Efficacy prediction marker for substance recognizing transferrin receptor

Abstract

To identify a factor that serves as an efficacy prediction marker for a substance recognizing transferrin receptor (TfR), or provide a method for predicting efficacy using the marker, or to provide an agent containing a substance recognizing TfR to be administered to a subject for whom efficacy has been predicted by the marker.SOLUTION: There are provided use of L-type amino acid transporter 1 (LAT1) or mTOR as an index for predicting the efficacy of a substance recognizing transferrin receptor (TfR); a method for predicting the efficacy of a substance recognizing TfR, the method comprising analyzing expression of LAT1 or expression of a gene encoding LAT1 in a sample derived from a subject, or analyzing activity of mTOR; and an agent comprising a substance recognizing TfR for administration to a subject for whom the efficacy of the agent has been predicted to be effective using LAT1 or mTOR as an index.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a marker for predicting the efficacy of a substance that recognizes a transferrin receptor, and the use thereof. [Background technology]

[0002] An antibody that specifically reacts with the human transferrin receptor (TfR) has been discovered (Patent Document 1), and the antitumor effects of this antibody (Non-Patent Document 1, Patent Documents 1-2) and the inhibitory effect of iron uptake into cells (Patent Document 3) have been clarified.

[0003] On the other hand, it is known that certain cells exhibit resistance to the effects of antibodies that specifically react with TfR (Non-Patent Document 2), leading to the hypothesis that this effect is determined by microenvironmental factors. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. WO2014 / 073641 [Patent Document 2] International Publication No. WO2023 / 204181 [Patent Document 3] International Publication No. WO2020 / 105621 [Non-patent literature]

[0005] [Non-Patent Document 1] Ogama Y, Kumagai Y, Komatsu N, et al. Phase 1 Clinical Trial of PPMX-T003, a Novel Human Monoclonal Antibody Specific for Transferrin Receptor 1, to Evaluate Its Safety, Pharmacokinetics, and Pharmacodynamics. Clin Pharmacol Drug Dev. 2023;12(6):579-587. doi:10.1002 / cpdd.1216 [Non-patent document 2] Kameda K, Yanagiya R, Miyatake Y, et al. The hepatic niche leads to aggressive natural killer cell leukemia proliferation through the transferrin-transferrin receptor 1 axis. Blood. 2023;142(4):352-364. doi:10.1182 / blood.2022018597 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to find a factor that serves as a predictive marker for the efficacy of a substance that recognizes TfR, to provide a method for predicting efficacy using the marker, or to stratify patients based on the predicted efficacy. Alternatively, an object of the present invention is to provide an agent containing a substance that recognizes TfR to be administered to a subject for whom efficacy has been predicted by the marker. [Means for solving the problem]

[0007] The following inventions are provided: [1] The use of L-type amino acid transporter 1 (LAT1) as an indicator for predicting the efficacy of substances that recognize the transferrin receptor (TfR). [2] The use according to [1], which comprises analyzing the expression of LAT1 or a gene encoding LAT1 in a sample derived from a subject for which the efficacy of the drug is predicted. [3] The use of mTOR as an indicator for predicting the efficacy of substances that recognize TfR. [4] The use according to [3], which comprises analyzing mTOR activity in a sample derived from a subject for predicting drug efficacy. [5] The use according to [4], wherein the analysis of mTOR activity includes analysis of the expression of at least one of the mTORC gene and the Myc gene. [6] The use according to any one of [1] to [5], wherein the substance that recognizes TfR is an antibody that inhibits the binding of transferrin to TfR. [7] The use according to any one of [1] to [6], wherein the substance that recognizes TfR is an antibody that recognizes human TfR. [8] The use described in [7], wherein the antibody that recognizes human TfR is an antibody that recognizes one or more amino acids at positions 629 to 633 of human TfR, or an antibody that inhibits the binding of other antibodies to one or more amino acids at positions 629 to 633 of human TfR. [9] The use according to any one of [6] to [8], wherein the antibody has a heavy chain first complementarity determining region (VH CDR1), a heavy chain second complementarity determining region (VH CDR2), and a heavy chain third complementarity determining region (VH CDR3) represented by SEQ ID NOs: 1, 2, and 3, respectively, and a light chain first complementarity determining region (VL CDR1), a light chain second complementarity determining region (VL CDR2), and a light chain third complementarity determining region (VL CDR3) represented by SEQ ID NOs: 4, 5, and 6, respectively.

[10] The use according to any one of [1] to [9], wherein the antibody is an antibody in which the heavy chain has SEQ ID NO: 7 and the light chain has SEQ ID NO: 8.

[11] The use according to any one of [1] to

[10] , wherein the medicinal effect is an anti-cancer effect.

[12] Analyzing the expression of LAT1 or the expression of a gene encoding LAT1 in a sample from a subject. A method for predicting the efficacy of substances that recognize TfR.

[13] analyzing mTOR activity in a sample from a subject; A method for predicting the efficacy of substances that recognize TfR.

[14] The method according to

[13] , wherein the analysis of mTOR activity includes analysis of the expression of at least one of the mTORC gene and the Myc gene.

[15] The use according to any one of

[12] to

[14] , wherein the substance that recognizes TfR is an antibody that inhibits the binding of transferrin to TfR.

[16] The method according to any one of

[12] to

[15] , wherein the substance that recognizes TfR is an antibody that recognizes human TfR.

[17] The method described in

[16] , wherein the antibody that recognizes human TfR is an antibody that recognizes one or more amino acids at positions 629 to 633 of human TfR, or an antibody that inhibits the binding of other antibodies to one or more amino acids at positions 629 to 633 of human TfR.

[18] The method according to any one of

[15] to

[17] , wherein the antibody has VH CDR1, VH CDR2, and VH CDR3 represented by SEQ ID NOs: 1, 2, and 3, respectively, and VL CDR1, VL CDR2, and VL CDR3 represented by SEQ ID NOs: 4, 5, and 6, respectively.

[19] The method according to any one of

[15] to

[18] , wherein the antibody has a heavy chain having SEQ ID NO: 7 and a light chain having SEQ ID NO: 8.

[20] The method according to any one of

[12] to

[19] , wherein the medicinal effect is an anti-cancer effect.

[21] An agent comprising a substance that recognizes TfR, the agent being intended for administration to a subject for whom the efficacy of the agent is predicted to be effective using LAT1 or mTOR as an index.

[22] A method for treating cancer, comprising administering a substance that recognizes TfR to a subject for whom the therapeutic effect of the agent is predicted to be effective using LAT1 or mTOR as an index. A substance that recognizes TfR, or an agent containing a substance that recognizes TfR, for administration to a subject for whom the therapeutic effect of the agent is predicted to be effective using LAT1 or mTOR as an index. Use of a substance that recognizes TfR, or use of an agent containing a substance that recognizes TfR, in the manufacture of an agent for administration to a subject for whom the therapeutic effect of the agent is predicted to be effective using LAT1 or mTOR as an index. [Effects of the Invention]

[0008] According to the present invention, a method useful for predicting the efficacy of a substance that recognizes TfR is provided. [Brief explanation of the drawings]

[0009] [Figure 1] ANKL cells in the spleen and bone marrow were resistant to treatment with the anti-TfR1 antibody PPMX-T003. Figure 1a shows the results of in vivo luciferase assays (IVLA) of two types of ANKL-PDXs (ANKL1-PDXs and ANKL3-PDXs) described in Non-Patent Document 2, performed before (IVLA1), early (IVLA2), and late (IVLA3) treatment with PPMX-T003. Figure 1b shows flow cytometry analysis of human CD45+ cells after hepatocyte removal using density gradient centrifugation in cell suspensions from the liver and spleen of ANKL1-PDXs described in Figure 1a before and early after treatment with PPMX-T003. Figure 1c shows hematoxylin-eosin (HE) staining and immunohistochemistry using an anti-human CD56 antibody. The squares in the low-magnification fields indicate the positions of the high-magnification fields. [Figure 2]Figure 2a shows a schematic diagram of the in vivo CRISPR screening targeting iron-requiring molecules. This procedure was performed three times independently, and three mice (mouse #1, mouse #2, and mouse #3) were analyzed. Figures 2b–d show the positive selection-Robust Ranking Algorithm (RRA) scores for all genes in (b) mouse #1, (c) mouse #2, and (d) mouse #3, respectively. The dots in the figure indicate the RRA values ​​of non-targeting sgRNAs. [Figure 3] Figure 3a shows the annotation of gene sets targeted by negatively selected sgRNAs that were dropped out by GSEA in the three mice evaluated. Five gene sets were commonly detected with negative selection-RRA scores below the cutoff value. Figures 3b-d show all annotated gene sets targeted by negatively selected sgRNAs in (b) Mouse #1, (c) Mouse #2, and (d) Mouse #3 using GSEA. Color values ​​indicate the false discovery rate (FDR), and red text indicates the five commonly annotated gene sets described in Figure 3a. [Figure 4] Figure 4a shows the negative selection RRA scores for all genes in mouse #1. Red, green, and yellow dots represent genes annotated as oxidative phosphorylation, DNA repair, and E2F targets in MSigDB HALLMARK, respectively. Figure 4b shows the ranking of commonly dropped sgRNAs targeting genes classified as DNA repair and E2F targets in the HALLMARK gene set in all three mice, along with their negative selection RRA scores. Figure 4c shows the results of measuring the percentage of γH2AX+ cells shown in Figure 5c. A total of three samples were analyzed for each treatment group. [Figure 5]Figure 5a shows a schematic diagram of sample preparation for connectivity scoring analysis. Figure 5b shows the ranking of connectivity scoring analysis using JFCR LinCAGE. Cytotoxic agents that cause DNA damage are written in red. Figure 5c shows the results of flow cytometry analysis of NK92 and KHYG1 cells exposed to PPMX-T003. Cells were cultured with or without 10 μg / mL PPMX-T003 for 24 hours before analysis. The relationship between cell cycle (DNA content) and DNA damage was plotted. [Figure 6] Figure 6a shows a schematic diagram of sample preparation for single-cell whole-transcriptome analysis. Figure 6b shows a heat map of the top 10 signature genes in each single-cell transcriptome analysis cluster. Figure 6c shows unsupervised clustering (UMAP) of the analyzed ANKL1 cells. Bar graphs indicate the cell populations in each cluster. Figure 6d shows the GSEA of the signature genes in Cluster 1. All annotation gene sets with an adjusted p-value of 0.25 or less are listed in HALLMARK from MSigDB. [Figure 7] Figures 7a-b show the expression profiles of MYC (a) and TFRC (b) in all spleen-derived cells analyzed by cluster. Each color represents the expression level in each cell type. Figure 7c shows the cell cycle scoring analysis results for all spleen-derived cells analyzed (left) and a schematic diagram showing cell cycle transitions (right). Figures 7d-e show the results of an apoptosis assay (d) and an MTT cell viability assay (e) in NK92 cells treated in vitro with various concentrations of rapamycin and 10 μg / mL PPMX-T003 for 48 hours. [Figure 8]Figures 8a-b show the GSEA results of comparing liver-derived (a) ANKL1 cells or (b) ANKL3 cells with normal NK cells using the KEGG pathway database. Pathways related to metabolism with adjusted p-values ​​below 0.25 were extracted. Figure 8c shows the GSEA plots of "FATTY_ACID_METABOLISM" (fatty acid metabolism) and "GLYCOLYSIS" (glycolysis) comparing liver-derived ANKL cells with normal NK cells. Figure 8d shows the GSEA plots of cysteine ​​and methionine metabolism in liver-derived ANKL cells compared with normal NK cells from a healthy volunteer. [Figure 9] Figure 9a shows the KEGG_CYSTEIN_AND METHIONINE_METABOLISM path view for Figure 8a (liver-resident ANKL1 cells vs. normal NK cells). Yellow diamonds, orange dashed lines, and purple dashed lines indicate the methionine, methionine cycle, and polyamine synthesis pathways, respectively. Figure 9b shows a volcano plot of the transcriptomes of liver-derived ANKL cells and normal NK cells. Red (increased) and blue (decreased) dots indicate SLC family genes, and annotated dots indicate amino acid transporters. Figures 9c–f show the results of in vitro cell proliferation assays of liver-derived ANKL cells and ANKL-derived cell lines cultured in single-amino-acid-deficient RPMI1640. Each bar indicates the relative proliferation value relative to conditions containing all amino acids. The red line indicates a relative proliferation value of 0.5, the cutoff value for these assays. A total of three samples were analyzed for each treatment group. [Figure 10]Figure 10a shows a volcano plot comparing the signature genes of Cluster 1 from the single-cell whole-transcriptome analysis in Figure 6 with those of other clusters. Red dots indicate SLC family genes, and underscores indicate genes encoding amino acid transporters. Figures 10b-c show violin plots for SLC7A5 (b) and SLC1A5 (c) from the single-cell whole-transcriptome analysis described in Figure 6. Figure 10d shows the results of an in vitro competitive proliferation assay of Cas9-overexpressing NK92 cells transfected with sgRNA-RFP plasmids via lentiviral vectors. The percentage of RFP fluorescence-positive cells (i.e., target gene knockout cells) was measured using flow cytometry 2 and 8 days after lentiviral transduction. The %RFP positive cells was calculated by dividing the number of RFP fluorescence-positive cells on day 8 by the number of cells on day 2. A total of three samples were analyzed for each treatment group. Figure 10e shows the results of Western blot analysis of Cas9-overexpressing NK92 cells transduced with non-targeting sgRNA (sgNT), sgSLC7A5, and empty (mock) RFP plasmid to verify the efficiency of LAT1 knockout by sgSLC7A5. [Figure 11] Figure 11a shows the schematic of the in vivo competitive proliferation assay of ANKL3 cells transfected with Cas9 and sgRNA. Figure 11b shows the results of an in vivo luciferase assay of ANKL3-PDX established using Cas9-overexpressing ANKL3 cells transfected with sgNT-RFP or sgSLC7A5-RFP plasmids. The assay was performed 7 days after ANKL3 cell inoculation. The luminescence intensity in the liver region was measured. Experiments were performed independently three times, and statistics were calculated using paired t-tests. Figure 11c shows the results of flow cytometry analysis of the percentage of RFP fluorescence-positive cells (i.e., target gene knockout cells) in liver-derived ANKL3 cells at the timing of the in vivo luciferase assay in Figure 11b. [Figure 12]Figures 12a-b show proliferation assays of ANKL-derived cell lines (a), liver-derived ANKL-PDX cells (b), and normal lymphocytes derived from peripheral blood of healthy volunteers (c) treated with various concentrations of JPH-203 for 48 hours (a) or 24 hours (b). A total of three samples were analyzed for each treatment group. Figure 12c shows the results of cell cycle assays of NK92 and KHYG1 cells treated with JPH-203. After 24 hours of incubation with 40 μM JPH-203, the cell cycle phase was measured using flow cytometry. A total of three samples were analyzed for each treatment group. Figure 12d shows the results of Western blot analysis of NK92 and KHYG1 cells treated with JPH-203. After 0, 6, and 24 hours of incubation with 40 μM JPH-203, the relative fluorescence intensity of each band compared to β-actin was calculated and indicated below the band. [Figure 13] FIG. 13 shows an uncropped photograph of the Western blotting shown in FIG. 12d. [Figure 14] Figures 14a-b show quantitative PCR results for TFRC transcription in (a) NK92 and (b) KHYG1 cells treated with 40 μM JPH-203 for 6 hours. Figure 14c shows the measurement results of FerroOrange-derived fluorescence shown in Figure 14d. Figure 14d shows the results of intracellular ferrous ion (Fe2+) staining using FerroOrange in NK92 and KHYG1 cells treated with control or 40 μM JPH-203. [Figure 15] Figure 15a shows the results of flow cytometry analysis of intracellular γH2AX expression in NK92 and KHYG1 cells cultured for 24 hours with or without 40 μM JPH-203 and / or 10 μg / mL PPMX-T003. A total of three samples were analyzed for each treatment group. Figure 15b shows the results of an in vivo luciferase assay of ANKL3-PDX treated sequentially with PPMX-T003, DMSO, or JPH-203. [Figure 16]Figure 16a shows unsupervised clustering of the pretreated liver-resident ANKL1 cells shown in Figure 6a. Figure 16b shows the results of cell cycle scoring analysis of liver-resident ANKL1 cells. Figure 16c shows a violin plot of SLC7A5 expression in liver-resident ANKL1 cells by cluster. Figure 16d shows flow cytometry analysis of ANKL1-PDX treated with JPH-203 or dimethyl sulfoxide (DMSO). A total of two mice per group were analyzed; all mice were exhausted and euthanized on day 13 for analysis. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention will be described in detail below. The features of the present invention described below can be combined in any combination.

[0011] <Definitions and General Techniques> Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meaning commonly understood by those of ordinary skill in the art. Generally, the nomenclature used in connection with and techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art.

[0012] The methods and techniques of the present invention are generally carried out according to conventional methods well known in the art, unless otherwise indicated, and as described in the various general and more specific references cited and discussed throughout the specification.

[0013] <Indicators for predicting drug efficacy and methods for predicting drug efficacy> One embodiment of the present invention relates to an index for predicting the efficacy of a substance that recognizes the transferrin receptor (TfR) and use thereof. Another embodiment relates to a method for predicting the efficacy of a substance that recognizes the TfR. Some embodiments of the present invention may not involve medical procedures on humans (e.g., diagnosing humans). Some embodiments of the present invention may also exclude procedures performed by physicians.

[0014] <tfr> In humans, transferrin receptor (TfR) is a single-pass transmembrane protein consisting of 760 amino acids, as shown in the following sequence, encoded by human chromosome 3 (SEQ ID NO: 9). This protein, also known as the CD71 antigen, is thought to be involved in cellular iron uptake and cell proliferation. The TfR of the present invention is not particularly limited in structure, and refers to all TfRs, including monomers, multimers, intact forms expressed in the cell membrane, soluble forms composed in the extracellular domain, truncated forms, mutation forms due to gene mutations or deletions, and forms that have undergone post-translational modifications such as phosphorylation.

[0015] human TfR (SEQ ID NO: 9) MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENADNNTKANVTKPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGEDFPAARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF

[0016] <Reacts and Reactivity> In this specification, unless otherwise specified, "react" and "reactivity" mean the same thing. That is, it means that an antibody recognizes an antigen, and "react" can be replaced with "recognize". This antigen may be intact TfR expressed on the cell membrane, a truncated form, or a solubilized form. It may also be TfR with a preserved three-dimensional structure or denatured TfR. As means for examining reactivity, flow cytometer (FACS), enzyme-linked immunosorbent assay (ELISA), western-blot, fluorescence microassay technology (FMAT), surface plasmon resonance (BIAcore), immunostaining, immunoprecipitation, etc. can be mentioned.

[0017] The antibody used for the flow cytometer may be an antibody labeled with a fluorescent substance such as FITC or biotin, or an unlabeled antibody. Depending on the presence or absence and type of labeling of the antibody used, fluorescently labeled avidin, fluorescently labeled anti-human immunoglobulin antibody, etc. can be used. Reactivity can be evaluated by adding a sufficient amount of anti-TfR antibody (usually the final concentration is 0.01 - 10 μg / mL) to the sample for reaction and comparing it with the reactivity of the negative control antibody and the positive control antibody.

[0018] <Substance that recognizes TfR> The substance that recognizes TfR is not particularly limited, and for example, at least one selected from the group consisting of nucleic acids (nucleic acid aptamers, decoy nucleic acids, etc.), ribozymes, antibodies and their fragments, peptides, cyclic peptides, and peptidomimetics can be used. For example, Non-Patent Document 3 describes an aptamer that mimics transferrin. Also, Non-Patent Document 4 describes a peptide targeting the transferrin receptor. In the present invention, such nucleic acids, peptides, etc. can be used.

[0019] [Antibody] In one aspect, the substance that recognizes TfR is an antibody (anti-TfR antibody). In the present invention, the following abbreviations (in parentheses) are used as necessary. Heavy chain (H chain), light chain (L chain), heavy chain variable region (VH), light chain variable region (VL), complementarity-determining region (CDR), first complementarity-determining region (CDR1), second complementarity-determining region (CDR2), third complementarity-determining region (CDR3), first heavy chain complementarity-determining region (VH CDR1), second heavy chain complementarity-determining region (VH CDR2), third heavy chain complementarity-determining region (VH CDR3), first light chain complementarity-determining region (VL CDR1), second light chain complementarity-determining region (VL CDR2), third light chain complementarity-determining region (VL CDR3).

[0020] The antibody is not particularly limited, but includes recombinant antibodies, monoclonal antibodies, polyclonal antibodies, etc. The antibody also includes antibody fragments as described below.

[0021] As used herein, the term "human antibody" refers to any antibody in which the variable and constant region sequences are human sequences. The term encompasses antibodies that have sequences derived from human genes but that have been altered to, for example, reduce potential immunogenicity, increase affinity, or remove cysteines that may cause undesired folding. The term also encompasses antibodies produced recombinantly in non-human cells that can provide glycosylation not typical of human cells. These antibodies can be prepared in a variety of ways.

[0022] As used herein, the term "humanized antibody" refers to an antibody of non-human origin in which amino acid residues characteristic of the antibody sequence of the non-human species are replaced with residues found at the corresponding positions in human antibodies. This "humanization" process is believed to reduce the immunogenicity of the resulting antibody in humans. It will be understood that antibodies of non-human origin can be humanized using techniques well known in the art (see, for example, Non-Patent Document 5). The antibody of interest can be engineered by recombinant DNA technology in which the CH1, CH2, CH3, hinge domain, and / or framework domain are replaced with corresponding human sequences. See, for example, WO 92 / 02190 and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and 5,777,085. As used herein, the term "humanized antibody" includes within its meaning chimeric human antibodies and CDR-grafted antibodies.

[0023] The sequence of the framework region (FR) in the variable region of an antibody is not particularly limited, as long as it does not substantially affect the specific binding to the corresponding antigen. Although it is preferable to use FRs from a human antibody, FRs from animal species other than humans (e.g., mouse or rat) can also be used.

[0024] In certain embodiments, the antibody comprises a constant region in addition to a variable region (e.g., an IgG antibody). The sequence of the constant region is not particularly limited, and for example, the constant region of a known human antibody can be used. The heavy chain constant region (CH) of a human antibody may be any that belongs to human immunoglobulin (hereinafter referred to as hIgG), but those of the hIgG class are preferred, and any subclass within the hIgG class, such as hIgG1, hIgG2, hIgG3, or hIgG4, can also be used. The light chain constant region (CL) may be any that belongs to hIg, and those of the κ class or λ class can be used. Constant regions from animal species other than humans (e.g., mice and rats) can also be used.

[0025] In the present invention, the terms "modified antibody" and "modified antibody" refer to an antibody in which one or more amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence of the variable region (CDR sequence and / or FR sequence) of a parent antibody.

[0026] In the present invention, the "parent antibody" refers to a TfR436 antibody having the amino acid sequences shown in SEQ ID NO: 7 for VH and SEQ ID NO: 8 for VL. These amino acid sequences may contain one or several (e.g., 1 to 8, preferably 1 to 5, more preferably 1 to 3, and particularly preferably 1 or 2) amino acids deleted, added, substituted, and / or inserted. Methods for introducing mutations into proteins are well known to those skilled in the art for preparing the amino acid sequence of an antibody with TfR-binding activity. For example, those skilled in the art can prepare modified antibodies functionally equivalent to antibodies with TfR-binding activity by introducing appropriate mutations into the amino acid sequence of an antibody with TfR-binding activity using site-directed mutagenesis (Non-Patent Documents 6-10). In this way, antibodies with TfR-binding activity in which one or several amino acids have been mutated in the variable or constant region of the antibody may also be used.

[0027] In one embodiment, the substance that recognizes TfR is an antibody that inhibits the binding of transferrin to TfR. The activity of inhibiting the binding of transferrin to TfR can be confirmed by any method known to those skilled in the art. A non-limiting example of such a method is one described in Patent Document 2, which includes the following procedure: HRP-labeled transferrin and a substance whose binding inhibitory activity is to be evaluated are reacted with immobilized sTfR; after washing, the color produced by HRP is measured at a predetermined absorbance using a color substrate, and the binding between transferrin and TfR is quantified. In this method, the color produced decreases with increasing dose of the substance whose binding inhibitory activity is to be evaluated, allowing the substance to be evaluated as having the activity of inhibiting the binding of transferrin to TfR.

[0028] In one embodiment, the substance that recognizes TfR is an antibody that recognizes human TfR. In a further embodiment, the antibody that recognizes TfR is an antibody in which the VH CDR1, VH CDR2, and VH CDR3 are represented by SEQ ID NOs: 1, 2, and 3, respectively, and the VL CDR1, VL CDR2, and VL CDR3 are represented by SEQ ID NOs: 4, 5, and 6, respectively. These amino acid sequences may have one or several (e.g., 1 to 8, preferably 1 to 5, more preferably 1 to 3, and particularly preferably 1 or 2) amino acids deleted, added, substituted, and / or inserted. For example, referring to Patent Document 1, it will be understood by those skilled in the art that even if VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 have amino acid sequences containing 1 to 8, preferably 1 to 5, more preferably 1 to 3, and particularly preferably 1 or 2 amino acid mutations (amino acid deletions, additions, substitutions, and / or insertions) in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, they can still have the same activity as those having no amino acid mutations.

[0029] In one embodiment, the antibody that recognizes TfR is an antibody that recognizes one or more amino acids at positions 629 to 633 of human TfR, or an antibody that inhibits the binding of other antibodies to one or more amino acids at positions 629 to 633 of human TfR. In a preferred embodiment, the antibody recognizes two or more consecutive or non-consecutive amino acids, three or more consecutive or non-consecutive amino acids, four or more consecutive or non-consecutive amino acids, or all of the amino acids at positions 629 to 633 of human TfR, or an antibody that inhibits the binding of other antibodies to two or more consecutive or non-consecutive amino acids, three or more consecutive or non-consecutive amino acids, four or more consecutive or non-consecutive amino acids, or all of the amino acids at positions 629 to 633 of human TfR. In a more preferred embodiment, the antibody recognizes the amino acids at positions 629, 630, and 633 of human TfR, or an antibody that inhibits the binding of other antibodies to the amino acids at positions 629, 630, and 633 of human TfR.

[0030] As used herein, "having activity equivalent to that of a parent antibody" means that the binding activity to TfR is equivalent. "Equivalent" does not necessarily mean that the activity is at the same level; the activity may be enhanced, or the activity may be decreased as long as the antibody still has activity. Examples of antibodies with decreased activity include antibodies that have 30% or more of the activity of the original antibody, preferably 50% or more, more preferably 80% or more, even more preferably 90% or more, and particularly preferably 95% or more.

[0031] Avidity refers to the recognition of an antigen. This antigen may be intact TfR expressed on the cell membrane, a truncated form, or a soluble form. It may also be TfR that retains its three-dimensional structure or a denatured TfR. For example, methods for examining avidity include flow cytometry (FACS), enzyme-linked immunosorbent assay (ELISA), western blotting, fluorescence microanalysis (FMAT), and surface plasmon resonance (BIAcore).

[0032] The Tf (transferrin)-TfR binding inhibitory activity of an antibody can be measured according to the method described in Non-Patent Document 2. The method can be simply explained as follows: A TfR solution is dispensed onto a substrate (such as a 96-well plate) and allowed to stand, solidified, and blocked. Next, an HRP-labeled Tf solution is dispensed, and an antibody is added and allowed to react at room temperature. Thereafter, the substrate is washed, and a color-developing reagent (such as TMB) is added to allow the reaction, and the absorbance is measured using a plate reader. By the above procedures, the Tf-TfR binding inhibitory activity of the antibody can be evaluated.

[0033] The antibody is not limited by its origin, and may be derived from any animal, such as a human antibody, a mouse antibody, or a rat antibody. It may also be a chimeric antibody, a humanized antibody, etc. One preferred embodiment of the antibody of the present invention is a human antibody, and one more preferred embodiment is a human antibody.

[0034] Antibodies may differ in amino acid sequence, molecular weight, isoelectric point, the presence or absence of sugar chains, or morphology depending on the antibody-producing cells, host, or purification method described below. For example, antibodies that are modified after translation from the amino acid sequences described herein are also included in the present invention. Furthermore, post-translational modifications at sites other than known post-translational modifications are also included in the present invention. Furthermore, when antibodies are expressed in prokaryotic cells, such as Escherichia coli, a methionine residue is added to the N-terminus of the original antibody amino acid sequence. Such antibodies may be used in the present invention. Post-translational modifications at sites other than known post-translational modifications are also included in the present invention.

[0035] [Antibody production] The antibody used in the present invention may be either a monoclonal antibody or a polyclonal antibody, which can be prepared by methods known to those skilled in the art.

[0036] Examples of antibodies include those produced in the blood of animals, those produced by hybridomas, those produced by hosts transformed using genetic engineering techniques with expression vectors containing antibody genes, those produced by screening the optimal antibody from a clone library using phage display and then producing the gene in CHO cells, and those obtained by directly obtaining human antibodies using transgenic mice that produce human antibodies.

[0037] To produce polyclonal antibodies, animals such as rabbits are immunized with an antigen to obtain serum, which can then be purified using, for example, ammonium sulfate precipitation, a protein A column, a protein G column, DEAE ion exchange chromatography, or an affinity column to prepare polyclonal antibodies.

[0038] Monoclonal antibodies can be produced, for example, as follows: An animal is immunized with an antigen, optionally together with an adjuvant. After confirming that the desired antibody level is elevated in the serum of the immunized animal, immune cells (e.g., spleen cells) are collected from the animal and fused with mammalian myeloma cells. Hybridomas obtained by cell fusion can be selected by culturing them in a conventional selective medium, such as HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). Hybridomas producing the desired antibody can then be screened by limiting dilution.

[0039] The production of antibodies by phage display is described below. (1) Antigen-reactive scFv from a phage display library Using phage display technology, libraries containing repertoires of antibodies with varying affinities for TfR can be provided. These libraries can then be screened to identify and isolate antibodies against TfR. Preferably, the phage library is an scFv phage display library generated using human VL and VH cDNAs prepared from mRNA isolated from human B cells. Methods for preparing and screening such libraries are known in the art. Genetic material is recovered from phage clones that show reactivity when screened using TfR as an antigen. Genetic analysis of the selected phage allows the determination of the VH and VL DNA sequences encoding the variable regions of human antibodies that bind to the antigen. Using the scFv sequences, the scFvs can be converted into IgG to obtain human antibodies.

[0040] (2) Conversion of scFv to IgG (production of human antibodies) Human antibodies are obtained by constructing expression vectors for the H or L chain, expressing them in host cells, and recovering and purifying the secreted supernatant. Human antibodies can also be obtained by expressing VH and VL in the same vector (tandem type). These methods are well known, and reference can be made to WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388, WO97 / 10354, etc.

[0041] Specifically, a full-length heavy chain gene can be obtained by linking the DNA encoding the VH to other DNA molecules encoding the heavy chain constant regions (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (see, for example, Non-Patent Document 11), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be the constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD, but is most preferably the constant region of IgG1 or IgG2. The IgG1 constant region sequence may be any of the various alleles or allotypes known to occur between different individuals, such as Gm(1), Gm(2), Gm(3), and Gm(17). These allotypes correspond to naturally occurring amino acid substitutions in the IgG1 constant region.

[0042] A full-length L chain gene (as well as a Fab light chain gene) can be obtained by linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see, for example, Non-Patent Document 11), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a κ or λ constant region. The κ constant region can be any of the various alleles known to occur between different individuals, such as Inv(1), Inv(2), or Inv(3). The λ constant region can be derived from any of the three λ genes.

[0043] The DNA encoding the H or L chain obtained as described above is inserted into an expression vector to produce an expression vector, which is then expressed in host cells. The secreted supernatant is then collected and purified to obtain a human antibody. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, cosmids, YACs, and EBV-derived episomes. Expression vectors and expression regulatory sequences may be selected to be compatible with the host cells used for expression. The antibody light chain gene and antibody heavy chain gene can be inserted into separate vectors, or both genes can be inserted into the same expression vector. The antibody gene is inserted into the expression vector using standard methods (e.g., ligation of the vector with complementary restriction sites on the antibody gene fragment, or blunt-end ligation if no restriction sites are present).

[0044] Convenient vectors encode functionally complete human CH or CL immunoglobulin sequences with appropriate restriction sites engineered to facilitate the insertion and expression of any VH or VL sequence, as described above. In such vectors, splicing typically occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C domain, as well as at splice regions present within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding region. The recombinant expression vector can also encode a signal peptide that facilitates secretion of the antibody chain from host cells. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0045] In addition to the antibody genes and control sequences, antibody expression vectors may contain additional sequences, such as sequences that control replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced. For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on host cells into which the vector has been introduced. Preferred selectable marker genes include the dehydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection / amplification), the neomycin phosphotransferase gene (for G418 selection), and the glutamate synthetase gene.

[0046] Host cells are transformed with the antibody gene expression vector prepared by the above method. The host cells may be any cells capable of producing antibodies, such as bacteria, yeast, animal cells, insect cells, or plant cells, but animal cells are preferred. Examples of animal cells include Chinese hamster ovary cells (CHO / dhfr(-) cells, CHO / DG44 cells), monkey-derived COS cells (Non-Patent Document 12), and SP2 / O cells (mouse myeloma) (Non-Patent Documents 13 and 14). Suitable methods for transformation include the lipofectin method (Non-Patent Documents 15 and 16), electroporation, the calcium phosphate method (Non-Patent Document 17), and the DEAE-Dextran method.

[0047] After culturing the transformant, human antibodies are isolated from the cells of the transformant or from the culture medium. Antibody isolation and purification can be achieved by using an appropriate combination of methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, affinity chromatography, ion exchange chromatography, and gel filtration chromatography.

[0048] [Antibody fragment] Antibody fragments can be produced based on antibodies or on the sequence information of the genes encoding them, including Fab, Fab', F(ab')2, scFv, and dsFv antibodies.

[0049] Fab is a fragment with a molecular weight of approximately 50,000, which is obtained by papain digestion of IgG in the presence of cysteine ​​and is composed of an L chain, an H chain variable region, and an H chain fragment consisting of a CH1 domain and part of the hinge region. In the present invention, it can be obtained by papain digestion of the above antibody. Alternatively, Fab can be prepared from a transformant transformed with an appropriate vector by incorporating DNA encoding a part of the H chain and L chain of the above antibody into the vector.

[0050] Fab' is a fragment with a molecular weight of approximately 50,000 that can be obtained by cleaving the disulfide bond between the H chains of F(ab')2, as described below. In the present invention, it can be obtained by digesting the above-mentioned antibody with pepsin and cleaving the disulfide bond using a reducing agent. Alternatively, like Fab, Fab' can also be prepared by genetic engineering using DNA encoding it.

[0051] F(ab')2 is a fragment with a molecular weight of approximately 100,000, obtained by digesting IgG with pepsin, in which fragments (Fab') consisting of an L chain, an H chain variable region, and an H chain fragment consisting of a CH1 domain and part of the hinge region are linked by disulfide bonds. In the present invention, it is obtained by digesting the above-mentioned antibody with pepsin. Furthermore, like Fab, it can also be prepared by genetic engineering using DNA encoding F(ab')2.

[0052] An scFv is an antibody fragment in which an Fv consisting of an H-chain variable region and an L-chain variable region is linked via a suitable peptide linker at the C-terminus of one chain to the N-terminus of the other to form a single chain. Examples of peptide linkers that can be used include the highly flexible (GGGGS)3 (SEQ ID NO: 10). For example, DNA encoding an scFv antibody can be constructed using DNA encoding the H-chain variable region and L-chain variable region of the antibody and DNA encoding the peptide linker, and this can be inserted into an appropriate vector. The vector can then be used to transform the resulting transformant, from which the scFv can be prepared.

[0053] dsFv is an Fv fragment in which Cys residues have been introduced at appropriate positions in the H-chain variable region and L-chain variable region, stabilizing the H-chain variable region and the L-chain variable region through disulfide bonds. The positions of the Cys residues to be introduced in each chain can be determined based on the three-dimensional structure predicted by molecular modeling. In the present invention, for example, the three-dimensional structure is predicted from the amino acid sequences of the H-chain variable region and L-chain variable region of the above-mentioned antibody, and DNA encoding the H-chain variable region and L-chain variable region into which mutations have been introduced based on such prediction is constructed. This DNA is then inserted into an appropriate vector, and a dsFv can be prepared from a transformant transformed with the vector.

[0054] Furthermore, antibody fragments can also be multimerized by linking scFv antibodies, dcFv antibodies, etc. using an appropriate linker, or by fusing streptavidin.

[0055] [Multispecific antibody] As long as the antibody recognizes TfR, it may be a multispecific antibody that simultaneously recognizes other targets (antigens), for example, a bispecific antibody.

[0056] Bispecific antibodies are antibodies that combine a molecule that recognizes TfR with a molecule that targets another antigen. Non-limiting examples include bispecific (mab)2, which is obtained by chemically crosslinking two monoclonal antibody molecules, bispecific F(ab')2, which is obtained by chemically crosslinking two Fab fragment molecules, quadroma, bsDb (bispecific diabody), scBsDb (single-chain bispecific diabody), scBsTaFv (single-chain bispecific tandem variable domain), Bite (bispecific T cell engager antibody), and DNL-F(ab)3 (docl-and-lock trivalent Fab) (Non-Patent Document 18).

[0057] <Medicinal effects> One of the medicinal effects of substances that recognize TfR is their anti-cancer effect (i.e., the effect of inhibiting the proliferation of cancer cells). The type of cancer is not particularly limited as long as it can be inhibited by a substance that recognizes TfR, and may be, for example, solid cancer (e.g., lung cancer, colon cancer, stomach cancer, bladder cancer, pancreatic cancer, prostate cancer, liver cancer, cervical cancer, uterine cancer, ovarian cancer, breast cancer, head and neck cancer, skin cancer, etc.) or blood cancer (e.g., leukemia, lymphoma, myeloma, etc.). Patent Documents 1, 2, 4, and 5 disclose the anti-cancer effect of substances that recognize TfR against various types of cancer (blood cancers such as leukemia (human erythroleukemia cells, acute myeloid leukemia cells, aggressive NK cell leukemia, etc.) and lymphoma (adult T-cell leukemia-lymphoma, etc.) (Patent Documents 1, 2, 4); solid cancers such as bladder cancer, stomach cancer, and pancreatic cancer (Patent Documents 1, 5), etc.). In some embodiments, the cancer is a cancer that expresses TfR. For example, as described in Patent Document 5, the expression of TfR may mean that even a slight staining due to TfR expression is observed in the cell membrane by immunostaining.

[0058] In one embodiment, the cancer is adult T-cell leukemia (ATL). In another embodiment, the cancer is an NK cell tumor. NK cell tumors include three disease types selected from aggressive NK-cell leukemia (ANKL), extranodal NK / T-cell lymphoma, nasal type (ENKL), and chronic lymphoproliferative disorders of NK cells (CLPD-NK). In one embodiment, the cancer is ANKL.

[0059] <lat1> As an index for predicting the drug efficacy of a substance that recognizes TfR, L-type amino acid transporter 1 (LAT1) can be used. LAT1 is an amino acid transporter that is specifically expressed in certain cancer cells and is known to be involved in the transport of amino acids such as methionine. As shown in this example, in cells where a growth inhibitory effect by a substance that recognizes TfR is shown, a high expression of the gene encoding LAT1 is observed. Also, by inhibiting LAT1, the cell growth inhibitory effect by a substance that recognizes TfR is suppressed. The expression of LAT1 can be analyzed according to any method known to those skilled in the art, and the method may be, for example, an immunological method such as ELISA using an antibody specific to LAT1, flow cytometry, Western blotting, etc.

[0060] As an index for predicting the drug efficacy of a substance that recognizes TfR, the gene encoding LAT1 can also be used. Examples of the gene encoding LAT1 include the SLC7A5 gene. The expression of the gene encoding LAT1 can be analyzed according to any method known to those skilled in the art, and the method may be, for example, RT-PCR method, real-time PCR method, Northern blotting method, dot blotting method, RNase protection assay method, microarray method, etc. using a primer or probe specific to the gene.

[0061] It can be predicted that the higher the expression of LAT1 or the gene encoding LAT1, the more effective the drug efficacy of the substance that recognizes TfR.

[0062] <Activity of mTOR> The activity of mTOR (mechanistic target of rapamycin or mammalian target of rapamycin) can be used as an indicator for predicting the efficacy of a substance that recognizes TfR. mTOR is a factor involved in regulating cell proliferation and metabolism through intracellular signal transduction. As shown in this example, high mTOR activity was observed in cells that exhibited the growth inhibitory effect of a substance that recognizes TfR. Furthermore, the cell growth inhibitory effect of a substance that recognizes TfR was suppressed by inhibiting mTOR.

[0063] Furthermore, as shown in this example, mTOR activity is reduced by inhibiting LAT1, suggesting that mTOR activity is regulated via the influx of amino acids into cells by LAT1. As described above, either LAT1 or mTOR can be used alone as an indicator for predicting the efficacy of a substance that recognizes TfR, but both can also be used in combination.

[0064] mTOR activity can be evaluated by methods known to those skilled in the art, for example, by analyzing the expression of the mTORC (mTOR complex) gene. mTORC may be at least one of mTORC1 and mTORC2, preferably mTORC1. Furthermore, since mTORC is known to promote the expression of the transcription factor Myc in cells, mTOR activity can also be evaluated, for example, by analyzing the expression of the Myc gene. Analysis of gene expression can be performed according to any method known to those skilled in the art, and the method may be similar to that described for analyzing the expression of the gene encoding LAT1. The higher the expression of the mTORC gene or Myc gene, the higher the mTOR activity can be evaluated, and therefore, the pharmacological effect of a substance that recognizes TfR can be predicted to be effective.

[0065] Alternatively, mTOR activity can be evaluated using a commercially available kit or the like, by measuring it according to the method specified in the kit. TM Examples include the mTOR Activity Kit (Merck, CBA055-1KIT).

[0066] mTOR activity can also be evaluated by analyzing the expression or phosphorylation of factors other than Myc that are present downstream of mTOR in mTOR-mediated intracellular signaling, or their genes, such as p70 / S6 kinase, 4E-BP1, and 4E-BP2.

[0067] <Subjects, samples> The subject for whom the efficacy of a drug is predicted may be one who requires or desires administration of a substance that recognizes TfR. Such subjects include, for example, those who have or have been diagnosed with cancer (e.g., solid cancer (e.g., lung cancer, colon cancer, stomach cancer, bladder cancer, pancreatic cancer, prostate cancer, liver cancer, cervical cancer, uterine cancer, ovarian cancer, breast cancer, head and neck cancer, skin cancer, etc.) or blood cancer (e.g., leukemia, lymphoma, myeloma, etc.)), and those who have or have been diagnosed with NK cell tumors (ANKL, ENKL, or CLPD-NK).

[0068] The subject may be a mammal such as a human, mouse, rat, etc., preferably a human or mouse, more preferably a human. The subject is not particularly limited by sex, age, etc.

[0069] Predicting the efficacy of a substance that recognizes TfR may involve analyzing factors indicative of efficacy in a subject-derived sample. The subject-derived sample may be any tissue, body fluid, etc., of the subject, such as any organ, organs, blood, plasma, serum, cerebrospinal fluid, pleural effusion, ascites, sweat, or urine, with the liver being a specific example. The tissue or body fluid may be isolated from the subject by any method, such as biopsy, blood sampling, or puncture. Alternatively, the subject-derived sample may be any cell derived from the subject's tissue, preferably a cancer cell, more preferably an ANKL cell. The cell may be obtained from tissue by any method, which can be appropriately selected depending on the type of cell of interest, such as by reacting minced tissue with a protease such as trypsin followed by centrifugation or filtration. The process of obtaining tissue or cells from a human body may not be included in the present invention.

[0070] <Agent> One embodiment of the present invention relates to an agent for administration to a specific subject, which comprises a substance that recognizes TfR.

[0071] The subject to be administered with the agent of the present invention is a subject for whom the pharmacological effect of a substance that recognizes TfR is predicted to be effective, based on at least one of LAT1 and mTOR as an index. A subject for whom the pharmacological effect of a substance that recognizes TfR is predicted to be effective may be, for example, a subject for whom the expression of each factor used as an index of pharmacological effect in a sample derived from the subject is higher than that of an appropriate control (e.g., normal (non-cancer cells) cells, a sample derived from a healthy subject, a sample derived from a subject that does not have cancer or has not been diagnosed with cancer, a sample derived from a subject for whom the pharmacological effect of a substance that recognizes TfR is not effective, etc.) or an arbitrarily set reference value.

[0072] The content of the substance that recognizes TfR in the agent of the present invention is not particularly limited, and may be, for example, 0.1 to 99.9 w / w %, 1 to 90 w / w %, or the like.

[0073] The agent of the present invention may contain a physiologically acceptable diluent or carrier in addition to the substance that recognizes TfR. Suitable carriers may be, for example, physiological saline, phosphate-buffered saline, phosphate-buffered saline glucose solution, buffered saline, etc., and are not particularly limited as long as the problem solved by the present invention can be solved. Alternatively, the substance that recognizes TfR may be lyophilized (freeze-dried) or frozen, and then reconstituted by adding an aqueous buffer solution or the like before use.

[0074] The dosage form or administration form of the agent of the present invention may be oral administration using tablets, capsules, granules, powders, syrups, etc., or parenteral administration using injections (subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.), transdermal, transmucosal, nasal, pulmonary, suppositories, etc., and is not particularly limited as long as the problem to be solved by the present invention can be solved.

[0075] The dosage of the agent of the present invention can be determined appropriately depending on symptoms, age, body weight, general condition, metabolic function, excretory function, etc., but for oral administration, the amount of the substance that recognizes TfR can be about 0.001 mg / kg to 1000 mg / kg per day for an adult. This can be administered once or in divided doses. For parenteral administration, the amount of the substance that recognizes TfR can be administered by subcutaneous injection, intramuscular injection, intravenous injection, etc., at a single dose of about 0.001 mg / kg to 1000 mg / kg. [Example]

[0076] The present invention will be further described below with reference to examples. Note that the following examples are intended to specifically explain the present invention, and the present invention should not be construed as being limited to the specific contents below.

[0077] <Materials, Subjects, and Methods> [Research approval] Experiments using patient-derived materials were approved by the Tokai University Clinical Research Review Board (H18-144). Animal experiments in this study were approved by the Tokai University Animal Experimentation Committee (221046). All experiments involving genetic modification were approved by the Tokai University Genetic Modification Experiment Safety Committee (22-009-27R2). Written informed consent was obtained from all patients and healthy volunteers.

[0078] The CDR sequences of the TfR436 antibody (PPMX-T003) are shown below. VH CDR1: SYGMH (SEQ ID NO: 1) VH CDR2: VISYDGSNKYYADSVKG (SEQ ID NO: 2) VH CDR3: DSNFWSGYYSPVDV (SEQ ID NO: 3) VL CDR1: TRSSGSIASNSVQ (SEQ ID NO: 4) VL CDR2: YEDTQRPS (SEQ ID NO: 5) VL CDR3: QSYDSAYHWV (SEQ ID NO: 6)

[0079] The VH and VL sequences of the TfR436 antibody (PPMX-T003) are shown below. TfR436 VH (SEQ ID NO: 7) DVQLVQSGGGVVQPGRSLRLSCAASGFPFKSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRGEDTAVYYCARDSNFWSGYYSPVDVWGQGTTVTVSS TfR436 VL (SEQ ID NO: 8) NFMLTQPHSVSESPGKTVTISCTRSSGSIASNSVQWYQQRPGSAPITVIYEDTQRPSGVPDRFSGSIDSSSNSASLTISGLQTEDEADYYCQSYDSAYHWVFGGGTKLAVL

[0080] [In vivo CRISPR screening of genes encoding iron-dependent molecules] Based on Non-Patent Document 19, 482 genes encoding iron-dependent molecules were selected. An sgRNA library containing sgRNAs targeting these genes and 1,004 control (non-targeting) sgRNAs was then constructed according to the method described in Non-Patent Document 20 (except for the use of the Ubi-RFP-sgRNA plasmid). The Ubi-RFP-sgRNA plasmid and sgRNA library oligonucleotide pool were provided by Professor Hirao of Kanazawa University (Table 1). Briefly, the Ubi-RFP-sgRNA plasmid was constructed from FG12 (Addgene, #14884) by cloning the RFP gene from pRSI12-U6-sh-HTS4-UbiC-TagRFP-2A-Puro (Addgene, #28289) into the GFP gene site and cloning the sgRNA expression cassette used in Non-Patent Document 20 into the siRNA expression cassette. The RNA oligonucleotide pool was ligated into BsmBI-digested Ubi-RFP-sgRNA plasmid using Gibson Assembly Master Mix (New England BioLabs, #E2611S). Next, the sgRNA library-inserted plasmid was transformed into 10-β electrocompetent E. coli (New England BioLabs, #C3020) using a Gene Pulser II (Bio-Rad; 1,000 V, 200 Ω, 25 μF). More than 100 colonies per sgRNA were collected, and the sgRNA library-containing plasmid was purified using NucleoBond Xtra Midi (Macherey-Nagel, #740410). The constructed sgRNA library plasmid was then transduced into ANKL1 cells using the lentiCas9-Venus plasmid (Addgene, #70267), followed by transfection with Venus. + Cells that were positive for both GFP and RFP fluorescence were sorted using flow cytometry 48 hours after each transduction procedure. 5 Sorted Venus (40 cells per sgRNA) + Patient-derived xenografts (PDXs) were established by intravenously injecting the RFP-positive cells into Nod / Shi-Scid, IL-2RγKO mice (NOG mice; In-Vivo Science). Genomic DNA was extracted from the remaining sorted cells (input) using the DNeasy Blood & Tissue Kit (Qiagen, #69504). After 14 days of in vivo expansion, ANKL1 cells were harvested from the liver and genomic DNA was extracted (output). The integrated sgRNA in genomic DNA was first amplified using Tks Gflex DNA polymerase (TaKaRa, R060A) with a pair of primers: 5'-GTCTAGAGAGGGCCTATTTCCCATGATTCC-3' (SEQ ID NO: 11) and 5'-CACCGACTCGGTGCCACTTTT-3' (SEQ ID NO: 12). The primers were then further amplified with a pair of primers: 5'-CAAGCAGAAGACGGCATACGAGATCXXXXXXTTTCTTGGGTAGTTTGCAGTTTT-3' (SEQ ID NO: 13) and 5'-AATGATACGGCGACCACCGAGATCTACACCACCGACTCGGTGCCACTTTT-3' (SEQ ID NO: 14) (where "X" represents the i7-index adapter sequence of Illumina TruSight Tumor 15, designated R701 to R706). A unique i7-index adapter sequence was added for each sample. The amplified, i7-indexed sgRNAs were then mixed and sequenced using the index primer 5'-TTTCAAGTTACGGTAAGCATATGATAGTCCATTTTAAAACATAATTTTAAAACTGCAAACTACCCAAGAAA-3' (SEQ ID NO: 15) and the sequence primer 5'-CGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3' (SEQ ID NO: 16) using DNBSEQ-G400 (MGI Tech).A reference FASTA-formatted file of the sgRNA library was created using Biostrings version 2.66.0 (Non-Patent Document 21). The resulting FASTQ-formatted file was then mapped, and sgRNA reads were counted using Rsubread version 2.12.3 (Non-Patent Document 22) and GenomicAlignments version 1.34.1 (Non-Patent Document 23). In total, 479 of 482 target sgRNAs were detected in the input sample. Positively and negatively selected sgRNAs were statistically analyzed using the "test" command in MAGeCK version 0.5.9.5 (Non-Patent Document 24). Gene Set Enrichment Analysis (GSEA) was performed using the "pathway" command in MAGeCK on the Hallmark GMT format file of the human Molecular Signatures Database (MSigDB) v2023.1 (https: / / www.gsea-msigdb.org / gsea / msigdb / index.jsp; data retrieved October 7, 2023). Gene sets with a false-discovery ratio of less than 0.25 were extracted as significant genes. This procedure was performed three times independently, and three NOG mice (mouse #1, mouse #2, mouse #3) were analyzed.

[0081] [Connectivity scoring analysis of differentially expressed gene sets in NK92 exposed to PPMX-T003] NK92 cells were preincubated for 18 hours and then equally divided into two dishes containing fresh medium. 10 μg / mL PPMX-T003 was then added to one of the dishes, and both samples were cultured for an additional 6 hours. After harvesting the cultured cells, total RNA was extracted using Sepasol-RNA I Super G (Nacalai Tesque, #09379-55) according to the manufacturer's protocol. A cDNA library was prepared using the TruSeq Stranded mRNA Sample Prep Kit (Illumina, #20020594). Sequencing was performed using the NovaSeq6000 (Illumina) and NovaSeq6000 S4 Reagent Kit v1.5 (Illumina, #20028312). The resulting FASTQ-formatted files were trimmed using Trimomatic version 0.38 (Non-Patent Document 25), followed by mapping using HISAT2 version 2.2.0. The mapped reads were counted using StringTie version 2.1.3b (Non-Patent Document 26) and EdgeR version 3.17 to obtain a gene signature that was differentially expressed between unexposed and PPMX-T003-exposed NK92 cells. Using the anticancer drug-related gene expression database (JFCR_LinCAGE; http: / / molpro.jfcr.or.jp / db / cs / index.html; accessed October 23, 2023) (Non-Patent Document 27), connectivity scoring analysis (Non-Patent Document 29) was performed according to the procedure described in Non-Patent Document 28 to identify drugs that induce gene expression changes similar to those of PPMX-T003.

[0082] [Single-cell RNA sequencing of ANKL1-expressing PDX-derived cells] Six ANKL1-PDXs were treated with PPMX-T003 at 10 mg / kg intravenously, three of which were injected twice a week for two weeks (a total of four injections). Treated and untreated ANKL1-PDXs were sacrificed, and human CD45 IgG was isolated from the spleens using a FACS Aria III. + ANKL cells were harvested. Single-cell cDNA libraries were constructed using the BD Rhapsody WTA Amplification Kit (Becton, Dickinson, #633801) and the BD Hu Single-Cell Sample Multiplexing Kit (Becton, Dickinson, #633781). Sequencing was performed using a NextSeq550 (Illumina). Data processing was performed using BD Rhapsody Sequence Analysis Pipeline version 1.11 provided by SevenBridge (Becton, Dickinson, October 29, 2022), resulting in 247,753,594 aligned reads from 28,021 predicted cells. Clustering and detection of signature genes were performed using Seurat version 4.3.0.1 in R version 4.2.2 (Non-Patent Document 30). GSEA of the signature genes was performed using clusterProfiler version 4.6.2 (Non-Patent Document 31).

[0083] [Cell culture] HEK293T cells were purchased from the RIKEN Cell Bank (#RCB2202) and cultured at 37°C and 5% CO2 in D-MEM (Fujifilm Wako, #043-30085) supplemented with 10% fetal bovine serum (FBS; Gibco, #26140079) and penicillin-streptomycin solution (Fujifilm Wako, #168-23191). Normal human lymphocytes were obtained from peripheral blood mononuclear cells of healthy volunteers and cultured in RPMI1640 (Fujifilm Wako, #189 02025, supplemented with 10% FBS) at 37°C and 5% CO2. NK92 cells were purchased from the American Type Culture Collection, and KHYG1 cells were purchased from the Japanese Collection of Research Bioresources. ANKL1 and ANKL3 were primary ANKL cells derived from two previously established ANKL PDX lines, ANKL1-PDX and ANKL3-PDX, as described in a previous study. ANKL1 cells were cultured in Leibovitz's L-15 medium (Gibco, #11415064) supplemented with 5% FBS, non-essential amino acids (Fujifilm Wako, #139-15651), 100 units / mL of human recombinant interleukin (IL)-2 (Peprotech, #200-02), and 0.1 mM 2-mercaptoethanol (Fujifilm Wako, #131-14572) at 37°C in a CO2-free incubator. Other cells were cultured in Artemis medium-1 (Nihon Techno Service, no catalog number) supplemented with 2% heat-inactivated human serum (KOHJIN BIO, #12181201) at 37°C and 5% CO .

[0084] [Antibodies and reagents] For flow cytometry analysis, the following antibodies were purchased from BioLegend: biotin-conjugated anti-human CD45 (#34004), FITC-conjugated anti-human CD45 (#368508), APC-conjugated anti-human CD45 (#304012), DyLight 649 goat anti-mouse IgG (#405312), and Alexa Fluor 488 goat anti-mouse IgG (#405319). All antibodies were used at a 1:500 dilution. Propidium iodide (PI; Sigma-Aldrich, #P4170) was used to stain DNA for cell cycle analysis and detection of dead cells. The antibody used for CD56 immunohistochemistry was purchased from Leica Biosystems (#CD56-504-L-CE). Antibodies used for Western blotting were purchased and diluted as follows: mouse anti-β-actin (Sigma-Aldrich, #A5441; diluted ×1,000), rabbit anti-CD71 (Proteintech, #10084; diluted ×1,000), rabbit anti-Myc (Cell Signaling Technology, #9402; diluted ×500), rabbit anti-phospho-p70 S6 kinase (Cell Signaling Technology, #9205S; diluted ×500), mouse anti-L-type amino acid transporter 1 (LAT1; Santa Cruz Biotechnology, #sc-37432; diluted ×500), HRP-conjugated goat anti-mouse IgG (Proteintech, #SA0001-1; diluted ×2,000), and HRP-conjugated goat anti-rabbit IgG (Proteintech, #SA00001-2; diluted ×2,000). The anti-human transferrin receptor 1 inhibitor antibody PPMX-T003 was provided by PerPersseus Proteomics, Inc. The LAT1-specific inhibitor JPH-203 was purchased from TargetMol (#TQ0081) and diluted in dimethyl sulfoxide. The mammalian target of rapamycin inhibitor rapamycin was purchased from AdipoGen (#AG-CN2-0025-C100) and diluted in dimethyl sulfoxide.

[0085] [Ankl-PDX animal care and breeding] Six-week-old female NOD / Shi-scid, IL-2Rγ KO (NOG) mice were purchased from In-Vivo Science and housed in a specific pathogen-free area of ​​the Tokai University Animal Facility, where temperature, humidity, and light levels were appropriately controlled. Mice aged 7 to 11 weeks were used for all experiments. All mice received humane care, and the humane endpoint was defined as mice that continuously lost more than 15% of their maximum body weight or showed significant wasting. The ANKL-PDX used in this study was established as detailed in Non-Patent Document 2. ANKL cells harvested from ANKL-PDX were intravenously injected into other NOG mice to replicate (passage) the ANKL-PDX.

[0086] [Collection of primary ANKL cells from ANKL-PDX] Livers and spleens were harvested from ANKL-PDXs 7–11 days after ANKL cell inoculation. Both organs were homogenized and filtered through a 70 μm cell strainer (Falcon, #352350). They were then enzymatically digested in HBSS(+) (Fujifilm Wako, #084-08965) containing 1 mg / mL collagenase A (Roche, #11088793001) and 100 U of DNase I (Roche, #10104159001) at 37°C for 15 min. Hepatocytes were removed by density gradient centrifugation using 35% Percoll (Sigma-Aldrich, #P1644) diluted in HBSS(-) (Fujifilm Wako, #085-09355). Finally, ANKL cells were isolated using an automated magnetically activated cell sorter (autoMACS; Myltenyi Biotech) with biotin-conjugated anti-human CD45 antibody and anti-biotin microbeads (Miltenyi Biotech, #130-090-485). The collected ANKL cells were stored in a deep freezer using CELLBANKER (ZENOGEN PHARMA #11910). The original method for collecting ANKL cells from PDXs is described in Non-Patent Document 2.

[0087] [Flow cytometry analysis and cell sorting] Cells were washed several times with phosphate-buffered saline containing 2% FBS and then incubated with an appropriate concentration of antibody solution for 30–60 minutes. The stained cells were then washed several times with phosphate-buffered saline containing 2% FBS. After staining for surface molecules, the cells were fixed and permeabilized with chilled 70% ethanol, blocked with 2% bovine serum albumin for 15 minutes, and then stained for intracellular antigens. Surface and intracellular molecule expression was analyzed using a FACS Lyric or LSR Fortessa (Becton, Dickinson). Cell sorting was performed using a FACS Melody or Aria III (Becton, Dickinson). Data processing and analysis were performed using FlowJo version 10 (Becton, Dickinson).

[0088] [Western Blot] Cells were directly lysed in 1x Laemmli sample buffer (60 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, 5% 2-mercaptoethanol, and 0.02% bromophenol blue) and then boiled in a heat block at 96°C for 10 minutes. The samples were subjected to 10-15% acrylamide gel electrophoresis. Proteins in the acrylamide gel were then transferred to a 0.2-µm polyvinylidene difluoride membrane (Fujifilm, #033-22453) using a HorizeBLOT 4M-R (ATTO, #2322470). The membrane was then blocked with 5% skim milk. The primary antibody was then diluted in 5% skim milk and incubated at room temperature for 2 hours. After washing the membrane with Tris-buffered saline containing 0.05% Tween 20, the secondary antibody was diluted in 5% skim milk and incubated at room temperature for 45 minutes. After washing the membrane, a sufficient amount of Immobilon Western Chemiluminescent HRP substrate (Merck Millipore, #WBKLS0100) was applied, and luminescence was detected using FUSION SOLO.7S.EDGE (Vilber Bio Imaging). For detailed materials and methods, see Non-Patent Document 32.

[0089] [Quantitative PCR] Total cellular RNA was extracted using Sepasol RNA I Super G (Nacalai Tesque, #09379 55) and reverse-transcribed using ReverTra Ace qPCR Master Mix with gDNA Remover (TOYOBO, #FSQ 301). PCR was performed using THUNDERBIRD SYBR qPCR Mix (TOYOBO, #QPS 201) and a StepOnePlus Real Time PCR System (Applied Biosystems). ACTB expression was selected as an internal control, and 2000 kJ / mL was used for data analysis. -ΔΔCT The primer sequences used for PCR were as follows: ACTB-Forward: 5'-CTCTTCCAGCCTTCCTTCCT-3' (SEQ ID NO: 17), ACTB-Reverse: 5'-AGCACTGTGTTGGCGTACAG-3' (SEQ ID NO: 18), MYC-Forward: 5'-CACCAGCAGCGACTCTGA-3' (SEQ ID NO: 19), MYC-Reverse: 5'-GATCCAGACTCTGACCTTTTGC-3' (SEQ ID NO: 20), TFRC-Forward: 5'-ACTTCTTCCGTGCTACTTCCAG-3' (SEQ ID NO: 21), TFRC-Reverse: 5'-ACTCCACTCTCATGACACGATC-3' (SEQ ID NO: 22), and TXNIP-Forward: 5'-CAGCAGTGCAAACAGACTTCGG-3' (SEQ ID NO: 23).

[0090] [Transduction using lentiviral vectors] HEK293T cells were co-transfected with the second-generation lentiviral packaging vectors psPAX2 (Addgene, #12260) and pMD2.G (Addgene, #12259) and plasmids containing the proposed genes (encoding sgRNA, Cas9, or luciferase) using PEI MAX (Polysciences, #24765-1). Forty-eight hours after transduction, the culture supernatant was collected and centrifuged at 10,000 × g for 4 hours to concentrate the lentivirus. Target cells were then infected with the concentrated lentiviral supernatant on RetroNectin (Takara Bio, #T100A)-coated culture plates and centrifuged at 1,000 × g for 90 minutes.

[0091] [sgRNA design and construction of related plasmids] Specific gene-targeting sgRNAs were designed using the online tool CHOPCHOP (http: / / chopchop.cbu.uib.no / ). Non-targeting sgRNA sequences were obtained from BRDN0001149198 (#80248; Addgene). Oligonucleotides were obtained from Eurofin Genomics and enzymatically digested with BsmBI (New England BioLabs, #R0580S) according to the manufacturer's protocol. Then, they were ligated into the FG12 (Addgene, #14884)-based sgRNA and mRFP expression plasmids obtained from Kanazawa University. The sequences of the sgRNAs are as follows: non-targeting sgRNA (sgNT): 5'-GTATTACTGATTGGGGGTATTACTGATTGGTGGG-3' (SEQ ID NO: 24), sgSLC7A5: 5'-TGGCGGTAGCGCTCACCATGGCGGTAGCGCTCACCA-3' (SEQ ID NO: 25), sgSLC1A5: 5'-CAGCGCCACCAAAGGACCAGGCCACCAAAGACGA-3' (SEQ ID NO: 26).

[0092] [CRISPR-Cas9 gene knockout] Cells were transduced sequentially with lentiCas9-Venus (Addgene, #70267) and sgRNA plasmids as described above, and analyzed by flow cytometry at least 48 hours after transduction.

[0093] [In vivo imaging assay] The firefly luciferase gene was cloned into CSII-CMV-MCS (Riken, #RDB04377) and digested with AgeI (New England Biolabs, #R3552S) and BamHI (New England Biolabs, #R3136S). Then, the vectors used to construct ANKL1 and ANKL3 cells (ANKL1 and ANKL3, respectively) were used. Luc and ANKL3 Luc On the day of analysis, 150 mg / kg of luciferin (Promega, #P1043) was injected intraperitoneally, and luminescence was measured using Living Image software (version 4.3.1; PerkinElmer) under 2.5% isoflurane inhalation.

[0094] [Histological analysis] Livers and spleens were collected from sacrificed ANKL-PDX mice, fixed in 4% formaldehyde, and embedded in paraffin. Sections were stained with hematoxylin and eosin. Immunohistochemistry was performed using a Bond Polymer Refine Detection (Leica, #DS9800) as described in non-patent literature 50. Images of tissue slides were scanned using CellSense software (Olympus), converted to virtual slide image format, and viewed using NDP.view2 (Hamamatsu Photonics).

[0095] [Transcriptome analysis using publicly available RNA-seq data] Transcriptome data for primary ANKL cells and normal NK cells used in Non-Patent Document 2 were obtained from Gene Expression Omnibus (GSE189722). FASTQ files were mapped to GRCh38.95 available on Ensembl (https: / / www.ensembl.org; accessed September 10, 2021) using HISAT2 version 2.2.0. Mapped reads were counted using StringTie version 2.1.3b. Differentially expressed genes were detected using DESeq2 version 1.38.3, and gene set enrichment analysis was performed using clusterProfiler version 4.6.2 using Hallmark GMT format files from Human MSigDB v2023.1.

[0096] [DNA damage detection assay] After culturing the cells with or without reagent for the appropriate period, they were harvested, fixed, and permeabilized with cold 70% ethanol. DNA damage was detected via detection of γH2AX using a DNA Damage Detection Kit (Dojindo, #343-09421) according to the manufacturer's protocol. In some analyses, DyLight649 goat anti-mouse IgG antibody was used as the secondary antibody instead of the secondary antibody in the kit. Fluorescence was detected using flow cytometry.

[0097] [Single amino acid depletion assay] Amino acid-depleted RPMI 1640 medium was purchased from Functional Peptide Institute, Inc. Purified amino acids were purchased from Fujifilm Wako as follows: L(+)-arginine (#019-04611), L-asparagine monohydrate (#019-04812), L-aspartic acid (#013-04832), L-glutamic acid (#070-00502), glycine (#073-00732), L-histidine (#086-00681), L-isoleucine (#123-00861), L-leucine (#126-0000). The amino acids used were: L-methionine (#135-01601), L(-)-phenylalanine (#163-01301), L(-)-proline (#163-04601), L-serine (#191-00401), L-threonine (#206-01321), L-tryptophan (#206-03381), L-valine (#220-00081), L-cysteine ​​(#039-20652), and L-tyrosine (#204-03561). L-glutamine was purchased from Gibco (#2530081). Single-amino acid-deficient RPMI 1640 medium was prepared by adding appropriate amounts of each amino acid to amino acid-depleted RPMI 1640 medium to the same concentration as in the original RPMI 1640 medium. NK92, KHYG1, ANKL1, and ANKL3 cells were cultured in single-amino-acid-deficient RPMI 1640 medium supplemented with 10% FBS and 100 units / mL recombinant human IL-2 at 37°C in 5% CO for 24 hours (ANKL1) or 48 hours (other cells). Cultured cells were harvested and counted using the MTT Cell Count Kit (Nacalai Tesque, #23506-80; NK92 and KHYG1) or trypan blue stain (Fujifilm Wako, #207-17081; ANKL1 and ANKL3) according to the manufacturer's protocol.

[0098] [In vitro proliferation competition assay of single gene knockout cells] Cas9-Venus overexpressing cells were transduced with the sgRNA RFP expression plasmid and cultured without further purification. Venus expression was analyzed by flow cytometry 5 and 8 days after transduction. + The percentage of RFP-positive cells in the cells was measured, and the relative abundance of knockout cells was calculated 8 days after transfection compared to 5 days after transfection. The lentivirus titer was adjusted 5 days after transfection when the percentage of RFP-positive cells reached 40-60%.

[0099] [In vivo proliferation competition assay of single gene knockout ANKL cells in the liver] An sgRNA RFP expression plasmid was introduced into ANKL1 and ANKL3 cells overexpressing Cas9-Venus, and RFP fluorescent-positive cells were purified with an efficiency of over 90% using flow cytometry 48 hours after transfection. The selected cells were intravenously inoculated into NOG mice and harvested from the liver 7 days after injection. The harvested cells were analyzed by flow cytometry to identify Venus-positive cells. + The percentage of RFP fluorescence-positive cells was calculated.

[0100] Cell proliferation and viability assays First, 3 × 10 cells were cultured in 800 μL of the appropriate medium containing the antibody or reagent. 4 cells (cell line) or 1 x 10 5 The cells (primary cells) were cultured at 37°C in 5% CO for 48 hours (cell lines) or 24 hours (primary cells). Then, the cells were transferred to 96-well plates and subjected to MTT assay or trypan blue staining to assess cell proliferation or viability according to the manufacturer's protocol, as described above.

[0101] [Live cell staining of intracellular ferrous ions and mitochondrial reactive oxygen species] FerroOrange and mtSOX deep red (Dojindo, #F374 and #MT14, respectively) were used to stain intracellular ferrous ions and mitochondrial reactive oxygen species (ROS) according to the manufacturer's protocols. Fluorescence of FerroOrange and mtSOX deep red was detected by light microscopy (IX83; Olympus) and flow cytometry, respectively.

[0102] [Cell cycle assay] After fixation and permeabilization, the collected cells were subjected to RNA digestion using 20 μg / mL RNase (NIPPON GENE, #313 01461). Cellular DNA was stained with 50 μg / mL PI, and cell cycle status was determined by measuring fluorescence by flow cytometry. To better separate the G0 / 1 and S phases of the cell cycle, ANKL-PDX were intraperitoneally inoculated with 50 mg / kg EdU 2 hours before sacrifice to detect DNA-synthesizing cells in liver-resident ANKL cells in vivo. EdU was labeled using the Click-iT Plus EdU Flow Cytometry Assay Kit (Thermo Fisher Scientific, #C10634) and detected by flow cytometry.

[0103] [Statistical analysis] All statistical analyses, except for deep sequencing-related analyses, were performed using GraphPad Prism 10.1.0 (GraphPad software). A two-tailed Welch t-test was used to analyze differences between two independent groups described by continuous variables. A two-tailed paired t-test was used to analyze differences between two groups with several dependencies. For analysis of differentially expressed genes or GSEA, the Benjamini-Hochberg method was used to adjust p-values. Survival analysis of ANKL-PDX was performed using the log-rank test. All experiments were independently repeated at least twice. The sample size for all experiments was determined empirically based on previous experimental experience using similar assays. Specific sample sizes and the number of independent experiments performed for each study are listed in the figures, brief figure descriptions, or in the <Materials, Subjects, and Methods> section. All assay samples were included in the analysis. In the figures, all results are indicated as follows: * = p < 0.05, ** = p < 0.01, and *** = p < 0.001.

[0104] <Result> Splenic and bone marrow ANKL cells are resistant to treatment with the anti-TfR1 antibody PPMX-T003. In vivo luciferase assays of two established ANKL-PDXs (ANKL1-PDXs and ANKL3-PDXs) (Non-Patent Document 2) demonstrated that luciferin luminescence from ANKL cells was detected in the spleen and bone marrow after PPMX-T003 injection, and that these cells ultimately repopulated in the liver following PPMX-T003 pharmacokinetic excretion from mice (Fig. 1a). Furthermore, significant ANKL cells were detected in cell suspensions of spleens from PPMX-T003-treated ANKL-PDXs by flow cytometry (Fig. 1b) and immunohistochemistry with anti-human CD45 antibody (Fig. 1c). These results suggest that the sensitivity of ANKL cells to PPMX-T003, reflecting their iron dependence, depends on the microenvironment in which the cells are located.

[0105] PPMX-T003 induces DNA double-strand breaks in ANKL cells in S phase, similar to conventional cytotoxic agents. To investigate the molecular mechanism of iron dependency in hepatic sinusoidal ANKL cells, we performed an in vivo CRISPR screen targeting genes encoding molecules that require iron for enzymatic activity. We designed an sgRNA library (10 sgRNAs per gene) targeting 482 genes of iron-dependent molecules, along with 1,004 non-targeting control sgRNAs, and evaluated whether these molecules contribute to in vivo cell proliferation and survival of ANKL cells in the liver (Table 1). Cas9-transfected ANKL1 cells and library sgRNA-transfected ANKL1 cells were purified and intravenously inoculated into NOG mice. After 14 days of in vivo growth, the cells were re-harvested from the liver. Genomic DNA was extracted and deep sequencing of the integrated sgRNAs was performed to detect sgRNAs that were positively or negatively selected during culture (Figure 2a). Deep sequencing analysis revealed that control sgRNAs were annotated above the positively selected sgRNAs in all cases, while sgRNAs targeting common essential genes such as CIAO1 and POLA1 (annotated by Depmap [https: / / depmap.org / portal / ]; accessed February 7, 2024) were negatively selected ("dropped out"; Tables 2-4; Figures 2b-d). These results demonstrate the reliability of the screening. A total of 2,884, 2,494, and 2,861 sgRNAs were dropped out in mouse #1, mouse #2, and mouse #3, respectively (Tables 2-4), suggesting their contribution to cell proliferation or survival of ANKL cells. Using HALLMARK from MSigDB, we performed GSEA of the genes targeted by the negatively selected sgRNAs in the three mice. We found that five gene sets (oxidative phosphorylation, DNA repair, E2F targets, mitotic spindle, and adipogenesis) were commonly dropped out with a false positive rate of less than 0.25 (Figures 3-4; Tables 5-7). In particular, the oxidative phosphorylation and DNA repair gene sets had low false positive rates in all three mice. The DNA repair and E2F target gene sets included various genes important for DNA repair, such as ERCC2 (contributing to nucleotide excision repair) and DGCR8 (contributing to repair of UV-induced DNA damage; Figure 4b).Furthermore, sgRNAs targeting genes encoding several DNA polymerases and ribonucleases required for DNA replication (POLA1, POLE, and RRM2) were significantly downregulated in all three mice (Fig. 4b). These results indicate that liver-resident ANKL cells depend on extracellular iron for two cellular functions: oxidative phosphorylation and DNA replication and repair.

[0106] [Table 1] TIFF2025160659000002.tif255154TIFF2025160659000003.tif255158TIFF2025160659000004.tif255159TIFF2025160659000005.tif255157TIFF2025160659000006.tif255157TIFF2025160659000007.tif255160TIFF2025160659000008.tif255159TIFF2025160659000009.tif255160TIFF2025160659000010.tif255158TIFF2025160659000011.tif255157TIFF2025160659000012.tif255157TIFF2025160659000013.tif255156TIFF2025160659000014.tif255158TIFF2025160659000015.tif255158TIFF2025160659000016.tif255158TIFF2025160659000017.tif255160TIFF2025160659000018.tif255160TIFF2025160659000019.tif255161TIFF2025160659000020.tif255159TIFF2025160659000021.tif255159TIFF2025160659000022.tif255160TIFF2025160659000023.tif255160TIFF2025160659000024.tif255161TIFF2025160659000025.tif255160TIFF2025160659000026.tif255161TIFF2025160659000027.tif255161TIFF2025160659000028.tif255161TIFF2025160659000029.tif255159TIFF2025160659000030.tif255159TIFF2025160659000031.tif255159TIFF2025160659000032.tif255160TIFF2025160659000033.tif255158TIFF2025160659000034.tif255162TIFF2025160659000035.tif255161TIFF2025160659000036.tif255161TIFF2025160659000037.tif255159TIFF2025160659000038.tif255160TIFF2025160659000039.tif255158TIFF2025160659000040.tif255160TIFF2025160659000041.tif255159TIFF2025160659000042.tif255158TIFF2025160659000043.tif255162TIFF2025160659000044.tif255157TIFF2025160659000045.tif255160TIFF2025160659000046.tif255159TIFF2025160659000047.tif255160TIFF2025160659000048.tif255159TIFF2025160659000049.tif255160TIFF2025160659000050.tif255157TIFF2025160659000051.tif255160TIFF2025160659000052.tif255160TIFF2025160659000053.tif255160TIFF2025160659000054.tif255162TIFF2025160659000055.tif255159TIFF2025160659000056.tif255159TIFF2025160659000057.tif255158TIFF2025160659000058.tif255158TIFF2025160659000059.tif255161TIFF2025160659000060.tif255161TIFF2025160659000061.tif255160TIFF2025160659000062.tif255160TIFF2025160659000063.tif255158TIFF2025160659000064.tif255163TIFF2025160659000065.tif255161TIFF2025160659000066.tif255162TIFF2025160659000067.tif255160TIFF2025160659000068.tif255160TIFF2025160659000069.tif255162TIFF2025160659000070.tif255159TIFF2025160659000071.tif255157TIFF2025160659000072.tif255161TIFF2025160659000073.tif255159TIFF2025160659000074.tif255162TIFF2025160659000075.tif255160TIFF2025160659000076.tif255161TIFF2025160659000077.tif255162TIFF2025160659000078.tif255158TIFF2025160659000079.tif255158TIFF2025160659000080.tif255157TIFF2025160659000081.tif255158TIFF2025160659000082.tif255160TIFF2025160659000083.tif255160TIFF2025160659000084.tif255159TIFF2025160659000085.tif255159TIFF2025160659000086.tif255158TIFF2025160659000087.tif255159TIFF2025160659000088.tif255160TIFF2025160659000089.tif255158TIFF2025160659000090.tif255160TIFF2025160659000091.tif255159TIFF2025160659000092.tif255160TIFF2025160659000093.tif255159TIFF2025160659000094.tif255161TIFF2025160659000095.tif255161TIFF2025160659000096.tif255162.

[0107]

Table 2

[0108]

Table 3

[0109]

Table 4

[0110]

Table 5

[0111]

Table 6

[0112]

Table 7

[0113] Next, we compared the differentially expressed gene profiles of PPMX-T003-treated NK92 cells (an ANKL-derived cell line) with those of tumor cells treated with various functionally well-known antitumor agents deposited in JFCR LinCAGE to assess their similarity in predicting the molecular mechanisms underlying the antitumor effects of PPMX-T003 (connectivity scoring analysis; Figure 5a). Among the drugs evaluated, traditional cytotoxic agents that induce DNA damage, such as anthracyclines and DNA intercalators, had high connectivity scores, indicating that the transcriptome changes induced by PPMX-T003 were similar to those induced by these agents (Figure 5b; Table 8). The expression of γH2AX (a well-known cytological marker of DNA double-strand breaks) and total cellular DNA content in ANKL-derived cell lines (NK92 and KHYG1) exposed to PPMX-T003 in vitro indicated that PPMX-T003 induces DNA damage through cell cycle progression (Figure 5c). Furthermore, γH2AX expression was also increased in ANKL1 cells derived from the liver of PPMX-T003-treated ANKL1-PDX, but not in cells derived from the spleen. These findings suggest that the antitumor effect induced by PPMX-T003 relies on replication-dependent DNA damage, which in part depends on liver-specific microenvironmental factors. Our previous GSEA also showed that liver-resident ANKL cells exhibited enriched DNA repair-related gene expression compared with spleen-resident cells, supporting the higher sensitivity of liver-resident ANKL cells to PPMX-T003 (Non-Patent Document 2).

[0114] [Table 8] TIFF2025160659000119.tif255165TIFF2025160659000120.tif255163TIFF2025160659000121.tif255165TIFF2025160659000122.tif255165TIFF2025160659000123.tif255166TIFF2025160659000124.tif255165TIFF2025160659000125.tif255163TIFF2025160659000126.tif255166TIFF2025160659000127.tif255165TIFF2025160659000128.tif255164TIFF2025160659000129.tif255166TIFF2025160659000130.tif255167TIFF2025160659000131.tif255165TIFF2025160659000132.tif255163TIFF2025160659000133.tif255166TIFF2025160659000134.tif255166TIFF2025160659000135.tif255165TIFF2025160659000136.tif255166TIFF2025160659000137.tif255164TIFF2025160659000138.tif255165TIFF2025160659000139.tif255165TIFF2025160659000140.tif255165TIFF2025160659000141.tif255165TIFF2025160659000142.tif255167TIFF2025160659000143.tif255165TIFF2025160659000144.tif255165TIFF2025160659000145.tif255168TIFF2025160659000146.tif255165TIFF2025160659000147.tif255165TIFF2025160659000148.tif255166TIFF2025160659000149.tif255167TIFF2025160659000150.tif255165TIFF2025160659000151.tif255167TIFF2025160659000152.tif255164TIFF2025160659000153.tif255164TIFF2025160659000154.tif255165TIFF2025160659000155.tif255167TIFF2025160659000156.tif255166TIFF2025160659000157.tif255165TIFF2025160659000158.tif255166TIFF2025160659000159.tif255164TIFF2025160659000160.tif255166TIFF2025160659000161.tif255165TIFF2025160659000162.tif255165TIFF2025160659000163.tif255165TIFF2025160659000164.tif255166TIFF2025160659000165.tif255167TIFF2025160659000166.tif255166TIFF2025160659000167.tif255165TIFF2025160659000168.tif255166TIFF2025160659000169.tif255165TIFF2025160659000170.tif255165TIFF2025160659000171.tif255166TIFF2025160659000172.tif255165TIFF2025160659000173.tif255163TIFF2025160659000174.tif255166TIFF2025160659000175.tif255167TIFF2025160659000176.tif255166TIFF2025160659000177.tif255164TIFF2025160659000178.tif255165TIFF2025160659000179.tif255165TIFF2025160659000180.tif255167TIFF2025160659000181.tif255168TIFF2025160659000182.tif255166TIFF2025160659000183.tif255166TIFF2025160659000184.tif255167TIFF2025160659000185.tif255167TIFF2025160659000186.tif255166TIFF2025160659000187.tif255168TIFF2025160659000188.tif255165TIFF2025160659000189.tif255167TIFF2025160659000190.tif255167TIFF2025160659000191.tif255167TIFF2025160659000192.tif255166TIFF2025160659000193.tif255166TIFF2025160659000194.tif255165TIFF2025160659000195.tif255167TIFF2025160659000196.tif255166TIFF2025160659000197.tif255168TIFF2025160659000198.tif255166TIFF2025160659000199.tif255166TIFF2025160659000200.tif255167TIFF2025160659000201.tif255167TIFF2025160659000202.tif255167TIFF2025160659000203.tif255167TIFF2025160659000204.tif255168TIFF2025160659000205.tif255167TIFF2025160659000206.tif255167TIFF2025160659000207.tif255167TIFF2025160659000208.tif255167TIFF2025160659000209.tif255166TIFF2025160659000210.tif255167TIFF2025160659000211.tif255168TIFF2025160659000212.tif255167TIFF2025160659000213.tif255168TIFF2025160659000214.tif255169TIFF2025160659000215.tif255169TIFF2025160659000216.tif255167TIFF2025160659000217.tif255167TIFF2025160659000218.tif255168TIFF2025160659000219.tif255166TIFF2025160659000220.tif255167TIFF2025160659000221.tif255168TIFF2025160659000222.tif255167TIFF2025160659000223.tif255168TIFF2025160659000224.tif255167TIFF2025160659000225.tif255167TIFF2025160659000226.tif255167TIFF2025160659000227.tif255166TIFF2025160659000228.tif255166TIFF2025160659000229.tif255166TIFF2025160659000230.tif255167TIFF2025160659000231.tif255165TIFF2025160659000232.tif255167TIFF2025160659000233.tif255167TIFF2025160659000234.tif255165TIFF2025160659000235.tif255167TIFF2025160659000236.tif255167TIFF2025160659000237.tif255165TIFF2025160659000238.tif255166TIFF2025160659000239.tif255169TIFF2025160659000240.tif255167TIFF2025160659000241.tif255168TIFF2025160659000242.tif255167TIFF2025160659000243.tif255166TIFF2025160659000244.tif255165TIFF2025160659000245.tif255169TIFF2025160659000246.tif255168TIFF2025160659000247.tif255168TIFF2025160659000248.tif255167TIFF2025160659000249.tif255168TIFF2025160659000250.tif255169TIFF2025160659000251.tif255167TIFF2025160659000252.tif255167TIFF2025160659000253.tif255167TIFF2025160659000254.tif255168TIFF2025160659000255.tif255168TIFF2025160659000256.tif255168TIFF2025160659000257.tif255168TIFF2025160659000258.tif255168TIFF2025160659000259.tif255167TIFF2025160659000260.tif255168TIFF2025160659000261.tif255167TIFF2025160659000262.tif255168TIFF2025160659000263.tif255169TIFF2025160659000264.tif255168TIFF2025160659000265.tif255167TIFF2025160659000266.tif255168TIFF2025160659000267.tif255170TIFF2025160659000268.tif255165TIFF2025160659000269.tif255168TIFF2025160659000270.tif255167TIFF2025160659000271.tif255166TIFF2025160659000272.tif255169TIFF2025160659000273.tif255166TIFF2025160659000274.tif255168TIFF2025160659000275.tif255168TIFF2025160659000276.tif255167TIFF2025160659000277.tif255167TIFF2025160659000278.tif255166TIFF2025160659000279.tif255168TIFF2025160659000280.tif255170TIFF2025160659000281.tif255168TIFF2025160659000282.tif255167TIFF2025160659000283.tif255168TIFF2025160659000284.tif255168TIFF2025160659000285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[0115] [PPMX-T003-sensitive ANKL cells are characterized by high mTORC1 / Myc / TfR1 activity] Next, we characterized the subpopulation of spleen-resident ANKL1 cells sensitive to PPMX-T003. Spleen-resident ANKL1 cells collected from ANKL1-PDXs before and after PPMX-T003 treatment were subjected to single-cell whole-transcriptome analysis (Fig. 6a). Unsupervised clustering analysis separated the cells into six clusters, suggesting that the population in Cluster 1 decreased after treatment (Fig. 6b-c). GSEA using HALLMARK from MSigDB revealed significantly elevated mTORC1 and Myc activity in cells belonging to Cluster 1 (Fig. 6d; Table 9). Furthermore, the expression levels of MYC and TFRC, a Myc target gene encoding TfR1, were specifically elevated in Cluster 1 (Fig. 7a-b). Cell cycle scoring analysis annotated Cluster 1 as a DNA-replicating cell cluster, which strongly correlated with the previously demonstrated antitumor mechanism of PPMX-T003 (Fig. 7c). These results suggest that the PPMX-T003-sensitive subpopulation of ANKL cells is characterized by high mTOR / Myc / TfR1 activity. This finding is reasonable, as liver-resident ANKL cells (sensitive to PPMX-T003) exhibit high mTOR / Myc / TfR1 activity (Non-Patent Document 2). Furthermore, we investigated the antitumor effect of PPMX-T003 on ANKL-derived cell lines in which mTORC1 was suppressed by sublethal doses of rapamycin. Apoptosis and cell viability assays revealed that the antitumor effect of PPMX-T003 was reduced in cells in which mTORC was suppressed (Figures 7d-e). This result also supports the above findings.

[0116] [Table 9]

[0117] [LAT1-mediated amino acid influx positively regulates ANKL cell proliferation] To identify extracellular factors regulating mTORC1 activity in ANKL cells, we reanalyzed bulk RNA-seq data (deposited in Gene Expression Omnibus; GSE189722) from ANKL1 and ANKL3 cells derived from the livers of healthy volunteers and peripheral NK cells, focusing on metabolic status. GSEA using the Kyoto Encyclopedia of Genes and Genomes and MSigDB-HALLMARK revealed that ANKL cells exhibited high amino acid metabolic activity but relatively low glucose and fatty acid metabolism (Fig. 8a–c). In particular, ANKL cells metabolized sulfur-containing amino acids (Fig. 8d). A pathway view of sulfur-containing amino acid metabolism (NPL 33) revealed that the transcription of genes related to the methionine cycle and polyamine synthesis, known as mTOR stimulators (NPL 34), was specifically upregulated in ANKL cells (Fig. 9a). Furthermore, mRNA expression analysis of solute carrier (SLC) family molecules demonstrated that various amino acid transporters were highly expressed in ANKL cells compared with primary NK cells (Figure 9b). When we examined the dependency of ANKL cells on extracellular amino acids by culturing them in vitro in RPMI-1640 medium lacking a single amino acid, all ANKL cells evaluated (ANKL-PDX-derived cells and cell lines) showed impaired growth (less than 50% growth compared with medium containing all amino acids) when single sulfur-containing amino acids were deficient (cysteine ​​and methionine; Figures 9c–f). These results suggest that ANKL cell proliferation is highly dependent on the influx of sulfur-containing amino acids. Furthermore, the single-cell whole transcriptome analysis data described in Figures 6–7 demonstrated that SLC7A5 and SLC1A5, encoding LAT1 and ASCT2, respectively, were upregulated in the PPMX-T003-sensitive cluster (cluster 1; Figure 10a). LAT1 is a cancer-specific L-type neutral amino acid transporter capable of transporting methionine (Non-Patent Documents 35-36), whereas ASCT2 is known as a cysteine ​​transporter (Non-Patent Document 37). Violin plots showed that SLC7A5 was more specifically expressed in cluster 1 than SLC1A5 (Figure 10b-c).SLC7A5 is annotated as an mTORC1-associated gene in the HALLMARK section of MSigDB (Table 9). In vitro proliferation competition assays using the CRISPR-Cas9 system in NK92 cells revealed that sgSLC7A5-RFP-transfected cells had significantly reduced viability (RFP-positive) compared with non-targeting control sgRNA (sgNT)-RFP-transfected cells, whereas sgSLC1A5-RFP-transfected cells did not (Figure 10d-e). Furthermore, we established ANKL-PDXs using sgSLC7A5-RFP-transfected or sgNT-RFP-transfected ANKL3 cells to assess the contribution of LAT1 to the proliferation of liver-resident ANKL cells (Figure 11a). Seven days after inoculation, in vivo luciferase assays and flow cytometry analysis demonstrated that LAT1-knockout cells exhibited reduced proliferation capacity (Figure 11b-c). These findings indicate that LAT1-mediated amino acid influx is important for controlling cell proliferation through the cell cycle in hepatic sinusoidal ANKL cells.

[0118] [LAT1-mediated amino acid influx is essential for the therapeutic effect of PPMX-T003 through positive regulation of mTOR / Myc activity] The relationship between LAT1 and mTORC / Myc activity and cell sensitivity to PPMX-T003 was examined. Inhibition of LAT1 using JPH-203 (Non-Patent Document 38) resulted in dose-dependent cell growth inhibition accompanied by G1 arrest (Fig. 12a-c). This growth inhibitory effect was observed specifically in tumor cells (Fig. 12b). Western blotting analysis of NK92 and KHYG1 cells treated with JPH-203 revealed downregulation of phosphorylated mTOR and p70S6K, which are major targets of mTORC1 contributing to cell proliferation, as well as Myc and TfR1, within 1 day (Figs. 12d and 13), suggesting that LAT1 positively regulates mTOR / Myc activity and TfR1 expression. Quantitative PCR analysis of ANKL-derived cell lines treated with JPH-203 also demonstrated downregulation of TFRC expression (Fig. 14a-b). Intracellular iron ions (Fe 2+ LAT1 inhibition significantly reduced iron levels without altering iron supplementation from the culture medium (Figure 14c-d), indicating a decrease in cellular iron demand. Additionally, LAT1 inhibition in ANKL-derived cell lines reduced γH2AX expression in response to PPMX-T003 exposure in vitro (Figure 15a) and reduced the antitumor effect of PPMX-T003 on liver-resident ANKL cells in vivo (Figure 15b). These findings suggest that LAT1-mediated regulation of mTOR / Myc activity via amino acid influx is a determinant of iron dependence for survival in ANKL cells and their resulting sensitivity to PPMX-T003. The single-cell transcriptome of liver-resident ANKL cells responding favorably to PPMX-T003 revealed a broader distribution of SLC7A5-expressing cells with active cell cycles than spleen-resident ANKL cells (Figures 6a, 7a, 16a-c), supporting these findings. Furthermore, the in vivo antitumor activity of JPH-203 itself was analyzed using ANKL3-PDX, but no clear therapeutic activity was observed (Fig. 16d).

[0119] LAT1-mediated influx of amino acids abundant in the liver sinusoids promotes cell cycle progression in ANKL cells through activation of mTOR / Myc and stimulates replication-associated DNA damage by PPMX-T003.

[0120] <Consideration> We previously reported that PPMX-T003 is a promising therapeutic agent for ANKL, particularly in the liver (Non-Patent Document 2). Based on this finding, a phase Ib / II clinical trial of PPMX-T003 for ANKL was initiated in April 2023 (jRCT2061230008). To strengthen the evidence of PPMX-T003's high efficacy, in this study we performed a detailed mechanistic analysis of the effects of PPMX-T003. PPMX-T003 induced DNA double-strand breaks in ANKL cells, particularly in the S phase of the cell cycle. Furthermore, our results suggest that LAT1-mediated amino acid influx is essential for the G1-S cell cycle transition of ANKL cells via mTOR / Myc activation, suggesting that abundant extracellular amino acids are an important determinant of the sensitivity of ANKL cells to PPMX-T003.

[0121] Previous studies have reported that the antitumor effects of iron chelators such as deferoxamine and deferasirox are caused by G1 arrest followed by p21-triggered apoptosis (39-49). Therefore, the cell death induced by specific TfR1 blockade via PPMX-T003 in ANKL is unique. Further studies of the mechanisms, including iron sensing, iron metabolism, and intracellular distribution of iron, are needed to clarify the differences between cell cycle regulation by iron chelation and specific TfR1 inhibition.

[0122] Our in vitro data suggest that ANKL cells are highly dependent on extracellular cysteine ​​rather than methionine for proliferation. However, in vivo CRISPR / Cas9 gene knockout analysis revealed that the methionine transporter LAT1 is more important for in vivo proliferation than the cysteine ​​transporter ASCT2. These contradictory data may stem from the existence of alternative cysteine ​​uptake pathways, as several molecules encoded by SLC family genes (e.g., SLC7A11, SLC7A9, and SLC3A1) are known to be cysteine ​​transporters. Further studies are underway to evaluate the dynamics of cysteine ​​in the hepatic sinusoids of ANKL-PDXs. LAT1 is a cancer-specific neutral amino acid transporter that has been widely investigated as a therapeutic target (35, 38, 49). In ANKL, we found that LAT1 plays a key role in excessive cell cycle progression in the hepatic sinusoids via activation of mTOR / Myc and enhances cellular iron requirement, thereby sensitizing cells to PPMX-T003. Therefore, to achieve a favorable therapeutic effect in PPMX-T003 treatment of ANKL, LAT1 activity should be maintained rather than inhibited. Single-cell transcriptome analysis revealed that liver-resident ANKL cells express SLC7A5 (the LAT1-encoding gene) regardless of cell cycle stage, whereas spleen-resident ANKL cells express SLC7A5 specifically during the S phase of the cell cycle, suggesting that LAT1 expression is regulated by some microenvironmental factors. To improve the therapeutic potential of PPMX-T003, it is necessary to elucidate the detailed molecular mechanism underlying the environmental regulation of LAT1 expression and subsequently discover drugs that regulate tumor LAT1 expression. Clinically, LAT1 may be a surrogate marker for the therapeutic efficacy of PPMX-T003, and this will be investigated in ongoing clinical trials. Unfortunately, JPH-203 monotherapy was ineffective against ANKL in our ANKL-PDX, despite the fact that LAT1-knockout ANKL cells were significantly unable to proliferate in vivo in the hepatic sinusoids (Fig. 16d).This may be due to the fact that the alternative function of LAT1 in liver-resident ANKL cells remains unclear, drug delivery problems with JPH203 to the hepatic sinusoids, overexpression of LAT1 due to environmental factors as mentioned above, and / or extremely high neutral amino acid concentrations in sinusoidal plasma. We are currently focusing on these concerns to investigate whether LAT1 may be a novel therapeutic target for ANKL.

[0123] In conclusion, PPMX-T003 induces DNA double-strand breaks in ANKL cells in a manner dependent on DNA replication during the S phase of the cell cycle, and its pharmacological efficacy is determined by LAT1-mediated amino acid influx via mTOR / Myc activation.

[0124] (Patent documents cited in the Detailed Description section) [Patent Document 4] International Publication WO2023 / 027164 [Patent Document 5] International Publication WO2023 / 107082

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Claims

1. Use of L-type amino acid transporter 1 (LAT1) as an indicator for predicting the efficacy of substances that recognize the transferrin receptor (TfR).

2. The use according to claim 1, comprising analyzing the expression of LAT1 or a gene encoding LAT1 in a sample derived from a subject for which a drug efficacy is to be predicted.

3. Use of mTOR as an index for predicting the efficacy of substances that recognize TfR.

4. The use according to claim 3, comprising analyzing mTOR activity in a sample from a subject for which a drug efficacy is to be predicted.

5. The use according to claim 4, wherein the analysis of mTOR activity comprises the analysis of the expression of at least one of the mTORC gene and the Myc gene.

6. The use according to any one of claims 1 to 5, wherein the substance that recognizes TfR is an antibody that inhibits binding between transferrin and TfR.

7. The use according to any one of claims 1 to 5, wherein the substance that recognizes TfR is an antibody that recognizes human TfR.

8. The use according to claim 7, wherein the antibody recognizing human TfR is an antibody that recognizes one or more amino acids at positions 629 to 633 of human TfR, or an antibody that inhibits the binding of other antibodies to one or more amino acids at positions 629 to 633 of human TfR.

9. The use according to claim 7, wherein the antibody has a heavy chain first complementarity determining region (VH CDR1), a heavy chain second complementarity determining region (VH CDR2), and a heavy chain third complementarity determining region (VH CDR3) represented by SEQ ID NOs: 1, 2, and 3, respectively, and a light chain first complementarity determining region (VL CDR1), a light chain second complementarity determining region (VL CDR2), and a light chain third complementarity determining region (VL CDR3) represented by SEQ ID NOs: 4, 5, and 6, respectively.

10. The use according to claim 7, wherein the antibody is an antibody whose heavy chain has SEQ ID NO: 7 and whose light chain has SEQ ID NO:

8.

11. The use according to any one of claims 1 to 5, wherein the medicinal effect is an anti-cancer effect.

12. Analyzing the expression of LAT1 or the expression of a gene encoding LAT1 in a sample from a subject; A method for predicting the efficacy of a substance that recognizes TfR.

13. analyzing mTOR activity in a sample from the subject; A method for predicting the efficacy of a substance that recognizes TfR.

14. The method of claim 12, wherein analyzing the activity of mTOR comprises analyzing the expression of at least one of the mTORC gene and the Myc gene.

15. The method according to any one of claims 12 to 14, wherein the substance that recognizes TfR is an antibody that inhibits the binding of transferrin to TfR.

16. The method according to any one of claims 12 to 14, wherein the substance that recognizes TfR is an antibody that recognizes human TfR.

17. The method according to claim 16, wherein the antibody recognizing human TfR is an antibody that recognizes one or more amino acids at positions 629 to 633 of human TfR, or an antibody that inhibits the binding of other antibodies to one or more amino acids at positions 629 to 633 of human TfR.

18. The method of claim 16, wherein the antibody has VH CDR1, VH CDR2, and VH CDR3 of SEQ ID NOs: 1, 2, and 3, respectively, and VL CDR1, VL CDR2, and VL CDR3 of SEQ ID NOs: 4, 5, and 6, respectively.

19. The method of claim 16, wherein the antibody is an antibody whose heavy chain has SEQ ID NO: 7 and whose light chain has SEQ ID NO:

8.

20. The method according to any one of claims 12 to 14, wherein the medicinal effect is an anti-cancer effect.

21. An agent comprising a substance that recognizes TfR, the agent being intended for administration to a subject for whom the efficacy of the agent is predicted to be effective using LAT1 or mTOR as an index.

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