embodiments, the cleavable linker is pH-sensitive, ie, sensitive to hydrolysis at certain pH values.

[0133] Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, an acid-labile linker (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, etc.) that is hydrolyzable in lysosomes can be used (see, for example, U.S. Pat. Nos. 5,122,366, 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 those found in blood, but are unstable below pH 5.5 or 5.0, the approximate pH of lysosomes. In some embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to a therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929)).

[0134] In yet other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). Various disulfide linkers are known in the art, including, for example, disulfide linkers 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-pyridyldithio)toluene), SPDB, and SMPT (see, e.g., Thorpe et al., 1987, Cancer Re. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel ed., Oxford U.S. Press, 1987. See also U.S. Pat. No. 4,880,935.

[0135] In yet other embodiments, the linker unit is non-cleavable and the drug is released by antibody degradation.

[0136] Typically, the linker is substantially insensitive to the extracellular environment. As used herein, "substantially insensitive to the extracellular environment" in the context of a 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 linkers in a sample of the antibody-drug conjugate compound are not cleaved. Whether a linker is substantially insensitive to the extracellular environment can be determined, for example, by incubating the antibody-drug conjugate compound with plasma 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.

[0137] Engineered anti-xlSLC1A4 monoclonal antibody

[0138] With respect to effector function, it may be desirable to modify the anti-xlSLC1A4 antibodies identified herein so as to enhance, for example, the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively, or additionally, one or more cysteine ​​residues may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and / or enhanced complement-mediated cell killing and enhanced antibody-dependent cellular cytotoxicity (ADCC) (see Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, BJ Immunol. 148:2918-2922 (1992)). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers, as described in Wolff et al. Cancer Research 53:2560-2565 (1993). Alternatively, antibodies can be engineered with dual Fc regions, thereby potentially having enhanced complement lysis and ADCC capabilities (see Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989)). International Publication No. WO 00 / 42072 (Presta, L.) describes antibodies with improved ADCC function in the presence of human effector cells, which contain amino acid substitutions in their Fc regions. Preferably, antibodies with improved ADCC contain substitutions at positions 298, 333, and / or 334 (Eu numbering of residues) in the Fc region. Preferably, the altered Fc region is a human IgGl Fc region comprising or consisting of substitutions at one, two or three of these positions, optionally in combination with one or more substitutions that increase CIq binding and / or CDC.

[0139] Antibodies with altered CIq binding and / or altered complement dependent cytotoxicity (CDC) are described in International Publication No. WO 99 / 51642, U.S. Pat. No. 6,194,551, U.S. Pat. No. 6,242,195, U.S. Pat. No. 6,528,624, and U.S. Pat. No. 6,538,124 (Idusogie et al.), which contain amino acid substitutions at one or more of amino acid positions 270, 322, 326, 327, 329, 313, 333, and / or 334 of their Fc region (Eu numbering of residues).

[0140] In some embodiments, the anti-xlSLC1A4 monoclonal antibodies of the present disclosure include glyco-engineered anti-xlSLC1A4 monoclonal antibodies.

[0141] As used herein, the term "glycoengineering" refers to any art-recognized method for altering the glycoform profile of a binding protein composition. Such methods include expressing the binding protein composition in genetically engineered host cells (e.g., CHO cells) that have been genetically engineered to express a heterologous glycosyltransferase or glycosidase. In other embodiments, glycoengineering methods involve culturing host cells under conditions that bias toward a particular glycoform profile.

[0142] As used herein, a "glyco-engineered antibody" includes (i) an antibody comprising a hypergalactosylated Fc fragment, (ii) an antibody comprising a hypomannosylated Fc fragment (which includes an afucosylated Fc fragment), and (iii) an antibody comprising a hypofucosylated Fc fragment (which includes an afucosylated Fc fragment). As used herein, a glyco-engineered fragment includes an Fc fragment with altered glycosylation selected from the group consisting of one or more of the following modified glycosylation: (i) hypergalactosylation, (ii) hypomannosylation, and (iii) hypofucosylation. Consequently, glycoengineered Fc fragments from anti-xlSLC1A4 monoclonal antibodies used in accordance with the present disclosure include examples of highly galactosylated, hypomannosylated, and hypofucosylated Fc fragments.

[0143] Those skilled in the art may refer to well-known techniques for obtaining anti-xlSLC1A4 monoclonal antibodies containing highly galactosylated Fc fragments, hypomannosylated Fc fragments, and hypofucosylated Fc fragments, which are known to bind to Fc receptors with higher affinity than non-modified Fc fragments.

[0144] Glycoengineered Anti-xlSLC1A4 Monoclonal Antibody The anti-xlSLC1A4 monoclonal antibody comprises a hypofucosylated Fc fragment, which may also be referred to as a "low-fucose" Fc fragment.

[0145] Antibody assays

[0146] As shown in the Examples herein, the present inventors have found that SLC1A4 is expressed in the cell membranes of various cancer tissues, including melanoma and liposarcoma.

[0147] As explained in more detail below, the growth of tumor cells expressing SLC1A4 is promoted, at least in part, through the supply of serine, which is internalized into cells by the SLC1A4 neutral amino acid transporter. As shown in the Examples herein, targeting SLC1A4 with the anti-xlSLC1A4 monoclonal antibodies of the present disclosure produces anti-tumor effects.

[0148] This means that cancers that express SLC1A4 are candidates for anti-cancer therapies that target SLC1A4.

[0149] As shown in the Examples herein, monoclonal antibodies directed against the extracellular loops of the human SLC1A4 protein of the present disclosure can be used to detect SLC1A4 on the surface of various SLC1A4 human cancer cells, and thus can distinguish between cancer cells that express SLC1A4 and cancer cells that do not express SLC1A4 on their surface. Any of the anti-xlSLC1A4 monoclonal antibodies of the present disclosure can be used to detect the presence of SLC1A4 on the cell surface of cancer cells to determine whether a subject has a cancer that expresses SLC1A4, or alternatively, to stage a cancer that expresses SLC1A4.

[0150] The present disclosure also relates to the in vitro use of a monoclonal antibody or antigen-binding fragment thereof according to the present disclosure for detecting SLC1A4 in a tumor sample, preferably in a cell sample.

[0151] The present disclosure provides an in vitro method for detecting the presence of SLC1A4 or cells expressing SLC1A4 in a sample, comprising: a) contacting the sample to be tested with an anti-xlSLC1A4 monoclonal antibody of the present disclosure; and b) detecting binding of said monoclonal antibody to SLC1A4 The present invention relates to the above method, which comprises the steps of:

[0152] The present disclosure further relates to an in vitro method for determining whether an individual suffering from a SLC1A4-expressing cancer is suffering from a SLC1A4-expressing cancer, comprising determining whether a tumor tissue sample previously obtained from the individual expresses SLC1A4 protein on the cell surface.

[0153] The present disclosure further relates to an in vitro method for determining whether an individual suffering from a serine-dependent cancer is suffering from a serine-dependent cancer, comprising determining whether a tumor tissue sample previously obtained from the individual expresses SLC1A4 protein on the cell surface.

[0154] The present disclosure further provides a method for determining whether an individual is afflicted with a SLC1A4-expressing cancer, comprising: a) administering an anti-xlSLC1A4 monoclonal antibody of the present disclosure labeled with a detectable molecule; and b) detecting the labeling of cancer cells with the labeled anti-xlSLC1A4 monoclonal antibody, wherein detection of the labeling of cancer cells indicates a cancer that expresses SLC1A4; The present invention relates to the above method, which comprises the steps of:

[0155] The methods described above can be performed via any in vivo immunoimaging technique known in the art.

[0156] 1. A method for imaging cancer tissue expressing SLC1A4 in a subject, comprising: a) administering to the subject a labeled anti-xlSLC1A4 monoclonal antibody according to the present disclosure; and b) Visualizing the expression of SLC1A4, for example by radioimmunoscintigraphy or by positron emission tomography (PET) Also provided herein is the above method, which comprises the steps of:

[0157] The present disclosure further provides a method for identifying a subject suitable for anti-tumor therapy using the anti-xlSLC1A4 monoclonal antibody disclosed herein, comprising: a) administering to the subject a labeled anti-xlSLC1A4 monoclonal antibody according to the present disclosure; and b) Visualizing the expression of SLC1A4, for example by radioimmunoscintigraphy or by positron emission tomography (PET) The process includes the steps of: wherein the presence of the labeled anti-xlSLC1A4 monoclonal antibody at the tumor site identifies the subject as suitable for anti-tumor therapy using the anti-xlSLC1A4 monoclonal antibody. Regarding the above method.

[0158] The present disclosure also provides a method for monitoring the effectiveness of an anti-tumor therapy in a subject, comprising: a) selecting a subject having a tumor that expresses SLC1A4, wherein the subject is being treated with an anti-tumor therapy; b) administering a labeled anti-xlSLC1A4 monoclonal antibody according to the present disclosure; c) imaging the localization of the administered labeled anti-xlSLC1A4 monoclonal antibody; and d) Determining tumor growth The process includes the steps of: wherein a decrease from baseline in uptake of the labeling signal indicates tumor regression and efficacy of the anti-tumor therapy. Regarding the above method.

[0159] In some embodiments, the anti-xlSLC1A4 monoclonal antibody is labeled with a positron emitter, and the visualizing or imaging step is performed by positron emission tomography (PET).

[0160] In embodiments in which the anti-xlSLC1A4 monoclonal antibody is labeled with a positron emitter, the antibody is conjugated to one or more moieties having the following structure (I): -L-Mz, wherein (i) L is a chelating moiety, M is a positron emitter, and z, independently at each occurrence, is 0 or 1, and wherein at least one of the z's is 1.

[0161] In another embodiment, the anti-xlSLC1A4 monoclonal antibody is labeled with a radioactive molecule, and the visualizing or imaging step is performed by, for example, radioimmunoscintigraphy.

[0162] Preferably, the suitable labelled isotope is: 110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 90 Y, 89 Zr, 94m Tc, 94 Tc, 99m Tc, 120 I, 123 I, 124 I, 125 I, 131 I, 32 P, 11 C. 13 N, 186 Re, 188 Re, 51 Mn, 52m Mn, 55 Co, 72 As, 75 Br,76 Br, 82m Rb, 83 It can be selected from among Sr, or other gamma-ray emitters, beta-ray emitters or positron emitters.

[0163] As shown in the examples herein, any SLC1A4-expressing cancer, such as melanoma or liposarcoma, can be treated with the anti-xlSLC1A4 monoclonal antibodies of the present disclosure, provided that tumor cells from the cancer tumor express SLC1A4 on their membranes, and therefore, the presence of SLC1A4 protein on the tumor cell membrane can be detected or determined according to any method.

[0164] Thus, the experimental data provided in the Examples herein demonstrate that the same anti-xlSLC1A4 monoclonal antibody is effective for treating multiple different types of cancer, provided that the SLC1A4 target protein is expressed on the tumor cell membrane.

[0165] In the field of anticancer active ingredients consisting of target-binding molecules, e.g., target-binding antibodies, it is not unprecedented for the same active ingredient to be effective in treating several different cancers. For example, the anti-PD1 antibody named pembrolizumab has been approved by the US Food and Drug Administration (FDA) as an active ingredient useful in the treatment of various types of cancer, provided that the cancers share the same physiological characteristics.

[0166] Thus, an individual suffering from a cancer that expresses SLC1A4 may be treated for the cancer with the anti-xlSLC1A4 monoclonal antibodies described herein if SLC1A4 membrane expression by tumor cells previously taken from the individual is detected or determined by a suitable method.

[0167] The level of membranous SLC1A4 expression can be based on in vitro immunohistochemical assessment of SLC1A4 expression by the cancer cells examined. In vivo immunoimaging of cancer patients using the appropriately labeled anti-xlSLC1A4 monoclonal antibody described herein can also be used to determine SLC1A4 expression by tumors.

[0168] therapeutic use

[0169] The present inventors have shown that the anti-xlSLC1A4 monoclonal antibodies of the present disclosure can be advantageously used to treat cancers that express SLC1A4.

[0170] The present inventors have demonstrated herein that the anti-xlSLC1A4 monoclonal antibody of the present disclosure is useful as a novel therapeutic tool for treating SLC1A4-expressing cancers, including serine-dependent cancers.

[0171] Thus, the present disclosure relates to the anti-xlSLC1A4 monoclonal antibody herein, an antigen-binding fragment thereof, or an immunoconjugate thereof for use as a drug.

[0172] The present disclosure also relates to the use of an anti-xlSLC1A4 monoclonal antibody, an antigen-binding fragment thereof, or an immunoconjugate thereof of the present disclosure for preparing a medicament.

[0173] The present disclosure also relates to an anti-xlSLC1A4, an antigen-binding fragment thereof, or an immunoconjugate thereof of the present disclosure for use in treating a cancer that expresses SLC1A4 in a subject.

[0174] The present disclosure also relates to the use of an anti-xlSLC1A4 of the present disclosure for the preparation of a medicament for treating a cancer that expresses SLC1A4.

[0175] The present disclosure also relates to a method of treating a cancer that expresses SLC1A4, comprising administering to an individual in need thereof an anti-xlSLC1A4 monoclonal antibody of the present disclosure.

[0176] The present disclosure also relates to a method of treating an SLC1A4-expressing cancer, comprising administering to an individual in need thereof a therapeutically effective amount of an anti-xlSLC1A4 monoclonal antibody of the present disclosure.

[0177] The present disclosure further provides a method of treating cancer in a subject, comprising: a) determining whether tumor cells of a subject express SLC1A4 in vitro or in vivo; and b) if SLC1A4 tumor cell expression is determined in step a), administering to the subject an anti-xlSLC1A4 monoclonal antibody or immunoconjugate thereof according to the present disclosure. The present invention relates to the above method, which comprises the steps of:

[0178] In some embodiments, step a) is performed in vitro, e.g., by providing a tumor sample previously obtained from the subject, e.g., a sample derived from a biopsy tumor sample.

[0179] In some other embodiments, step a) is performed in vitro, for example, by immunoimaging techniques, such as those described elsewhere in this disclosure.

[0180] The anti-xlSLC1A4 monoclonal antibodies, antigen-binding fragments or immunoconjugates thereof of the present disclosure may be used alone or in combination with any other suitable agent.

[0181] Although the present inventors do not wish to be bound by any particular theory, they believe that a single type of cancer, such as lung cancer, may have multiple different phenotypes depending on the subject affected. For example, the present inventors believe that some lung cancer subjects may have lung cancer that expresses SLC1A4, while other lung cancer subjects may have lung cancer that does not express SLC1A4. In fact, only subjects affected by lung cancer that expresses SLC1A4 are eligible for therapeutic treatment with the anti-xlSLC1A4 monoclonal antibody of the present disclosure. Alternatively, the present inventors believe that the anti-xlSLC1A4 monoclonal antibody of the present disclosure can be used to treat a wide variety of different cancers, provided that the cancer expresses SLC1A4.

[0182] Examples of cancers that express SLC1A4 include, but are not limited to, melanoma and liposarcoma.

[0183] In some embodiments, the SLC1A4-expressing cancer may be selected from the group consisting of bone cancer, brain cancer, ovarian cancer, breast cancer, lung cancer, colorectal cancer, osteosarcoma, skin cancer, malignant melanoma, melanoma, prostate cancer, and liposarcoma.

[0184] The term "liposarcoma" or "LPS" has its common meaning in the art and refers to soft tissue sarcoma of mesenchymal origin as revised in the World Health Organization classification ICD10C49.9. The term "liposarcoma" also refers to well-differentiated liposarcoma (WD-LPS) and dedifferentiated liposarcoma (DD-LPS). The term "liposarcoma" also refers to malignant mesenchymal neoplasms, a type of soft tissue sarcoma, a group of lipomatous tumors ranging in severity from slow-growing to aggressive and metastatic. Liposarcoma most often arises in the lower extremities or retroperitoneum, but can also occur in the upper extremities, neck, abdominal cavity, spermatic cord, breast, vulva, and axilla. The term "liposarcoma" also refers to dedifferentiated and well-differentiated liposarcoma.

[0185] In one embodiment, the antibodies, fragments thereof, and immunoconjugates of the present disclosure are particularly suitable for, but not limited to, the treatment of the following cancers, which have been determined to consist of cancers that express SLC1A4: Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, Childhood Cancers, AIDS-Related Cancers, Kaposi Sarcoma, AIDS-Related Lymphoma, Primary CNS Lymphoma, Anal Cancer, Astrocytomas, Atypical Teratoid / Rhabdoid Tumors Tumor, Basal Cell Carcinoma, Skin Cancer (Nonmelanoma), Bile Duct Cancer, Bladder Cancer, Bone Cancer, Ewing Sarcoma Family of Tumors, Osteosarcoma, and Malignant Fibrous Histiocytoma, Brain Stem Glioma, Atypical Teratoid / Rhabdoid Tumor, Embryonal Tumors, Germ Cell Tumors, Craniopharyngioma, Ependymoma, Breast Cancer, Bronchial Tumor Tumors, Burkitt Lymphoma, Non-Hodgkin Lymphoma, Carcinoid TumorTumor), Gastrointestinal Carcinoma, Cardiac (Heart) Tumors, Primary Lymphoma, Cervical Cancer, Cholangiocarcinoma, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myeloid Leukemia (CML) Myelogenous Leukemia, Chronic Myeloproliferative Neoplasms, Colon Cancer, Colorectal Cancer, Craniopharyngioma, Cutaneous T-Cell Lymphoma, Mycosis Fungoides and Sezary Syndrome, Ductal Carcinoma in Situ (DCIS) Carcinoma In Situ, Embryonal Tumors, Endometrial Cancer, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Eye Cancer, Intraocular Melanoma, Retinoblastoma, Fallopian Tube Cancer, Fibrous Histiocytoma of Bone, Malignant Tumors and Osteosarcoma, Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor Gastrointestinal Stromal Tumor (GIST)Tumors, Germ Cell Tumor, Ovarian Disease, Testicular Disease, Gestational Trophoblastic Disease, Glioma, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular (Liver) Cancer, Histiocytosis, Langerhans Cell, Hodgkin Lymphoma, Hypopharyngeal Cancer, Islet Cell Tumors, Pancreatic Neuroendocrine Tumors, Kaposi Sarcoma, Kidney Cell Histiocytosis, Renal Cell Histiocytosis Histiocytosis), Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia, Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Chronic Lymphocytic Leukemia (CLL). Lymphocytic, Chronic Myelogenous (CML), Hairy Cell, Lip Cancer, Oral Cavity Cancer, Liver Cancer (Primary), Lung Cancer, Non-Small Cell Lymphoma, Small Cell Lymphoma), Hodgkin macroglobulinemia (Hodgkin Macroglobulinemia, Non-Hodgkin's MacroglobulinemiaMacroglobulinemia, Waldenstrom's Disease, Male Breast Cancer, Melanoma, Merkel Cell Carcinoma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Midline Tract Carcinoma Involving NUT Gene, Mouth Cancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma / Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplasia Syndromes, Myelodysplastic / Myeloproliferative Neoplasms Neoplasms, Myelogenous Leukemia, Chronic Myeloid Leukemia (CML), Myeloid Leukemia, Acute Myeloma Leukemia (AML), Myeloma, Multiple Myeloproliferative Neoplasms, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer (whole mouth), Oral Cavity Cancer, Lip and Oropharyngeal Cancer Cancer, osteosarcoma and malignant fibrous histiocytoma of boneand Malignant Fibrous Histiocytoma of Bone, Ovarian Cancer, Low Malignant Potential Tumor, Pancreatic Cancer, Pancreatic Neuroendocrine Tumors (Islet Cell Tumors), Papillomatosis, Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm / Multiple Myeloma, Pleuropulmonary Blastoma Blastoma, Pregnancy and Breast Cancer, Primary Central Nervous System (CNS) Lymphoma, Primary Peritoneal Cancer, Prostate Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer, Renal Pelvis and Ureter, Transitional Cell Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Rhabdomyosarcoma, Uterine Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell CarcinomaCarcinoma, Squamous Neck Cancer with Occult Primary, Metastatic Cancer, Stomach (Gastric) Cancer, T-Cell Lymphoma, Testicular Cancer, Throat Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Unknown Primary Cancer, Transitional Cell Cancer of the Ureter and Renal Pelvis, Urethral Cancer, Uterine Cancer, Endometrioma, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer Cancer, Waldenstrom Macroglobulinemia, and Wilms Tumor.

[0186] In some embodiments, the anti-xlSLC1A4 monoclonal antibody, antigen-binding fragment thereof, or immunoconjugate of the present disclosure, particularly the antibody-drug conjugate according to the present disclosure, is used in combination with one or more other active agents to prevent or treat a subject suffering from a cancer that expresses SLC1A4, which is a serine-dependent cancer.

[0187] When used to treat cancers that express SLC1A4, the anti-xlSLC1A4 monoclonal antibodies, antigen-binding fragments thereof, or immunoconjugates, particularly ADCs, of the present disclosure can be used in combination with conventional cancer therapies, such as surgery, radiation therapy, chemotherapy, or a combination thereof.

[0188] In one aspect, other therapeutic agents useful in combination cancer therapy with the anti-xlSLC1A4 monoclonal antibody, antigen-binding fragment thereof, or antibody-drug conjugate according to the present disclosure can include one or more other active agents aimed at depriving tumor cells that express SLC1A4 of serine.

[0189] Active agents that cause serine deprivation from tumor cells expressing SLC1A4 include inhibitors of the interleukin-6 (IL-6) signaling pathway, which can be selected from an IL-6 inhibitor, an IL-6 receptor inhibitor, an IL-6 / IL-6 receptor complex inhibitor, a gp130 inhibitor, and a STAT3 inhibitor.

[0190] The term "interleukin-6 (IL-6) signaling inhibitor" refers to a compound or agent that selectively blocks or inactivates the IL-6 signaling pathway. As used herein, the term "selectively blocks or inactivates" refers to a compound that preferentially binds to and blocks or inactivates IL-6, the IL-6 receptor, the IL-6 / IL-6R complex, STAT3, and / or gp130. In particular, the term "selectively blocks or inactivates" refers to a compound that blocks the interaction of IL-6 with its IL-6 receptor, the interaction of IL-6 with gp130, the interaction of IL-6 receptor with gp130, the interaction of the IL-6 / IL-6 receptor complex with gp130, or the interaction of STAT3 phosphorylation. Typically, IL-6 signaling inhibitors may include, but are not limited to, polypeptides, aptamers, antibodies or portions thereof, antisense oligonucleotides, i.e., siRNA or shRNA, or ribozymes.

[0191] The term "IL-6 / IL-6R complex" or "IL-6 / IL-6 receptor complex" collectively refers to the complex formed by the soluble form of IL-6 and the membrane-bound form of IL-6 receptor α. In some embodiments, "IL-6 / IL-6R complex" refers to the complex formed by (i) the soluble form of IL-6 secreted by a subject's cancer cells and (ii) the membrane-bound form of IL-6 receptor α on myoblasts of the same subject.

[0192] In some embodiments, the IL-6 signaling inhibitor that may be used in combination with the anti-xlSLC1A4 monoclonal antibody of the present disclosure may be an antibody itself or an antigen-binding portion thereof directed against one of IL-6, IL-6 receptor, STAT3, and / or gp130.

[0193] In some other embodiments, the IL-6 signaling inhibitor that can be used in combination with the anti-xlSLC1A4 monoclonal antibody of the present disclosure can be an IL-6 expression inhibitor, an IL-6 receptor expression inhibitor, a STAT 3 expression inhibitor, and / or a gp130 expression inhibitor.

[0194] In some embodiments, an expression-inhibiting agent for use in a method according to the present disclosure can be an antisense oligonucleotide.

[0195] In some embodiments, the IL-6 signaling inhibitor of the present disclosure can be a chemical compound.

[0196] In some embodiments, the chemical compounds of the present disclosure include compounds that inhibit IL-6 expression, IL-6R expression, STAT expression, STAT3 phosphorylation, and / or gp130 expression.

[0197] In some embodiments, chemical compounds that can be used in combination with the anti-xlSLC1A4 monoclonal antibodies of the present disclosure can be blocking anti-gp130 compounds, compounds that inhibit the phosphorylation of STAT3, or compounds that inhibit the expression of STAT3.

[0198] Some of the blocking anti-gp130 compounds have been shown, for example, by Wu et al., Mol Cancer Ther. 2016 Nov;15(11):2609-2619.

[0199] In some embodiments, the gp-130 inhibitor of the present disclosure can be bazedoxifene.

[0200] In some embodiments, the STAT3 inhibitor is SH5-07, APTSTAT3-9R, C188-9, BP-1-102, Niclosamide (BAY2353), STAT3-IN-1, WP1066, Cryptotanshinone, Stattic, Resveratrol (SRT501), S31-201, HO-3867, Napabucasin (BBI608), Brevilin A, Artesunate (WR-256283), Bosutinib (SKI-606), TPCA-1, SC-43, Ginkgolic Acid acid C17:1, Ochromycinone (STA-21), Colivelin, Cucurbitacin IIb, GYY4137, Scutellarin, Kaempferol-3-O-rutinoside, Cucurbitacin I, SH-4-54, Nifuroxazide, and Pimozide.

[0201] In some embodiments, the STAT3 inhibitor can be C188-9, Stattic, or a combination thereof.

[0202] As used herein, "bazedoxifene" has its general meaning in the art and consists of a gp130 inhibitor having the formula (1): 1-[[4-[2-(hexahydro-1H-azepin-1-yl)ethoxy]phenyl]methyl]-2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol monoacetate (salt) has the molecular formula C 30 H 34 N2O3-C2H4O2 and has CAS number 198481-33-3.

[0203] As used herein, "C188-9" is a potent inhibitor of STAT3 that binds to STAT3 with high affinity. C188-9 can be defined by reference to CAS number 432001-19-9.

[0204] As used herein, a "Stattic" is a small molecule compound that inhibits STAT3 activation. Stattic can be defined by reference to CAS number 19983-44-9.

[0205] Pharmaceutical Composition

[0206] For administration, the anti-xlSLC1A4 monoclonal antibody, antigen-binding fragment thereof, or antibody-drug conjugate of the present disclosure is preferably formulated as a pharmaceutical composition.

[0207] Pharmaceutical compositions comprising the anti-xlSLC1A4 monoclonal antibody, antigen-binding fragment thereof, or antibody-drug conjugate of the present disclosure can be formulated according to known methods for preparing pharmaceutically useful compositions, whereby the therapeutic molecule is combined in admixture with a pharmaceutically acceptable carrier.

[0208] A composition is said to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well known to those skilled in the art (see, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995)). The formulation may further include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface, etc.

[0209] Administration route

[0210] The form of the pharmaceutical composition, the route of administration, the dosage and the regimen will naturally depend on the condition to be treated, the severity of the condition, the age, weight and sex of the patient, etc.

[0211] The pharmaceutical compositions of the present disclosure can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration, and the like.

[0212] Preferably, the pharmaceutical composition comprises one or more pharmaceutically acceptable vehicles / carriers for an injectable formulation, which may in particular be isotonic, sterile, saline (such as mono- or di-sodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or mixtures of these salts), or a dried, in particular lyophilized, composition, which, in some cases, makes it possible to constitute an injectable solution upon addition of sterile water or saline.

[0213] The dose used for administration can be adapted as a function of various parameters, in particular as a function of the mode of administration used, the pathology involved, or alternatively as a function of the desired duration of treatment.

[0214] To prepare a pharmaceutical composition, an effective amount of the antibody may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0215] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the formulation must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0216] Solutions of the active compounds as free bases or pharmaceutically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0217] Pharmaceutical Carrier Substances

[0218] The anti-xlSLC1A4 monoclonal antibodies of the present disclosure can be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and are formed with inorganic acids such as hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like.

[0219] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of injectable compositions can be brought about by the use in the composition of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0220] Sterile injectable solution is prepared by incorporating the required amount of active compound into a suitable solvent, and optionally with various other ingredients as listed above, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains basic dispersion medium and other ingredients as listed above.For the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, which obtains powder of active ingredient and any desired additional ingredient from the solution thereof that has been previously sterilized and filtered.

[0221] The preparation of more or highly concentrated solutions for direct injection is also contemplated in the present invention, where the use of DMSO as a solvent is envisioned to result in very rapid penetration, delivering high concentrations of active agent to small tumor areas.

[0222] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, although drug release capsules and the like can also be used.

[0223] For example, for parenteral administration in an aqueous solution, the solution should be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous vehicles that can be used will be known to those of skill in the art in light of the present disclosure. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous solution or injected at the proposed injection site (see, e.g., "Remington's Pharmaceutical Sciences," 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dose for each individual.

[0224] The anti-xlSLC1A4 monoclonal antibodies of the present disclosure can be formulated into a therapeutic mixture compatible with administration of the monoclonal antibody in a dosage range of about 0.1 mg to about 10 mg per kg of body weight, advantageously in a dosage range of about 0.5 mg to about 5 mg per kg of body weight, for example, a dosage of about 1 mg per kg of body weight.

[0225] Thus, the anti-xlSLC1A4 monoclonal antibodies of the present disclosure may be formulated into a therapeutic mixture that can include from about 8 milligrams to about 800 milligrams, advantageously from about 4 milligrams to about 400 milligrams, e.g., about 80 milligrams per dose.

[0226] In addition to compounds formulated for parenteral administration, e.g., intravenous or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solid forms for oral administration; sustained-release capsules; and other forms currently in use.

[0227] In some 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.

[0228] Nanocapsules generally can entrap compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymer overloading, such ultrafine particles (sized around 0.1 μm) are generally designed using polymers that can be degraded in vivo. Biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles can be easily fabricated.

[0229] Liposomes are formed from phospholipids dispersed in an aqueous medium and spontaneously form 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 between 200 and 500 Å, which contain aqueous solution in their cores. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0230] This specification will be further illustrated by the accompanying figures and the following examples, however, these examples and figures should not be construed in any way as limiting the scope of the present disclosure.

[0231] Example

[0232] a. Materials and Methods

[0233] A.1. Cell culture:

[0234] Nine cell lines, each expressing different levels of the SLCA14 transporter, were used: (i) human DD-liposarcoma IB111 and IB115, (ii) human melanoma SKMEL 5, A375, and A431, (iii) human breast cancer SKBR3, (iv) human ovarian cancer SKOV3, (v) human leukemia U937, and (vi) human pancreatic cancer HPAC.

[0235] The cell lines were cultured in (i) DMEM (for IB111, IB115, A375, and SKMEL-5) or (ii) DMEM-F12 medium with 10% decomplemented fetal calf serum and 1% PenStrep (streptomycin and penicillin) (for SKBR3, A431, SKOV3, SKBR3, and HPAC). Additionally, IB115 was transduced with shSLC1A4 (a short hairpin nucleic acid directed against SLC1A4 mRNA) to verify antibody specificity. The shRNA is the nucleic acid sequence of SEQ ID NO:54, targeting the nucleic acid sequence of SEQ ID NO:53.

[0236] To determine the therapeutic effect of antibodies on cells, 2,000 IB111 or IB115 cells and 500 SKMEL or HPAC cells were seeded in a 96-well plate (day 0). On days 1 and 4, increasing concentrations of antibody (0.00015 μg / mL to 150 μg / mL) were added to physiological medium (EMEM, 1% dialyzed serum, containing the seven nonessential amino acids: proline, alanine, asparagine, aspartic acid, glutamic acid, glycine, and serine). Confluency was monitored and analyzed using Incucyte Systems for Live-Cell Imaging and Analysis. After 7 days, sulforhodamine coloration was performed, and DO was measured at 560 nm.

[0237] A.2. Western Blot Assay

[0238] The Western blot assay was performed as disclosed below. 2 μg of antibody is loaded per well (non-reduced: 2 μg + Laemli, reduced: 2 μg + Laemli + Bmercapto-ethanol, boiled at 95° C. for 10 minutes), Gel migration is performed in precast gels Gebagel 4-12% at 160 V for 1 h (Reference: 15G-0412-10 GeneBio Application).

[0239] After transfer, the gel is stained with Gelcode blue safe Protein stain (reference number 1860957 Thermo Scientific) and washed with water.

[0240] A.3. Quantitative RT-PCR:

[0241] RNA samples were aliquoted onto a 384-well plate and analyzed using a LightCycler 480. 商標 The protocol consisted of a 10-minute preincubation step at 95°C for polymerase activation, followed by 45 cycles of amplification. Quantitative analysis of gene expression was performed by the ΔΔCt method, and the β-actin gene was used as a reference for normalization.

[0242] A.4. PDX and transplanted mouse models of liposarcoma

[0243] Swiss nude mice were inoculated with human dedifferentiated 20LPS fragments (20-30 mm 2 ) was implanted subcutaneously.

[0244] In the transplantation model, the IB111 and IB115 liposarcoma cell lines were injected subcutaneously into the right flank of nude mice (10 × 10 6 pieces).

[0245] The tumor is 100 mm 3 When tumor size reached 100 mg / kg, mice were injected intravenously with vehicle or 2-20 mg / kg antibody every other week. Tumors were measured every other week using calipers.

[0246] A.5. Fluorescence Activated Cell Sorting (FACS)

[0247] 300,000 cells were placed in a 96R well plate and fixed with 4% paraformaldehyde for 15 minutes at room temperature. The cells were then incubated with primary antibodies diluted in PBS-BSA 2% for 1 hour at room temperature. Secondary antibodies were used at 1 / 100 and incubated on the cells for 1 hour. Positive cells were detected by CytoFLEX. 登録商標 Flow cytometry (Beckman) was used to measure the variability of the variability, and the results were analyzed using FlowJoint. 登録商標 analyzed by software.

[0248] A.6. Tissue microarray (TMA)

[0249] A total of 1,405 formalin-fixed, paraffin-embedded cancer specimens were arranged in multiple TMAs, which were obtained from the Institut du Cancer de Montpellier (ICM). Cell surface SLC1A4 expression was detected using an IHC assay with the following criteria: (i) cell color intensity: no staining (negative [-]), light brown (weakly positive [+]), brown (moderately positive [++]), and chocolate brown (strongly positive [+++]); (ii) number of positive cells: less than 10% (-), 10–25% (+), 26–49% (++), and 50% or more (+++). Finally, an H score was calculated according to the two results above using the following formula: 3 × (strongly stained percentage) + 2 × (moderately stained percentage) + weakly stained percentage, giving a range of 0–300 (Figure 11).

[0250] A.7. Internalization Assays

[0251] For the constant exposure Incucyte internalization assay, liposarcoma cell lines IB111 and IB115 and human melanoma cell line SKMEL5 were seeded into Corning well flat-bottom plates and allowed to adhere overnight at 37°C. IgG or anti-SLC1A4 antibodies were conjugated to Incucyte fabfluor dye according to the manufacturer's protocol (Sartorius). Media containing Fabluor-labeled antibodies (10 μg / mL) was added to the cells. The cells were transferred to an Incucyte Imager / Incubator system, and fluorescence was monitored over time. Fluorescence intensity was analyzed using Incucyte software, and the total fluorescence intensity of the well was divided by the confluent area determined by simultaneous phase contrast imaging. Data were exported and graphed using Prism.

[0252] Example 1: Binding of anti-SLC1A4 monoclonal antibodies to cells expressing SLC1A4

[0253] 1.A. SLC1A4 protein and mRNA expression in various human cancer cell lines

[0254] Several human cancer cell lines were tested for their ability to express SLC1A4 target proteins: (i) SK-MEL-5 melanoma cell line; (ii) SK-MEL-28 melanoma cell line; (iii) HPAC pancreatic cancer cell line; (iv) A2058 melanoma cell line; (v) MW2664 melanoma cell line; and (vi) IB115 sarcoma cell line.

[0255] The results are shown in Figure 1 (Figure 1A and Figure 1B). Figure 1A shows that all cancer cell lines tested express SLC1A4, except for the HPAC pancreatic cell line.

[0256] The results shown in Figure 1B and Figure 1C show that SLC1A4 mRNA expression was significantly higher in the SK-MEL-5 melanoma cell line than in the A375 melanoma cell line and the IB111 or IB115 liposarcoma cell lines, whereas no SLC1A4 mRNA expression was detected in the HPAC pancreatic cancer cell line.

[0257] 1.B. Generation of Monoclonal Antibodies Directed Against the External Loops of SLC1A4

[0258] The binding of the monoclonal antibodies of the present disclosure to the external loops of the SLC1A4 protein is depicted schematically in Figures 2A and 2A-2.

[0259] Western blotting confirmed the efficient generation of anti-SLC1A4 monoclonal antibodies. For each of the B5.1, E10.2, D6.2, G11.4, B8.3, and A10 antibodies, we confirmed the efficient generation of monoclonal antibodies containing either (i) an aggregated form of both heavy and light chains (non-reducing conditions) or (ii) a dissociated form of each heavy and light chain (reducing conditions, as shown in Figure 2B).

[0260] 1.C. Binding of anti-SLC1A4 monoclonal antibodies to human cancer cells

[0261] The binding of anti-SLC1A4 monoclonal antibodies (in which the human Fc fragment was replaced by a rabbit Fc domain), namely B5.1, E10.2, D6.2, G11.4, B8.3 and A10 monoclonal antibodies, to various cancer cell lines was assayed.

[0262] As shown in Figure 3, each of the tested antibodies bound to cancer cells expressing SLC1A4. As expected, none of the anti-SLC1A4 antibodies bound to HPAC pancreatic cells, which do not express SLC1A4.

[0263] Similar results were obtained with human anti-SLC1A4 monoclonal antibodies (mAbs) containing the same variable region and a human IgG1 Fc domain. The results in Figure 4 show that the fully human anti-SLC1A4 monoclonal antibodies E10, D6, B8, and G11 bound to the SLC1A4-expressing melanoma cell line SK-MEL-5 (Figures 4A, 4B, and 4F), whereas no detectable binding occurred to HPAC pancreatic cells, which do not express SLC1A4 (Figures 4C and 4D). Similarly, the results in Figures 4E to 4H show that the fully human anti-SLC1A4 monoclonal antibodies E10, D6, and B8 bound to the SLC1A4-expressing sarcoma cell lines IB111 and IB115 (Figure 4E), the SKBR3 breast cancer cell line (Figure 4F), the A375 and A431 melanoma cell lines (Figure 4G), the SKOV3 ovarian cancer cell line (Figure 4H), and the U937 leukemia cell line (Figure 4H).

[0264] Example 2: In vitro anti-proliferative effects of monoclonal anti-SLC1A4 antibodies

[0265] The in vitro antitumor effects of rabbit Fc-engineered human anti-SLC1A4 monoclonal antibodies B5, A10, B8, D6, E10, and G11 were assayed in the human liposarcoma IB115 cell line.

[0266] The results show that all anti-SLC1A4 monoclonal antibodies tested have significant anti-proliferative effects on human sarcoma cells.

[0267] The in vitro antitumor effects of fully human anti-SLC1A4 monoclonal antibodies B8, D6, G11, and E10 were assayed in the human liposarcoma IB115 cell line. The in vitro antitumor effects of fully human anti-SLC1A4 monoclonal antibodies B8, D6, and E10 were also assayed in the human liposarcoma IB111 cell line, the human melanoma cell line SKMEL5, and the human pancreatic cancer HPAC.

[0268] The results are illustrated in Figure 5, where the tumor cell killing capacity of human monoclonal antibodies B8, D6, E10 and G11 was assayed.

[0269] The results in Figures 5 and 6A-6D demonstrate that the anti-SLC1A4 human monoclonal antibodies of the present disclosure possess high tumor cell killing ability.

[0270] Example 3: In vivo antitumor properties of anti-SLC1A4 human monoclonal antibodies

[0271] The in vivo antitumor properties of a human anti-SLC1A4 monoclonal antibody were evaluated in (i) PDX mice xenografted with human dedifferentiated liposarcoma cells derived from patients with liposarcoma, and (ii) in 10x10 6 The cells were assayed in an allograft mouse model of liposarcoma in which they were injected subcutaneously into the right flank.

[0272] The results in Figures 7 and 9 show that all tested anti-SLC1A4 human monoclonal antibodies, i.e., B8, D6, and E10, can inhibit tumor growth in the sarcoma cell lines IB111 and IB115 and increase mouse survival (Figures 8 and 10).

[0273] The results show the inhibition of tumor growth over time for each of the antibodies tested.

[0274] Example 4: Examination of SLC1A4 expression in human cancers

[0275] Cell surface expression of SLC1A4 in human samples was assessed on different TMAs using the fully human anti-SLC1A4 monoclonal antibody D6.

[0276] The results in Figure 11 show that SLC1A4 is strongly expressed in melanoma, breast cancer, and liver cancer, while it is weakly expressed in endometrial cancer, for example. The same results were observed using transcriptome and proteome data from the cancer cell line encyclopaedia database.

[0277] The results confirm the clinical relevance of using anti-SLC1A4 antibodies in a variety of human cancers.

[0278] Example 5: Internalization dynamics of anti-SLC1A4 human monoclonal antibodies

[0279] The internalization of human anti-SLC1A4 monoclonal antibodies B8, D6, and E10 was assayed in human liposarcoma IB111 and IB115 cell lines and human melanoma SKMEL5 cell line by monitoring the localization and intensity of fluorescently labeled anti-SLC1A4 monoclonal antibodies B8, D6, and E10.

[0280] The results in Figure 12 show that the full time course of anti-SLC1A4 antibody internalization is faster in the human melanoma SKMEL5 cell line (Figure 12C) compared to the IB111 and IB115 liposarcoma cell lines (Figures 12A and 12B).

[0281] The results indicate that anti-SLC1A4 antibodies are likely internalized continuously and slowly. This data supports the future deployment of anti-SLC1A4 antibodies as antibody-drug conjugates.

[0282] [Table 1] JPEG2025540190000002.jpg187170JPEG2025540190000003.jpg174170JPEG2025540190 000004.jpg204170JPEG2025540190000005.jpg236170JPEG2025540190000006.jpg59170

[0283] In the event of a discrepancy between the sequences disclosed in the above-mentioned sequence listing and the attached sequence listing (e.g., according to WIPO Standard ST.26), the correct sequence is the sequence disclosed in the above-mentioned table included in this disclosure.

Claims

1. A monoclonal antibody directed against an extracellular loop of the human SLC1A4 protein, a) a monoclonal antibody or antigen-binding fragment thereof, (i) a heavy chain, the variable domain of which is VH-CDR1 having the sequence set forth as SEQ ID NO: 1; VH-CDR2 having the sequence set forth as SEQ ID NO: 2; and VH-CDR3 having the sequence set forth as SEQ ID NO: 3 Including, and / or (ii) a light chain, the variable domain of which comprises: VL-CDR1 having the sequence set forth as SEQ ID NO: 5; VL-CDR2 having the sequence set forth as SEQ ID NO: 6; and VL-CDR3 having the sequence set forth as SEQ ID NO: 7 Including, the monoclonal antibody or antigen-binding fragment thereof, b) a monoclonal antibody or an antigen-binding fragment thereof, (i) a heavy chain, the variable domain of which is VH-CDR1 having the sequence set forth as SEQ ID NO: 9; VH-CDR2 having the sequence set forth as SEQ ID NO: 10; and VH-CDR9 having the sequence set forth as SEQ ID NO: 11 Including, and / or (ii) a light chain, the variable domain of which comprises: VL-CDR1 having the sequence set forth as SEQ ID NO: 13; VL-CDR2 having the sequence set forth as SEQ ID NO: 14; and VL-CDR3 having the sequence set forth as SEQ ID NO: 15 the monoclonal antibody or antigen-binding fragment thereof, c) a monoclonal antibody or an antigen-binding fragment thereof, (i) a heavy chain, the variable domain of which is VH-CDR1 having the sequence set forth as SEQ ID NO: 17; VH-CDR2 having the sequence set forth as SEQ ID NO: 18; and VH-CDR9 having the sequence set forth as SEQ ID NO: 19 Including, and / or (ii) a light chain, the variable domain of which comprises: VL-CDR1 having the sequence set forth as SEQ ID NO: 21; VL-CDR2 having the sequence set forth as SEQ ID NO: 22; and VL-CDR3 having the sequence set forth as SEQ ID NO: 23 Including, the monoclonal antibody or antigen-binding fragment thereof, d) a monoclonal antibody or an antigen-binding fragment thereof, (i) a heavy chain, the variable domain of which is VH-CDR1 having the sequence set forth as SEQ ID NO: 25; VH-CDR2 having the sequence set forth as SEQ ID NO: 26; and VH-CDR9 having the sequence set forth as SEQ ID NO: 27; Including, and / or (ii) a light chain, the variable domain of which comprises: VL-CDR1 having the sequence set forth as SEQ ID NO: 29; VL-CDR2 having the sequence set forth as SEQ ID NO: 30; and VL-CDR3 having the sequence set forth as SEQ ID NO: 31 Including, the monoclonal antibody or antigen-binding fragment thereof, e) a monoclonal antibody or antigen-binding fragment thereof, (i) a heavy chain, the variable domain of which is VH-CDR1 having the sequence set forth as SEQ ID NO: 33; VH-CDR2 having the sequence set forth as SEQ ID NO: 34; and VH-CDR9 having the sequence set forth as SEQ ID NO: 35 Including, and / or (ii) a light chain, the variable domain of which comprises: VL-CDR1 having the sequence set forth as SEQ ID NO: 37; VL-CDR2 having the sequence set forth as SEQ ID NO: 38; and VL-CDR3 having the sequence set forth as SEQ ID NO: 39 Including, the monoclonal antibody or antigen-binding fragment thereof, f) a monoclonal antibody or an antigen-binding fragment thereof, (i) a heavy chain, the variable domain of which is VH-CDR1 having the sequence set forth as SEQ ID NO: 41; VH-CDR2 having the sequence set forth as SEQ ID NO: 42; and VH-CDR9 having the sequence set forth as SEQ ID NO: 43 Including, and / or (ii) a light chain, the variable domain of which comprises: VL-CDR1 having the sequence set forth as SEQ ID NO: 45; VL-CDR2 having the sequence set forth as SEQ ID NO: 46; and VL-CDR3 having the sequence set forth as SEQ ID NO: 47 Including, The monoclonal antibody or antigen-binding fragment thereof comprising The monoclonal antibody is selected from the group consisting of:

2. (i) a heavy chain variable domain having at least 85% amino acid identity with the heavy chain variable domain of SEQ ID NO: 4 and comprising a VH-CDR1 of SEQ ID NO: 1, a VH-CDR2 of SEQ ID NO: 2, and a VH-CDR3 of SEQ ID NO: 3; and / or (ii) a light chain variable domain having at least 85% amino acid identity with the light chain variable domain of SEQ ID NO: 8 and comprising a VL-CDR1 of SEQ ID NO: 5, a VL-CDR2 of SEQ ID NO: 6, and a VL-CDR3 of SEQ ID NO:

7. The monoclonal antibody of claim 1, comprising:

3. (i) a heavy chain variable domain having at least 85% amino acid identity with the heavy chain variable domain of SEQ ID NO: 12 and comprising a VH-CDR1 of SEQ ID NO: 9, a VH-CDR2 of SEQ ID NO: 10, and a VH-CDR3 of SEQ ID NO: 11; and / or (ii) a light chain variable domain having at least 85% amino acid identity with the light chain variable domain of SEQ ID NO: 16 and comprising a VL-CDR1 of SEQ ID NO: 13, a VL-CDR2 of SEQ ID NO: 14, and a VL-CDR3 of SEQ ID NO:

15. The monoclonal antibody of claim 1, comprising:

4. (i) a heavy chain variable domain having at least 85% amino acid identity with the heavy chain variable domain of SEQ ID NO: 20 and comprising a VH-CDR1 of SEQ ID NO: 17, a VH-CDR2 of SEQ ID NO: 18, and a VH-CDR3 of SEQ ID NO: 19; and / or (ii) a light chain variable domain having at least 85% amino acid identity with the light chain variable domain of SEQ ID NO: 24 and comprising a VL-CDR1 of SEQ ID NO: 21, a VL-CDR2 of SEQ ID NO: 22, and a VL-CDR3 of SEQ ID NO:

23. The monoclonal antibody c) of claim 1, comprising:

5. (i) a heavy chain variable domain having at least 85% amino acid identity with the heavy chain variable domain of SEQ ID NO: 28 and comprising a VH-CDR1 of SEQ ID NO: 25, a VH-CDR2 of SEQ ID NO: 26, and a VH-CDR3 of SEQ ID NO: 27; and / or (ii) a light chain variable domain having at least 85% amino acid identity with the light chain variable domain of SEQ ID NO: 32 and comprising a VL-CDR1 of SEQ ID NO: 29, a VL-CDR2 of SEQ ID NO: 30, and a VL-CDR3 of SEQ ID NO:

31. The monoclonal antibody d) of claim 1, comprising:

6. (i) a heavy chain variable domain having at least 85% amino acid identity with the heavy chain variable domain of SEQ ID NO: 36 and comprising a VH-CDR1 of SEQ ID NO: 33, a VH-CDR2 of SEQ ID NO: 34, and a VH-CDR3 of SEQ ID NO: 35; and / or (ii) a light chain variable domain having at least 85% amino acid identity with the light chain variable domain of SEQ ID NO: 40 and comprising a VL-CDR1 of SEQ ID NO: 37, a VL-CDR2 of SEQ ID NO: 38, and a VL-CDR3 of SEQ ID NO:

39. The monoclonal antibody e) of claim 1, comprising:

7. (i) a heavy chain variable domain having at least 85% amino acid identity with the heavy chain variable domain of SEQ ID NO: 44 and comprising a VH-CDR1 of SEQ ID NO: 41, a VH-CDR2 of SEQ ID NO: 42, and a VH-CDR3 of SEQ ID NO: 43; and / or (ii) a light chain variable domain having at least 85% amino acid identity with the light chain variable domain of SEQ ID NO: 48 and comprising a VL-CDR1 of SEQ ID NO: 45, a VL-CDR2 of SEQ ID NO: 46, and a VL-CDR3 of SEQ ID NO:

47. The monoclonal antibody e) of claim 1, comprising:

8. The monoclonal antibody according to any one of claims 1 to 7, which is a human monoclonal antibody.

9. An antigen-binding fragment of the antibody according to any one of claims 1 to 8.

10. An immunoconjugate comprising an antibody according to any one of claims 1 to 8 conjugated to a therapeutic agent, preferably a cytotoxic agent.

11. An immunoconjugate comprising an antibody according to any one of claims 1 to 8, linked to a detectable molecule.

12. An in vitro method for using a monoclonal antibody according to any one of claims 1 to 8 or an antigen-binding fragment thereof according to claim 9 for detecting SLC1A4 or cells expressing SLC1A4 in a sample, preferably in a cell sample.

13. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 8, or an antigen-binding fragment thereof according to claim 9, or an immunoconjugate according to claim 10, in combination with a pharmaceutically acceptable carrier.

14. An antibody according to any one of claims 1 to 8, or an antigen-binding fragment thereof according to claim 9, or an immunoconjugate according to claim 10, for use as a medicament.

15. An antibody according to any one of claims 1 to 8, or an antigen-binding fragment thereof according to claim 9, or an immunoconjugate according to claim 10, or a pharmaceutical composition according to claim 13, for use in treating a cancer that expresses SLC1A4.

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