Methods of treating KRAS mutant cancers

Abstract

To provide methods of treating a KRAS mutant cancer in an individual.SOLUTION: Provided are a method including administering to an individual identified as having a KRAS mutant cancer a therapeutically effective amount of an agent that inhibits cardiotrophin-like cytokine factor 1 (CLCF1)-ciliary neurotrophic factor receptor (CNTFR) signaling, or a composition therefor.SELECTED DRAWING: Figure 2-1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 931,608, filed November 6, 2019. No. 62 / 898,249, filed September 10, 2019. and claims the benefit of US Provisional Patent Application No. 20060122634, filed on May 1, 2006, which applications are incorporated herein by reference in their entirety.

[0002] Statement of government support This invention was made under contract number R01 CA225103 awarded by the National Cancer Institute. This invention was made with government support. The government has certain rights in this invention.

[0003] preface Lung cancer is the leading cause of cancer-related deaths worldwide. The subgroup accounts for 85-90% of cases, and lung adenocarcinoma (LUAD) is the most common NS. It is a histological subtype of CLC. Approximately 30% of LUAD cases have KRAS mutations. However, these patients currently have few targeted treatment options. In LUAD subtypes characterized by alterations in Drug resistance remains a major limitation. Monoclonal antibody-based immunotherapeutic agents are also available. It could dramatically improve the options available and have a significant impact on survival for some patients. Despite these advances, innovative approaches to lung cancer treatment, particularly those based on currently available drugs, remain limited. Continued efforts to address mechanisms of carcinogenesis not targeted by existing drugs There is a significant clinical need.

[0004] Cancers arise and progress within a microenvironment that is itself altered as a result of the tumorigenic process. Stromal cells in contact with cancer cells directly signal to tumor cells, or may act indirectly by recruiting other stromal components to promote tumor progression. An important aspect of this process is the secretion of growth factors and cytokines that stimulate cancer-associated fibrosis. CAFs have distinct characteristics in different tumors and tissues. It is a diverse population of interstitial cells that

[0005] CAFs act as tumor suppressors of cancer cells in vivo by secreting soluble factors that stimulate tumor cell proliferation. Supports the growth of cancer cells (e.g., lung cancer cells). One such soluble factor is cardiotoxin. clopidogrel-like cytokine factor 1 (CLCF1). CLCF1 is structurally related to The interleukin (IL)-6 family of hematopoietic and neuropoietic cytokines (IL-6 , IL-11, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), Oncos It belongs to the family of interstitial cells. The resulting CLCF1 binds to the receptor for this protein, the GPI-anchored CNTF receptor. It is received as a growth signal by tumor cells expressing the CNTFR. When bound to either soluble or non-soluble CNTFR, the signaling β receptor gp130 (transmembrane 1 A heterodimer of the LIF receptor (LIFR) and a 30 kDa glycoprotein is induced. This is due to the activation of intracellular signaling pathways such as the JAK / STAT pathway and the MAPK / ERK pathway. Bring on the Cade.

[0006] The RAS family of genes, including HRAS, KRAS, and NRAS, are involved in human cancers. It is a commonly found oncogene and has a highly similar structure consisting of a chain of 188–189 amino acids. The sequences and structural features of these three proteins are They are highly conserved except for their carboxyl-terminal domains and post-translational lipid modifications. RAS, KRAS, and NRAS are regulated in a similar manner within cells. is a molecular switch in the signaling pathways that regulate cell proliferation, differentiation, and survival in mammalian cells. It encodes a monomeric GTPase that functions as a switch. It constitutively activates RAS. Mutations that cause KRAS are found in 20% to 25% of all human cancers. In its active state, it binds to GTP and has a unique enzymatic activity that cleaves the terminal phosphate of nucleotides. When GTP is converted to GDP, KRAS becomes inactive. The conversion rate is usually slow, but it is activated by the presence of an accessory GTPase-activating protein (GAP). Second, KRAS increases the amount of bound nucleotide (GDP) Binding to guanine nucleotide exchange factors (GEFs) (such as SOS) forces release Upon GTP binding, the switch I region (residues 30-40) and the switch II region are activated. Several residues in the II region (residues 60–70) are involved in the binding of KRAS effector proteins. These switches allow the molecules to adopt conformations that allow for In mammalian cells, endogenous KRAS protein is regulated by GAPs and GEFs. The protein is primarily in the GDP state, and activation is transient. Common oncogenic mutations in interfere with GTP hydrolysis, resulting in the protein being inactive. It remains in the GTP state and continues to transmit signals to effector pathways. The RAS acts as a molecular on / off switch. When turned on, it releases growth factors and It activates proteins required for the propagation of signaling from other receptors, such as c-Raf and PI3K. Member and activate. Summary of the Invention

[0007] Methods of treating KRAS mutant cancer in an individual are provided. In certain embodiments, The method comprises administering to an individual identified as having a KRAS mutant cancer an effective amount of a cardiovascular Trophin-like cytokine factor 1 (CLCF1)-ciliary neurotrophic factor receptor (CNTF R) administering an agent that inhibits signal transduction. KRAS mutant cancers include KRAS mutant non-small cell lung cancer (NSCLC), e.g., KRAS For example, the present disclosure relates to KRAS-mutated lung cancer, such as KRAS-mutated lung adenocarcinoma (LUAD). Kits that find use in practicing the methods are also provided. [Brief explanation of the drawings]

[0008] [Figure 1]Tumor growth in human LUAD is increased by CLCF1 and reduced by CNTFR knockdown. Panel A: 72 hours of CLCF1 treatment increases cell viability after serum starvation of LUAD cell lines A549, H23, and H358 in a concentration-dependent manner compared to untreated controls. Panels B and D: Recombinant human CLCF1 phosphorylates STAT3 (Y705) in A549, H23, and H358 in both a concentration-dependent ([CLCF1] = 10 nM) and (Panels C and D) time-dependent (15 min post-treatment) manner. Panel E: qRT-PCR measurement of CNTFR knockdown by shCNTFR or control shGFP (four biological replicates each). **P<0.01, ***P<0.001 using one-way analysis of variance (ANOVA). Data are presented as mean ± SD. Panel F: Proliferation of A549 after knockdown with the indicated shRNAs. Two-way ANOVA. Panel G: Proliferation rate of LUAD cells after CNTFR knockdown on day 7 (four independent biological replicates with three technical replicates per group). One-way ANOVA. Panel H: Representative photographs of colony formation assays in A549 and H23. Panel I: Quantification of colony number from Panel H. Four independent biological replicates with three technical replicates per group. ***p<0.001 using one-way ANOVA. Data are presented as mean ± SD. Panel J: Representative images of spheres from cells grown in anchorage-independent conditions in A549 and H23. Panel K: Quantification of sphere number (three biological replicates). One-way ANOVA. Panel L: Quantification of tumor volume of A549 xenografts with the indicated shRNAs. *P<0.05 using two-way ANOVA. Data are presented as mean ± SEM. Panel M: Quantification of tumor volume at the final time point in the indicated LUAD cell line xenografts. Whiskers identify maximum and minimum values, boxes indicate the 75th and 25th percentiles, and lines indicate median values. One-way ANOVA. Panel N: Representative hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for phosphohistone H3 (PH3) and cleaved caspase-3 (CC3) in A549 xenografts. Scale bar: 50 μm.Panel O: Quantification of PH3- and CC3-positive foci in A549, H23, and H2009 xenografts. *P<0.05, ***p<0.001 using one-way ANOVA. Data are presented as mean±SEM. [Figure 2] Generation of CNTFR receptor decoys using yeast display. Panel A: i) CNTFR signals through the β receptor, gp130, and LIFR. ii) CLCF1 complexes with CNTFR, activating the β receptor. iii) Soluble CNTFR allows gp130 and LIFR to heterodimerize even in cells lacking CNTFR expression. iv) Engineered soluble CNTFR (eCNTFR), which does not bind to the β receptor, can function as an antagonist. Panel B: Schematic of yeast-displayed CNTFR and overlaid flow cytometry dot plots showing binding of yeast-displayed wtCNTFR to CLCF1-His at 10 nM (cyan) and 0 nM (red). Panel C: Flow cytometry histograms of the initial CNTFR library and intermediate sorted populations compared to wtCNTFR (WT), measuring binding to 0.5 nM CLCF1. Only the gated population of yeast expressing CNTFR is shown. Panel D: Binding curves of affinity-matured yeast-displayed CNTFR mutants with various concentrations of CLCF1, and the measured apparent Kd values. Panel E: Representative overlaid flow cytometry dot plots for sort 2 (red), sort 4 (blue), and sort 6 (orange), showing enrichment of non-LIFR binders. Panel F: Y177H and K178N, isolated from negative screening for LIFR-Fc, additively decrease LIFR-Fc binding. The measured apparent Kd values ​​represent binding affinity for CLCF1. Data are presented as mean (n=3 independent replicates) ± SD. *P<0.05, **P<0.01, ***P<0.001. [Figure 3]Characterization of eCNTFR constructs. Panel A: 3D structure prediction of wtCNTFR (yellow) and eCNTFR (blue) was performed using the Phyre2 server (Protein Homology / analogY Recognition Engine V2.0) and shows the locations of four mutations from affinity maturation (blue), two mutations that reduce LIFR binding (green), and two mutations that reduce gp130 binding (magenta). Insets show the aromatic cluster and conserved residues in CNTFR for cytokine binding (red) and the mutations from affinity maturation (blue). Binding affinities of soluble wtCNTFR and eCNTFR constructs were compared for (Panel B) CLCF1, (Panel C) gp130-Fc and LIFR-Fc, (Panel D) CNTF, and (Panel E) mouse CLCF1. Kd values ​​were calculated as needed. Data are presented as the mean (n = 3 independent replicates) ± SD. ***P < 0.001 compared to the corresponding wtCNTFR construct. Panel F: Competition assay using ELISA to measure the ability of eCNTFR-Fc to block binding between wtCNTFR-Fc and CLCF1-His, LIFR-His, and gp130-His. When LIFR-His and gp130-His were included, CLCF1 (10 nM) was also added to induce complex formation. Panels G and H: eCNTFR-Fc inhibits STAT3 phosphorylation (Y705) in A549 and H23 cells. Panels I and J: eCNTFR-Fc inhibits CLCF1-induced cell survival in serum-starved A549 and H23 cells. Data are presented as mean ± SD (n = 3 independent replicates). **P < 0.01, ***P < 0.001 compared to the corresponding non-eCNTFR-Fc-treated control. [Figure 4]Genotype specificity of eCNTFR-Fc in LUAD. Panel A: Viability of cell lines after treatment with 2.5 μM eCNTFR-Fc (three independent biological replicates with four technical replicates per group). Panel B: Western blot of A549 and H23 treated with serum, CLCF1, eCNTFR-Fc, CLCF1 + eCNTFR-Fc, or eCNTFR-Fc + serum after 24 h of serum starvation. Panel C: Quantification of the Western blot from Panel B. Panel D: Ras-GTP levels assessed by Ras-GTP ELISA in cell lysates from A549 and H23 treated with serum, CLCF1, eCNTFR-Fc, CLCF1 + eCNTFR-Fc, or eCNTFR-Fc + serum after 24 h of serum starvation. Two biological replicates are shown. Data are expressed as the mean (n = 3 independent replicates) ± SD. [Figure 5]Effect of eCNTFR-Fc in a preclinical xenograft model. Panel A: Blood clearance and sequestration of CLCF1 following intraperitoneal (ip) administration of 10 mg / kg eCNTFR-Fc in non-tumor-bearing NOD / SCID / gamma mice. Serum samples were collected post-injection, and unbound CLCF1 was measured by ELISA using eCNTFR-Fc as a capture agent. Vehicle-treated mice were used to determine baseline CLCF1 levels. Panel B: Quantification of tumor volume for A549 xenografts [n=8 tumors excluding PBS (n=6 tumors at final time point)]. *P<0.05, **P<0.01, ***P<0.001, ns=not significant using two-way ANOVA. Panel C: Quantification of tumor volume at final time point for A549 xenografts. Panel D: Waterfall plot showing percent tumor change from baseline for A549 xenografts. Panel E: Tumor volume quantification of the patient-derived xenograft 727 (PDTX727) model (n=16 tumors). Panel F: Quantification of tumor volume at the final time point of PDTX727 and a representative image of a PDTX727 tumor. Scale bar, 10 mm. Unpaired, two-tailed Student's t-test. Panel G: Tumor volume quantification at the final time point of the PDTX model. Panel H: Representative H&E staining from an A549 xenograft and IHC for phospho-histone H3 (PH3) and cleaved caspase-3 (CC3). Scale bar, 50 μm. Panel I: Quantification of PH3- and CC3-positive foci. One-way ANOVA. Panel J: Representative H&E staining from a PDTX xenograft and IHC for PH3 and CC3. Scale bar, 50 μm. Panel K: Quantification of PH3- and CC3-positive foci. Unpaired, two-tailed Student's t-test. Panel L: Representative IHC of phospho-ERK (P-ERK) and phospho-S6RP (P-S6) in A549 xenografts and (Panel M) PDTX. Panel N: Western blot of A549 xenografts. Panel O: Quantification of Western blots. Data are presented as mean ± SEM. [Figure 6]Effect of eCNTFR-Fc in an autotopic, KRAS-inducible genetically engineered mouse model. Panel A: Representative 2D axial microCT (μCT) images of cross sections of mouse lungs at cervical vertebra 8 from KRASG12D / P53f / f(KRAS;P53) mice treated with PBS or eCNTFR-Fc (10 mg / kg) three times a week for 4 weeks (day 28), starting 8 weeks after delivery of 5 × 106 pfu of Cre-expressing adenovirus (day 0). The red outline encircles the heart, and red arrows indicate representative tumor nodules. Panel B: μCT tumor burden quantification using ImageJ software. Arbitrary units (AU). Panel C: Representative H&E images of lungs 28 days after the start of treatment. Scale bar, 1 mm. Panel D: Effect of treatment on tumor burden (%) and (Panel E) tumor foci. ***P<0.001 using an unpaired two-tailed Student's t-test. Data are expressed as mean ± SEM. Panel F: Representative IHC of PH3 and CC3 from a GEM model. Panel G: Quantification of PH3- and CC3-positive foci. ***P<0.001 using an unpaired two-tailed Student's t-test. Panel H: Representative IHC of phospho-ERK (P-ERK), phospho-S6RP (P-S6), and phospho-STAT3 (P-STAT3) 28 days after treatment initiation. Panel I: Kaplan-Meier analysis of survival to ethical endpoint in mice from the same experiment (n=11 mice per group). Log-rank test. Panel J: CLCF1 ELISA performed on patient plasma samples and normal controls. Mutations of interest = KRAS G12C, KRAS G12V, and EGFR mutant / KRAS wt. [Figure 7] Expression of CLCF1 across 40 cancer types. CLCF1 expression is plotted as log2-normalized transcripts per million (TPM) on the x-axis. Data were downloaded from a public repository (TCGA). Figures are plotted as mean-ranked log2(TPM+1). Blue line indicates the 75% quantile of CLCF1 expression across all samples. Abbreviations: The Cancer Genome Atlas, TCGA. DETAILED DESCRIPTION OF THE INVENTION

[0009] Before describing the methods and kits of the present disclosure in more detail, it is important to note that the methods and kits are not intended to be limiting unless otherwise specified. The present invention is not limited to the specific embodiments set forth herein, and as such may, of course, vary. It should be understood that the scope of the methods and kits is limited to the appended claims. The terminology used herein is intended to describe specific embodiments only and is limited by the terms and conditions set forth herein. It should also be understood that the present disclosure is for illustrative purposes only and is not intended to be limiting.

[0010] When a range of values ​​is provided, the value between the upper and lower limits of the range shall be Each intervening value to the tenth of the lowest unit unless specifically indicated, and its stated range It is understood that any other stated or intervening value within the range is encompassed by the methods and kits. The upper and lower limits of these smaller ranges are independently included within the smaller ranges. and subject to any specifically excluded limits within the stated ranges. Where a stated range includes one or both of the limits, Ranges excluding either or both of the inclusive limits are also included in the methods and kits. .

[0011] Certain ranges are presented herein with the numerical values ​​preceded by the term "about." A term refers to the exact number it precedes, and to any number that is close or Used herein to provide literal support for approximate numbers. In determining whether a particular stated number is close or approximate, The number that is mentioned but not stated is the number that is specifically stated (in the text where it is presented). It may be a number that provides a substantial equivalent (in the context).

[0012] Unless otherwise defined, all technical and scientific terms used herein are defined by the The same meaning as commonly understood by a person skilled in the art to which the method and kit pertain. Any methods and kits similar or equivalent to those described herein may be used by the present invention. Representative exemplary methods and kits that can be used to practice or test the methods and kits include: The methods and kits are described here.

[0013] All publications and patents cited herein are to be construed as being incorporated by reference in their entirety. All such claims are hereby incorporated by reference as if specifically and individually indicated to be incorporated by reference. The methods and / or materials in connection with which the publications are incorporated and cited are disclosed or omitted. The disclosures of which are incorporated herein by reference are for illustrative purposes only. The disclosure is for the purpose of this document, and the publication date provided may differ from the actual publication date. Any disclosures or information contained herein that are not accurate or complete may be necessary to support the disclosure of such information. This should not be construed as an admission that the person does not have the right to

[0014] As used in this specification and the appended claims, the singular forms "a," "an," and "an" are used interchangeably. and "the" are intended to include plural referents unless the context clearly dictates otherwise. It is understood that the claims may be drafted to exclude any optional element. Thus, this statement is intended to be used "exclusively" in connection with the recitation of claim elements. Literal justification for using exclusive language such as "only" or for using "negative" limitations It is intended to serve as a

[0015] Certain of the methods and kits are described in the context of separate embodiments for clarity. It will be appreciated that the above features may also be provided in combination in a single embodiment. Various features of the methods and kits are described in the context of a single embodiment for brevity. The features may be provided separately or in any suitable subcombination. All combinations are specifically encompassed by this disclosure and any combinations thereof are permissible. Insofar as it encompasses any process and / or composition that can be used in the present invention, the entirety of each combination is included. All of the compounds disclosed herein are herein disclosed as if they were individually and explicitly disclosed. All subcombinations listed in the embodiments describing such variables are also possible. Each such subcombination is also specifically encompassed by the methods and kits of the present invention. Each and every application is disclosed herein to the same extent as if each application were individually and explicitly disclosed.

[0016] As will be apparent to those skilled in the art upon reading this disclosure, the individual components described and illustrated herein may be Each of the embodiments may be combined with any of the other embodiments without departing from the scope or spirit of the disclosed methods. Individual features that can be easily separated from or combined with any of the features of the embodiments of Any recited method has elements and features. or in any other order which is logically possible.

[0017] method The present disclosure provides methods of treating KRAS mutant cancer in an individual. In embodiments, the method comprises administering to an individual identified as having a KRAS mutant cancer an effective amount of: Cardiotrophin-like cytokine factor 1 (CLCF1)-ciliary neurotrophic factor receptor ( The present disclosure relates to administering an agent that inhibits KRAS (Knockout Receptor Binding Factor) signaling. Targeting the CLCF1-CNTFR signaling axis in atypical cancers is a significant antitumor agent Based in part on the surprising discovery, demonstrated for the first time herein, that Details regarding embodiments of the disclosed methods will now be described.

[0018] KRAS-mutated cancer As summarized above, the agent is administered to an individual identified as having a KRAS mutant cancer. "KRAS mutant cancer" refers to cancers whose initiation and / or maintenance is due to the KRAS (human :UniProtKB-P01116) In certain embodiments, one or more KR AS mutations constitutively activate KRAS in KRAS-mutant cancer cells, followed by and its downstream Raf / MEK / ERK1 / 2 and / or PI3K / PIP3 / AK Activates the T survival pathway.

[0019] As used herein, "cancer" includes one or more cancer cells, and "cancer cells" include By neoplastic cell phenotype, we mean cells that exhibit a neoplastic cell phenotype, which includes, for example, abnormal cell growth, abnormal cellular growth, Loss of cell proliferation, density-dependent growth inhibition, anchorage-independent growth capacity, and immunocompromised nonhuman the ability to promote tumor growth and / or development in a human animal model, and / or The cells may be characterized by one or more suitable indicators of cell transformation. "Cancer cell" is used interchangeably with "tumor cell," "malignant cell," or "cancerous cell." These include cancer cells of solid tumors, semi-solid tumors, primary tumors, metastatic tumors, and the like.

[0020] In certain embodiments, the individual has a solid tumor, a semi-solid tumor, a primary tumor, a metastatic tumor According to some embodiments, the individual has a KRAS mutant cancer characterized by the presence of The body is prone to breast cancer, glioblastoma, neuroblastoma, head and neck cancer, stomach cancer, ovarian cancer, and skin cancer. cancer (e.g., basal cell carcinoma, melanoma, etc.), lung cancer, colorectal cancer, prostate cancer, Glioma, bladder cancer, endometrial cancer, kidney cancer, leukemia (e.g., T-cell acute lymphoblastic leukemia (T-ALL, acute myeloid leukemia (AML), etc.), liver cancer (e.g., primary or hepatocellular carcinoma (HCC), such as recurrent HCC, B-cell malignancies (e.g., non-Hodgkin's Lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, etc.), pancreatic cancer, thyroid cancer, and KRAS mutant cancers selected from any combination of these and any of their subtypes In certain embodiments, the individual's KRAS mutant cancer is human pancreatic ductal adenocarcinoma ( PDAC), non-small cell lung cancer, colorectal cancer, and / or cholangiocarcinoma. In any embodiment, the KRAS mutant cancer is characterized by the presence of CLCF1 in the tumor microenvironment. Non-limiting examples of cancers exhibiting CLCF1 expression are shown in Figure 7. show.

[0021] In certain embodiments, the KRAS mutant cancer is KRAS mutant lung cancer. Non-limiting examples of KRAS mutant lung cancers that can be treated according to the methods of the present disclosure include KRAS KRAS mutant small cell lung cancer (SCLC) and KRAS mutant non-small cell lung cancer (NSCLC) If the individual has KRAS mutant NSCLC, in some embodiments, the individual The patient has KRAS-mutated lung adenocarcinoma (LUAD).

[0022] According to some embodiments, the KRAS mutant cancer is KRAS mutant pancreatic cancer. Non-limiting examples of KRAS mutant pancreatic cancers that can be treated according to the methods of the present disclosure include KRAS This includes AS-mutated human pancreatic ductal adenocarcinoma (PDAC).

[0023] KRAS-mutant cancers are characterized by one or more of a variety of KRAS mutations. Non-limiting examples of KRAS mutations include: These include insertions, deletions, mutations that induce one or more amino acid substitutions, etc. Therefore, KRAS mutant cancers are characterized by a single gene encoding human KRAS (UniProtKB-P01116). The amino acid sequences of the nucleotides of the nucleotide ... In certain embodiments, the KRAS mutant cancer is a cancer of human KRAS 12, 13, 61 , 117, and 146. Examples include KRA S-mutant cancers may contain one or more of the following amino acid substitutions in human KRAS: :G12A, G12C, G12D, G12R, G12S, G12V, G13D, Q61H , Q61K, K117N, and A146T. According to some embodiments, the KRAS mutation Atypical cancers contain substitutions at position 12 of KRAS. KRAS-mutant cancers contain substitutions at position 12. In this case, KRAS mutant cancers are G12A, G12C, G12D, G12R, G12S, and and G12V (in this context). As used herein, "consisting of" means that the amino acid substitution is the only KRAS mutation in KRAS-mutated cancer. If the KRAS mutant cancer contains a substitution at position 12, then the KRAS AS mutant cancers were selected from G12A, G12C, G12D, G12S, and G12V. In certain embodiments, the amino acid sequence may comprise or consist of an amino acid substitution KRAS mutant cancers contain or consist of the amino acid substitution G12A. In this embodiment, the KRAS mutant cancer comprises or includes the amino acid substitution G12C. According to some embodiments, the KRAS mutant cancer comprises an amino acid substitution G12 In certain embodiments, the KRAS mutant cancer comprises or consists of , comprising or consisting of the amino acid substitution G12R. KRAS mutant cancers contain or consist of the amino acid substitution G12S. In this embodiment, the KRAS mutant cancer comprises or includes the amino acid substitution G12V. It consists of: [Table 1]

[0024] As used herein, a drug is administered to an individual who has been "identified" as having a KRAS mutant cancer. The administration of the drug means that the individual has KRAS mutant cancer or a subtype thereof. Pre-administration knowledge, for example, whether the individual's cancer is G12A, G12C, G12D, G12R, or G1 2S, G12V, G13D, Q61H, Q61K, K117N, or A146T, etc. KRAS mutant cancers containing or consisting of an amino acid substitution at position 12 of KRAS This means that an agent is administered to an individual based at least in part on the knowledge that the agent will

[0025] In certain embodiments, the methods of the present disclosure provide for the treatment of a patient identified as having KRAS mutant cancer. Identifying an individual as having a KRAS mutant cancer further includes identifying the individual. , e.g., a report indicating that an individual's cancer is a KRAS mutant cancer or a subtype thereof For example, if an individual's cancer is G12A, G12C, or G12 D, G12R, G12S, G12V, G13D, Q61H, Q61K, K117N, and is a KRAS gene that contains or consists of an amino acid substitution at position 12 of KRAS, such as A146T. This may include receiving and reviewing a report indicating an AS-mutated cancer. According to embodiments, identifying an individual as having KRAS mutant cancer includes determining whether the individual has: determining that the individual's cancer is a KRAS mutant cancer; Various approaches can be taken to determine whether , assaying a cancer biopsy sample for one or more KRAS mutations. Suitable assays include the detection of the gene or mRNA encoding KRAS in an individual's cancer cells. Sequencing the transcripts (e.g., Illumina, Oxford Nanopo Technologies, Pacific Biosciences, etc. (using readily available nucleic acid sequencing systems), KR for one or more mutations of interest Mutation-specific amplification primers were used to interrogate the gene or mRNA transcript encoding AS. PCR is performed using a PCR product that specifically binds to one or more specific mutant KRAS proteins. using an antibody-based assay using one or more antibodies that combine Any other suitable method for determining whether an individual's cancer contains one or more KRAS mutations. Assays include, but are not limited to:

[0026] In certain embodiments, the agent is administered to a patient with a particular type of KRAS mutant cancer. For example, according to some embodiments, the drug is administered to KR Only in individuals identified as having KRAS mutant cancers containing an amino acid substitution at position 12 of AS In certain embodiments, the drug is administered The agent is selected from the group consisting of G12A, G12C, G12D, G12S, and G12V. The drug will be administered only to individuals identified as having a KRAS-mutated cancer containing the amino acid substitution identified. According to some embodiments, the agent is selected from the group consisting of G12A, G12C, G12D, G12S, and and G12V. and administering the drug to individuals identified as such, and administering the drug to individuals who have plasma CLCF1 concentrations above a threshold plasma CLCF1 concentration. As used herein, the term "anticoagulant" refers to a drug administered to a subject only if the subject is identified as having an LCF1 concentration. "Administered only to" means that the individual meets certain criteria, e.g., the type of KRAS mutation(s). This means that the drug will not be administered to an individual unless certain criteria are met, such as the patient's blood pressure, plasma CLCF1 concentration, etc. do.

[0027] Individuals with KRAS mutant cancer can vary. In certain embodiments, The individual is a "mammal" or "mammalian," and these terms apply to animals of the order Carnivora (e.g., dogs). and cats), rodents (e.g., mice, guinea pigs, and rats), and primates To describe organisms within the class Mammalia, including (e.g., humans, chimpanzees, and monkeys) In some embodiments, the individual is a human. In some embodiments, the individual is treated with an animal model (e.g., a mouse model) of cancer, e.g., a KRAS mutant cancer. models, primate models, etc.).

[0028] Drugs Drugs administered to individuals identified as having KRAS mutant cancers are intended to treat cardiotrophic Ciliary neurotrophic factor receptor (CNTFR) signaling The agent can be any agent that inhibits (eg, reduces or blocks) neural transmission. Drugs that can be used include small molecules, protein-based drugs (e.g., peptides, Antibodies, engineered ligands, engineered receptors, etc. Drugs include, for example, The agent may be detectably labeled, such as with an in vivo imaging agent. For example, it may be further conjugated to polyethylene glycol (PEG). Fusion to a domain (or fragment thereof), conjugation to PEG, etc., can be used to, for example, deliver to a subject It may find utility to increase the serum half-life of the drug upon administration.

[0029] "Small molecule" means a compound with a molecular weight of 1000 atomic mass units (amu) or less. In some embodiments, the small molecule is 750 amu or less, 500 amu or less, 400 amu or less, amu or less, 300 amu or less, or 200 amu or less.

[0030] According to some embodiments, the agent is an antibody. The term "antibody" refers to an antibody or immunoglobulin of any isotype (e.g., IgG (e.g., For example, IgG1, IgG2, IgG3, or IgG4), IgE, IgD, IgA, I gM, etc.), whole antibodies (e.g., antibodies composed of a tetramer, which further comprise heavy chains and single-chain antibody; Fv, single-chain F v(scFv), Fab, F(ab')2, Fab', (scFv')2, and dia. Fragments of antibodies that retain specific binding to a target, including but not limited to, antibodies (e.g., antibodies containing IgG, ... For example, whole antibodies or single-chain antibody fragments; chimeric antibodies; monoclonal antibodies, human antibodies, Humanized antibodies (e.g., humanized whole antibodies, humanized antibody fragments, etc.), as well as antigen-binding portions of antibodies and non-antibody proteins or fragments thereof (e.g., antibody Fc regions or fragments thereof). This includes fusion proteins containing

[0031] Drugs that bind to CNTFR In certain embodiments, the method specifically binds to CNTFR and inhibits CNTFR-mediated signal transduction. Such agents include administering drugs that inhibit signal transduction. molecules, antibodies, CNTFR ligands (e.g., engineered CNTFR ligands), etc. Non-limiting examples of such agents include those that specifically bind to CNTFR and bind to CNTFR and its and ligands such as CLCF1, CNTF, and NP. CNTFR is a neurotransmitter that binds to ciliary neurotrophic factor (CNTF) and cardiotrophin-like Cytokine receptor factor 1 (CLCF1) and other ligands such as neuropoietin (NP) The ligand-specific component of the three-component receptor for CNTFR. Binding occurs via the transmembrane components of the receptor, gp130 and the leukemia inhibitory factor receptor (LIFR). Human CNTFR, CNTF, CLCF1, and N The wild-type amino acid sequence of P is shown in Table 2. [Table 2]

[0032] According to some embodiments, the agent is a CNTFR or a ligand-CNTFR complex. It specifically binds to the subunit (e.g., gp130 or LIFR) and CNTFR. Inhibits the interaction between the ligand and the CNTFR complex subunit.

[0033] In certain embodiments, the agent is an engineered CNTFR ligand. As used herein, an "engineered CNTFR ligand" refers to a polypeptide that binds to CNTFR. a peptide, A "mutant" is an engineered variant of a wild-type CNTFR ligand, such as a P ligand. The selected CNTFR ligand exhibits one or more of the following properties compared to the corresponding wild-type CNTFR ligand: For example, an engineered CNTF ligand may have a mutation similar to wild-type CNTF. The CLCF1 ligands of the present disclosure may contain one or more mutations compared to wild-type CLCF1. As used throughout this disclosure, "mutation" refers to a sequence that is a subset of a sequence that may contain one or more mutations compared to the sequence. "Mutation" or "mutations" refers to the mutations that occur in the corresponding wild-type polypeptide. one or more amino acid substitutions in the polypeptide compared to the polypeptide, an amino acid deletion (e.g., truncation), one or more amino acid insertions, or any of these. may include a combination of:

[0034] According to some embodiments, when the agent is an engineered CNTFR ligand, the agent increased binding affinity for CNTFR compared to the corresponding wild-type CNTFR ligand In certain embodiments, the agent is an engineered CNTFR ligand that exhibits , an engineered CNTFR ligand, the agent may be a corresponding wild-type CNTFR ligand. The engineered CNTF compared its binding affinity to the CNTFR- and CNTFR-containing complexes. gp130, LIFR, or both for complexes containing R ligands and CNTFR These are engineered CNTFR ligands that result in decreased binding affinity for one of the two. According to an embodiment, when the agent is an engineered CNTFR ligand, the agent is a corresponding exhibits increased binding affinity for CNTFR compared to a wild-type CNTFR ligand; and , binding affinity to the corresponding wild-type CNTFR ligand and complexes containing CNTFR g for the engineered CNTFR ligand and CNTFR-containing complexes compared to Engineered CNTs resulting in reduced binding affinity of p130, LIFR, or both FR ligand. "Increased binding affinity" or "higher binding affinity" refers to the The CNTFR ligands bind more tightly to the CNTFR than the corresponding wild-type CNTFR ligands. Close bonding (lower K D (as indicated by the value). For example, In certain embodiments, when the CNTFR ligand is a mutant CLCF1 ligand The binding affinity of the CLCF1 ligand to CNTFR is 20 nM or less K D Has value do.

[0035] As used herein, the first molecule is, for example, about 10 5 M -1 or greater affinity or K a (i.e., the equilibrium association constant for a particular binding interaction in units of 1 / M) to the second molecule A first molecule "specifically binds" to a second molecule if they bind or associate. In this embodiment, the first molecule is about 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 1 0 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 , or 10 13 M -1 More than K a "High affinity" binding is defined as binding to a second molecule with a 7 M -1 , small At least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least Also 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 , or it K exceeds a Alternatively, affinity refers to the binding of a specific binding interaction in units of M. The equilibrium dissociation constant (K D ) (e.g., 10 -5 M~10 -13 M or less) In certain embodiments, specific binding can be defined as about 10-5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, or about 10 -9 M, 10 -10 M, 1 0 -11 M or 10 -12 K below M D This means that the target molecule binds to the target molecule. The binding affinity of the first molecule to the target can be determined using conventional techniques, e.g., competitive ELISA. (enzyme-linked immunosorbent assay), equilibrium dialysis, surface plasmon resonance (SPR) technology (e.g., BIAcore2000 using the general procedures outlined by the manufacturer) It can be easily determined by the use of immunoassay equipment, radioimmunoassay, etc. do.

[0036] In certain embodiments, CNTF ligands are more potent than the corresponding wild-type CNTFR ligands. CNTFR ligands that exhibit increased binding affinity to R are more sensitive to CLCF1 ligands ("mutants"). Some experiments have shown that CLCF1 is a variant of CLCF1, which may be referred to as "variant CLCF1" or "engineered CLCF1." In embodiments, such CLCF1 ligands comprise a nucleotide sequence at amino acid positions 86, 96, 148, 16 9, 180, or any combination thereof, wherein The sequence is the same as SEQ ID NO: 3. By way of example, such a CLCF1 ligand may be SEQ ID NO: 3 Compared with the CLCF1 ligand having the amino acid sequence shown in Selected from H148R, W169L, K180R, and any combination thereof CLCF1 mutations that exhibit increased binding affinity to CNTFR may be present. Non-limiting examples of variants, as well as strategies for identifying additional such variants, are provided in USS N16 / 465,726, the disclosure of which is incorporated in its entirety for all purposes. is incorporated herein by reference.

[0037] In some embodiments, the CNTFR ligand is a CNTFR ligand and CNTF This results in a decrease in the binding affinity of gp130 to complexes containing R. In this case, such a ligand may be located at amino acid positions 22, 169, 180, or any of these. wherein the numbering is the sequence As an example, such a CLCF1 ligand is the same as SEQ ID NO: 3. Compared with the CLCF1 ligand with the amino acid sequence, Y22C, W169L, K180 R, and any combination thereof. This resulted in a decrease in the binding affinity of gp130 to the F1 mutant and CNTFR-containing complexes. Non-limiting examples of CLCF1 variants, as well as methods for identifying additional such variants The strategy is described in USSN 16 / 465,726, the disclosure of which is hereby incorporated by reference for all purposes. and is hereby incorporated by reference in its entirety for the purposes of this application.

[0038] In some embodiments, in direct binding assays, fluorophores or radioactive Isotope-conjugated CNTFR ligands, gp130, or LIFR, or CNTs containing N- or C-terminal epitope tags for detection by labeled antibodies The equilibrium binding constant (K D ) is measured If labeling or tagging is not feasible or desired, competitive binding may be used. Unlabeled CNTs that allow detection of 50% of the maximum signal of the labeled competitor using the assay The half-maximal inhibitory concentration (IC) is the amount of LIFR ligand, gp130, or LIFR. 50 )of The measured IC 50 Value to K D The value can be calculated do.

[0039] The amino acid sequences of two non-limiting examples of CNTFR ligands of the present disclosure are provided below in Table 3. It is served. [Table 3]

[0040] Exemplary CNTFR ligands in Table 3 are engineered CLCF1 mutants. The variant exhibits increased binding affinity for CNTFR compared to wild-type CLCF1. The 2 mutant also inhibited the binding of gp130 and CNTFR to complexes containing the mutant and CNTFR. In some embodiments, the CNTFR ligand results in a decrease in the binding affinity of the CNTFR ligand to the LIFR. The ligands are CNTFR ligands shown in Table 3. In some embodiments, such ligands CNTFR ligands can be fusion proteins (e.g., fused to an Fc domain), conjugates (e.g., conjugated to PEG, drugs, etc.), or It exists in the combination of

[0041] In certain embodiments, the CNTFR ligands of the present disclosure bind to CNTFR and For the CNTFR ligands shown in Table 3, the activity was 70% or more, 75% or more, 80% or more, and 85% or more. % or more, 90% or more, 95% or more, 99% or more, or 100% amino acid sequence identity In some embodiments, such a CNTFR ligand is a fusion protein ( e.g., fused to an Fc domain), conjugates (e.g., PEG, drugs, etc. conjugated to), or combinations thereof.

[0042] In certain embodiments, the CNTFR ligand is a CLCF1 variant that binds to CNTFR. The variants are selected from L86F, Q96R, H148R, and any combination thereof. wherein the CLCF1 variant comprises an amino acid substitution selected from the amino acid sequences set forth in SEQ ID NO:6. 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more of the acid sequence In some embodiments, the amino acid sequence identity may be greater than or equal to 99%, greater than or equal to 100%, or both. Such CNTFR ligands may be fused to fusion proteins (e.g., Fc domains). conjugates (e.g., conjugated to PEG, drugs, etc.) , or a combination thereof.

[0043] In certain embodiments, the CNTFR ligand is a CLCF1 variant that binds to CNTFR. The variants are Y22C, L86F, Q96R, H148R, F151A, K154A, and W 169L, K180R, and any combination thereof. wherein the CLCF1 variant has a sequence similar to that of SEQ ID NO:7 but is at least 70% similar to that of the amino acid sequence set forth in SEQ ID NO:7. Above, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or contains 100% amino acid sequence identity. In some embodiments, such a CNTF R ligands can be fused proteins (e.g., fused to an Fc domain), conjugates, or other suitable proteins. conjugated to PEG, drugs, etc.), or combinations thereof It exists in the mix.

[0044] Drugs that bind to CLCF1 In certain embodiments, the agent that inhibits CLCF1-CNTFR signaling is It is a drug that specifically binds to CLCF1 and inhibits signal transduction via CNTFR. Such agents may be, for example, small molecules, antibodies, or CLCF1 receptor (e.g., engineered Non-limiting examples of such agents include CLCF1 receptors, soluble CLCF1 receptors, and the like. 1 and inhibits the interaction between CLCF1 and CNTFR. .

[0045] According to some embodiments, the agent that specifically binds to CLCF1 is a soluble CNTF A "soluble CNTFR polypeptide" is a CNTFR polypeptide that is integrated into the cell membrane. The wild-type human CNTFR amino acid sequence (Uni ProtKB-P26992) are provided in Table 4 below. [Table 4]

[0046] According to certain embodiments, the soluble CNTFR polypeptide comprises a soluble CNTFR polypeptide. Polypeptides with one or more mutations prevent them from being incorporated into cell membranes. The one or more mutations can be located in any suitable region(s) of the CNTFR polypeptide. In certain embodiments, the soluble CNTFR polypeptide expresses wild-type CNTFR. It contains one or more mutations in the domain that anchors it to the vacuole membrane that confer solubility. is a glycosylphosphatidylinositol (GPI) that anchors proteins to the cell membrane. It contains a lipidation site (S342) that is post-translationally modified. It anchors CNTFR to the cell membrane. The native human CNTFR domain can be defined as consisting of amino acids 343 to 372. , where the numbering is the same as SEQ ID NO: 8 (shown as underlined in Table 4). Under these conditions, this portion of CNTFR is enzymatically released to release CNTFR from the cell membrane. According to some embodiments, the soluble CNTFR polypeptide of the present disclosure is modified. Contains a substitution mutation at S342 that prevents post-translational modification by GPI, thereby conferring solubility Wild-type human CNTFR also contains a signal peptide consisting of amino acids 1 to 22 of SEQ ID NO:8. peptides (underlined in Table 4).

[0047] According to certain embodiments, the CNTFR domain that anchors the CNTFR to the cell membrane is One or more amino acid substitutions that eliminate the ability of the CNTFR polypeptide to be anchored to the cell membrane Alternatively or additionally, the soluble C The NTFR polypeptide abolishes the ability of the CNTFR polypeptide to be anchored to the cell membrane. It may also include truncations that anchor the CNTFR to the cell membrane (e.g., domain), thereby conferring solubility. The CNTFR polypeptide lacks the CNTFR domain that anchors CNTFR to the cell membrane. For example, a soluble CNTFR polypeptide may be a polypeptide comprising amino acids 343 to 349 of SEQ ID NO: 8. 372 may be missing.

[0048] In addition to optionally containing one or more mutations that confer solubility, the soluble CNTF of the present disclosure The R polypeptide may comprise one or more polypeptides that confer one or more other desirable properties to the polypeptide. Other desirable properties of interest include the ability to differentiate wild-type CNTF receptors, e.g., For example, CLC is expressed as a receptor having the amino acid sequence set forth in SEQ ID NO: 8 or a mature form thereof. Higher binding affinity for F1, CLCF compared to one or more other CNTFR ligands 1, modified (e.g., higher) specificity for the ligand-CNTFR complex subunit modified (e.g., reduced) to the target (e.g., gp130, LIFR, etc.) These include, but are not limited to, binding affinity.

[0049] "Higher binding affinity" or "increased binding affinity" refers to the increased binding affinity of a soluble CNTFR polypeptide. Tighter binding (lower binding) of CNTF to CLCF1 compared to wild-type CNTF receptor IK D "Lower binding affinity" or "lower binding affinity" means exhibiting a higher binding affinity (as indicated by a higher binding affinity value). or "reduced binding affinity" means that a soluble CNTFR polypeptide has a reduced binding affinity to wild-type CNTF. Compared to the receptor, a molecule (e.g., a receptor such as LIFR, gp130, or both) less tightly bound (higher K D is meant to indicate the

[0050] A molecule of interest, e.g., a ligand-C such as CLCF1 or LIFR, gp130, etc. CNTFR ligand binding agents (e.g., soluble CNTFR ligands) for the CNTFR complex subunits Methods for measuring the binding affinity of TFR polypeptides are available. For example, see Table Surface plasmon resonance (SPR) technology (e.g., using a BIAcore™ 2000 instrument) KinExA® Kinetic Exclusion Assay (Sapidyne Instruments) ments), biolayer interferometry (BLI) technology (e.g., ForteBio Oc tet®), or other similar assays / techniques, to identify CNTFR ligand binding agents. In the context of this disclosure, Suitable techniques for measuring binding affinity are described, for example, in Hunter, SA and Cochran, JR (2016) Methods Enzymol.580:21 -44.

[0051] In some embodiments, in direct binding assays, fluorophores or radioactive CNTFR polypeptide conjugated to an isotope or for detection with a labeled antibody CNTFR polypeptides containing N- or C-terminal epitope tags for Equilibrium binding constant (K D ) can be measured. If labels or tags are not feasible or If quantification is not desired, use a competitive binding assay to measure the amount of quantification that produces 50% of the maximum signal of the labeled competitor. The half-maximal inhibitory concentration (IC) is the amount of unlabeled CNTFR polypeptide that becomes detectable. 50 ) can then be determined. 50 Value to K D The value can be calculated Cut.

[0052] As summarized above, in certain embodiments, the soluble CNTFR polypeptides of the present disclosure The CNTF receptor is a wild-type CNTF receptor, e.g., a receptor having the amino acid sequence set forth in SEQ ID NO:8. The potential for CLCF1-CNTFR complex subunits compared with the intact form or its mature form one or more that alter (e.g., decrease) the binding affinity of a soluble CNTFR polypeptide "CLCF1-CNTFR complex subunit" refers to a subunit of the CLCF1-CNTFR complex in which CNTFR is a CL Ligand-C refers to a protein that associates with wild-type CNTFR when it binds to CF1. Non-limiting examples of NTFR complex subunits include LIFR and gp130. In certain embodiments, the one or more mutations are in LIFR, gp130, or a One or more mutations that decrease the binding affinity of the soluble CNTFR polypeptide to both The soluble CNTFR polypeptide acts as an agonist when bound to CLCF1. This may prevent CNTFR-mediated signaling from occurring, thereby reducing CNTFR-mediated signaling (e.g., reducing cell proliferation). (It can be done).

[0053] In some embodiments, the soluble CNTFR polypeptide is a CLCF1-CNTFR complex. Soluble CNTFR polypeptides exhibit reduced binding affinity to the combined subunits. The binding affinity of the dextromethorphan derivatives was ≥100 nM in the presence of 10 nM CLCF1. D It has a value.

[0054] In certain embodiments, the soluble CNTFR polypeptide has a reduced ability to bind to LIFR. a CNTFR polypeptide having an amino acid sequence set forth in SEQ ID NO:8, which has a binding affinity to the CNTFR polypeptide; mutations at amino acid positions 177, 178, or both (e.g., amino acid An exemplary mutation at position 177 is Y177H. Another exemplary mutation at position 177 is Y177H. An exemplary mutation at position 178 is Y177A. An exemplary mutation at position 178 is K178N. A typical mutation is K178A. Such mutations are inhibitors of CNTFR signaling. CLCF1-CNTFR complex, whereas soluble CNTFR polypeptide Soluble CNTFR polypeptides with unchanged affinity for the subunits are CLC agonists by its ability to recruit, for example, LIFR and gp130 upon binding to F1. In certain embodiments, the soluble CNTFR polypeptides of the present disclosure has the mutations Y177H and K178N, or the mutations Y177A and K178A, or Contains the mutations Y177H and K178A, or the mutations Y177A and K178N.

[0055] According to certain embodiments, the soluble CNTFR polypeptide is set forth in SEQ ID NO:8. Decreased binding to gp130 compared to a CNTFR polypeptide having the amino acid sequence have good binding affinity and have mutations at amino acid positions 268, 269, or both (e.g., An exemplary mutation at position 268 is T268A. An exemplary mutation at position 269 is T268A. The mutation is D269A. In certain embodiments, the soluble CNTFR polypeptide of the present disclosure Tide contains the mutations T268A and D269A.

[0056] As summarized above, soluble CNTFR polypeptides are similar to wild-type CNTF receptors, e.g., For example, C is a receptor having the amino acid sequence set forth in SEQ ID NO: 8 or its mature form. Altering the binding affinity and / or specificity of a soluble CNTFR polypeptide for LCF1. According to certain embodiments, the present invention may include one or more mutations that enhance (e.g., increase) For example, if a soluble CNTFR polypeptide exhibits increased binding affinity for CLCF1 The binding affinity of the soluble CNTFR polypeptide to CLCF1 is 10 nM or less K DIt has a value.

[0057] In some embodiments, the soluble CNTFR polypeptide exhibits binding to CLCF1. It contains one or more mutations that increase affinity and / or specificity. Such a soluble CNTFR polypeptide has the amino acid sequence set forth in SEQ ID NO:8. Compared to the corresponding CNTFR polypeptide, amino acid positions 110, 174, 237, and 287 or any combination thereof. An exemplary mutation is R110Q. An exemplary mutation at position 174 is T174P. 237 An exemplary mutation at position 237 is S237F. Another exemplary mutation at position 237 is S237Y. An exemplary mutation at position 287 is I287F. In certain embodiments, the soluble CNT The FR polypeptide contains the mutations R110Q, T174P, S237F / S237Y, and I 287F or any combination thereof (e.g., each).

[0058] In some embodiments, the soluble CNTFR polypeptide is an amino acid sequence set forth in SEQ ID NO:8. Compared to the CNTFR polypeptide having the amino acid sequence 77, 178, 237, 268, 269, 287, or any combination thereof. It includes variations (e.g., amino acid substitutions).

[0059] In certain embodiments, the soluble CNTFR polypeptide has the mutations R110Q, T17 4P, Y177H / Y177A, K178N / K178A, S237F / S237Y, T 268A, D269A, and I287F, or any combination thereof (e.g., , respectively).

[0060] Soluble CNTFR polypeptides according to one embodiment have the amino acid sequence set forth in Table 5 below: In Table 5, mutations are shown in bold / underlined. In this example, the soluble The CNTFR polypeptide is a wild-type CNTF receptor having the amino acid sequence set forth in SEQ ID NO:8. Compared to the human IgG1 receptor, it contains a C-terminal truncation of amino acids 343 to 372. In embodiments, such soluble CNTFR polypeptides contain a signal peptide (see Table 5). (indicated by underline) [Table 5]

[0061] According to certain embodiments, the soluble CNTFR polypeptide of the present disclosure is SEQ ID NO:8 Or, 70% or more, 75% or more, 80% or more of amino acids 23 to 342 of SEQ ID NO: 9 or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100% identity. or a fragment thereof, for example, 250 to 319 amino acids, ~260 amino acids, 260-270 amino acids, 270-280 amino acids, 2 80-290 amino acids, 290-300 amino acids, 300-310 amino acids or a fragment having a length of 310 to 319 amino acids. In addition, such CNTFR polypeptides may be used in combination with one or more ligand-CNTFR complexes. of binding affinity to a subunit (e.g., LIFR, gp130, or both) reduction, increased binding affinity / specificity for CNTFR1, CNTFR ligands (e.g. CNTF, NP, etc.), and any combination thereof. The desired features may include one or more of:

[0062] In some embodiments, the soluble CNTFR polypeptide is R110Q, T174P , Y177H, K178N, S237F, T268A, D269A, and I287F and one or more (e.g., each) amino acid substitutions at amino acid 23 of SEQ ID NO:9. 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more for 342 %, 99% or more, or 100% identity.

[0063] According to some embodiments, the soluble CNTFR polypeptides of the present disclosure comprise an Fc domain. Such fusion proteins are described in more detail below. The amino acid sequence of an exemplary soluble CNTFR polypeptide fused to an Fc domain is: The Fc domains are listed in Table 6 below (Fc domains are underlined and signal peptides are italicized). (indicated in bold). [Table 6]

[0064] According to certain embodiments, the soluble CNTFR polypeptide of the present disclosure is SEQ ID NO:1. 70% or more, 75% or more, 80% or more, 85% or more of amino acids 23 to 578 of 0 Amino acid sequences with 90% or more, 95% or more, 99%, or 100% identity, or or fragments thereof, e.g., 450 to 555 amino acids, 500 to 555 amino acids , 525-555 amino acids, 540-555 amino acids, or 550-555 In certain embodiments, such soluble C The NTFR polypeptide contains a signal peptide (shown in italics in Table 6). Does not include.

[0065] According to certain embodiments, the soluble CNTFR polypeptide-Fc fusion comprises R110 Q, T174P, Y177H, K178N, S237F, T268A, D269A, and and one or more (e.g., each) amino acid substitutions of I287F and I287F, and 70% or more, 75% or more, 80% or more, 85% or more of amino acids 23 to 578 of 0 Amino acid sequences with 90% or more, 95% or more, 99% or more, or 100% identity or fragments thereof, e.g., 450-555 amino acids, 500-555 amino acids 525-555 amino acids, 540-555 amino acids, or 550-5 In certain embodiments, such soluble fragments include fragments having a length of 55 amino acids. The CNTFR polypeptide contains a signal peptide (shown in italics in Table 6). ) is not included.

[0066] Fusion Proteins and Conjugates In certain embodiments, the agent administered to the individual (e.g., a drug described elsewhere herein) The heterologous moiety (e.g., any of the agents used) is stably associated (e.g., fused, conjugated or otherwise attached).

[0067] In some embodiments, the polypeptide fused to the heterologous polypeptide is a drug. The heterologous polypeptide of interest may be an Fc domain or a fusion protein. Fc domains (e.g., human or mouse Fc domains), albumin, transferrin, XT EN, homoamino acid polymer, proline-alanine-serine polymer, elastin-like peptide These include, but are not limited to, thiazol-2, thiazol-3, thiazol-4, thiazol-5, thiazol-6, thiazol-7, thiazol-8, thiazol-9, thiazol-10, thiazol-11, thiazol-12, thiazol-13, thiazol-14, thiazol-15, In certain embodiments, the heterologous polypeptide is a polypeptide of the same type that is not fused to the heterologous polypeptide. The stability and / or safety of polypeptide agents when administered to an individual may be improved compared to polypeptide agents. or increase serum half-life. In certain embodiments, any of the polypeptide agents or fused to a human Fc domain (e.g., a full-length human Fc domain or a fragment thereof). A fusion protein is provided comprising a polypeptide as described elsewhere herein. Non-limiting examples of human Fc domains that can be fused to any of the agents are listed in Table 7 below. a human IgG1 Fc domain having the sequence (SEQ ID NO: 11) as set forth in . [Table 7]

[0068] According to certain embodiments, the drug is conjugated to a chemical moiety. Conjugates are provided. Chemical moieties of interest include polyethylene glycol (P EG), anti-cancer drugs, detectable labels, and combinations thereof. Not limited to.

[0069] The targeted anticancer drugs include drugs that inhibit cell growth and / or kill cancer cells. Such agents can be varied and include cytostatic and cytotoxic agents ( For example, killing of target cell tissue with or without being internalized by the target cell. In certain embodiments, the therapeutic agent may include an enediyne, lecithin, or a steroid. Xytropsin, duocarmycin, taxane, puromycin, dolastatin, The cytotoxic agent is selected from the group consisting of itansonoids, and vinca alkaloids. In some embodiments, the cytotoxic agent is paclitaxel, docetaxel, CC-1065, CPT-11 (SN-38), topotecan, doxorubicin, morpholino-doxorubicin rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin maytansin, combretastatin, calicheamicin, maytansin, maytansin DM1, me auristatin E or auristatin F, such as itansine DM4, DM-1, or auristatin F Uristatin or other dolastatin derivatives, AEB (AEB-071), AEVB ( 5-benzoylvaleric acid-AE ester), AEFP (antibody-endostatin fusion protein) Protein), MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin cin F), pyrrolobenzodiazepines (PBDs), eleutherobin, netropsin, and and any combination thereof. According to certain embodiments, the agent is HTI-2 Hemiasterlin and hemiasterlin analogs such as 86 (e.g., their entire contents are disclosed in reference No. 7,579,323, WO2004 / 026, the disclosures of which are incorporated herein. 293, and U.S. Patent No. 8,129,407), abrin, brucine, sik Toxin, diphtheria toxin, batrachotoxin, botulinum toxin, Shiga toxin, endotoxin, P seudomonas exotoxin, Pseudomonas endotoxin, tetanus toxin, pertussis toxin , anthrax toxin, cholera toxin, falcarinol, fumonisin B1, fumonisin B2, afla Toxins, maurotoxin, agitoxin, charybdotoxin , margatoxin, slotoxin, sila Toxin (scyllatoxin), hefutoxin, calciseptin, taicatoxin (taicatoxin), calcicludin, geldanamycin, gelonin, lotaust Larin, ochratoxin A, patulin, ricin, strychnine , trichothecene, zearalenone and tetradotoxin Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, The nonbinding active fragment of diphtheria toxin, exotoxin A chain (Pseudomonas aeruginosa nosa-derived), ricin A chain, abrin A chain, modeccin A chain, alginate Fasarcin, Aleurites fordii protein, Dia Anti-inflammatory protein, Phytolaca americana protein (PAPI, P APII, and PAP-S), Momordica charantia inhibitor, Kuru Curcin, crotin, Sapaonaria officinalis inhibitor agents, gelonin, mitogellin, restrictocin ctocin, phenomycin, enomycin cin) and trichothecenes.

[0070] The detectable label may be used for any intended use (e.g., in vitro and / or in vivo studies). and / or clinical applications). The enzymes used to generate detectable products include radioisotopes, enzymes (e.g., horseradish peroxidase), and These include enzymes such as phospholipases and alkaline phosphatases, fluorescent proteins, and paramagnetic atoms. In certain embodiments, the CNTFR ligand is coupled to a specific binding partner of a detectable label. conjugated (e.g., via a detectable label including avidin / streptavidin) conjugated to biotin so that detection can occur via

[0071] According to certain embodiments, the agent is suitable for near-infrared (NIR) optical imaging, single photon Spectral Emission Computed Tomography (SPECT) / CT imaging, Positron Emission Tomography ( Labeling agents used in in vivo imaging such as PET (positron emission tomography) and nuclear magnetic resonance (NMR) spectroscopy Labeling agents used for such purposes include fluorescent labels and radioisotopes. In certain embodiments, the labeling agent may include, but is not limited to, two or more images. Multimodal in vivo imaging using a sizing technique It is a scavenging agent (e.g., Thorp-Greenwood and Coogan (2 011) See Dalton Trans. 40:6129-6143).

[0072] In certain embodiments, the labeling agent is an imaging agent used in near-infrared (NIR) imaging applications. an in vivo imaging agent, the agent being Kodak X-SIGHT dye, Pz 247 , DyLight 750 and 800 Fluors, Cy 5.5 and 7 Flu ors, Alexa Fluor 680 and 750 Dyes, IRDye 680 and 800CW Fluors. According to certain embodiments, the labeling agent is an in vivo imaging agent used in SPECT imaging applications, the agent comprising: 99m Tc, 111 In, 123 In, 201 Tl, and 133 Xe is selected. In certain embodiments, the labeling agent is suitable for positron emission tomography (PET) imaging applications. The in vivo imaging agent used is 11 C. 13 N, 15 O. 18 F, 64 Cu, 62 Cu, 124 I, 76 Br, 82 Rb, and 68 Ga.

[0073] Linkers used in the conjugates of the present disclosure include ester linkers, amide linkers, Maleimide or maleimide-based linkers, valine-citrulline linkers, hydrazo linker, N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB ) linker, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate carboxylate (SMCC) linker, vinyl sulfone-based linker, tetraethylene including polyethylene glycol (PEG), such as, but not limited to, polyethylene glycol linkers containing propanoic acid, linkers containing caproleic acid, and the like Examples of linkers include linkers containing any combination of

[0074] Numerous strategies are available for linking drugs to chemical moieties of interest via linkers. For example, a chemical moiety of interest can be synthesized by covalently attaching a linker to the chemical moiety. The linker may be derivatized with a "chemical handle" on the drug, where the linker reacts with a "chemical handle" on the drug. The functional groups on the linker can be varied and can be used to attach chemical bonds to the drug. According to one embodiment, the chemical handle on the drug may be selected based on its compatibility with the drug. , provided by incorporating an unnatural amino acid with its chemical handle into a drug. Such unnatural amino acids can be prepared, for example, through chemical synthesis or recombinant techniques, e.g., Orthogonal amino acids suitable for incorporating unnatural amino acids during translation in host cells Aminoacyl-tRNA synthetase-tRNA pairs can be used to incorporate them into drugs. .

[0075] The functional groups of the unnatural amino acids present in the drug are azide, alkyne, alkene, and amino. oxy, hydrazine, aldehyde, nitrone, nitrile oxide, cyclopropene, norbornane ene, isocyanide, aryl halide, boronic acid, or other suitable functional group and the functional group on the linker is selected to react with the functional group of the unnatural amino acid ( and vice versa).

[0076] Administration As summarized above, the methods of the present disclosure are directed to treating KRAS mutant cancer in an individual. "Treating" or "treatment" means any method of treating a patient with a KRAS mutant cancer. It means at least an improvement in symptoms, where improvement is an improvement in the parasites associated with the KRAS mutant cancer being treated. It is used broadly to refer to at least some relief of a condition (e.g., a symptom). Therefore, treatment also may be beneficial in individuals with KRAS mutant cancer or at least the symptoms that characterize it. To avoid further suffering, it is important to know if you have KRAS mutant cancer or at least the symptoms associated with it. , completely inhibited, e.g., prevented from occurring or stopped, e.g., terminated This also includes situations in which

[0077] The agent that inhibits CLCF1-CNTFR signaling is administered to the individual in a therapeutically effective amount. In some embodiments, a therapeutically effective amount of the agent (e.g., in a pharmaceutical composition comprising it) is administered. (those present in the table) may be administered alone (e.g., in monotherapy) or in combination with one or more further treatments. When administered in one or more doses in combination with other therapeutic agents (e.g., in combination therapy), and determining the symptoms of the KRAS mutant cancer in the individual based on the individual's symptoms in the absence of treatment with the agent. at least about 5%, at least about 10%, at least about 15%, at least at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least At least about 50%, at least about 60%, at least about 70%, at least about 80%, at least The amount is effective to reduce the risk of rheumatoid arthritis by about 90% or more. For example, the disclosed method may be effective in reducing the proliferation of cancer cells of a KRAS mutant cancer when the agent is administered in an effective amount. Inhibiting growth, metastasis, and / or invasiveness.

[0078] Dosing depends on the severity and responsiveness of the KRAS mutant cancer being treated. The schedule can be calculated from measurements of drug accumulation in the patient's body. The physician in charge can determine the optimum dosage, method of administration and repetition rate. Amounts may vary depending on the relative potency of the individual agents and generally vary in vitro and in vivo. EC that has been shown to be effective in animal models 50 can be estimated based on Generally, the dosage is 0.01 μg to 100 g per kg of body weight, and is administered daily, weekly, or It may be given more than once a month, or once a year. The treating physician will determine the measured dwell time and Based on the concentration of the drug in the body fluids or tissues, a dosing repetition rate can be estimated. After successful treatment, the subject may undergo maintenance therapy to prevent recurrence of the disease state. It may be desirable to administer the drug in a dose ranging from 0.01 μg to 100 g per kg of body weight. maintenance dose, once or more daily, once every few months, once every 6 months, once a year, or any other The dose is administered at a suitable frequency.

[0079] The therapeutic methods of the present disclosure involve the use of a single type of Alternatively, the method may involve administering a drug to an individual, such as by inhibiting CLCF1-CNTFR signaling. CLCF1-CNTFR signaling was inhibited by the administration of a cocktail of different drugs. This may involve administering more than one type of agent that inhibits, for example, CNTFR-specific a first agent (e.g., a compound described herein) that binds to and inhibits signaling through CNTFR; (any of the engineered ligands described above) and CNTs that specifically bind to CLCF1. A second agent that inhibits signaling through the FR (e.g., an engineered antibody described herein) The method may involve administration of a soluble CNTFR polypeptide (any of the soluble CNTFR polypeptides).

[0080] The agent may be administered by any available method and route suitable for drug delivery, including in vivo and ex vivo methods. The compounds may be administered to an individual using various methods, including in vivo methods, and systemic and local routes of administration. Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, and topical. Intended applications include intraocular, intravenous, intraarterial, oral, and other enteral and parenteral routes of administration. Routes of administration may be combined as needed, or may be combined depending on the specific drug and / or The dosage may be adjusted depending on the desired effect. In some embodiments, the agent can be administered parenterally, e.g., intravenously, In some embodiments, the agent is administered intra-arterially, for example, for systemic delivery. Administered by injection (e.g., intravenous infusion) or at a local site (e.g., intratumoral injection) will be done.

[0081] The drug can be incorporated into a variety of formulations for administration to an individual. More specifically, the drug can be prepared into pharmaceutical compositions by combining with suitable pharmaceutically acceptable excipients or diluents. It can be formulated into tablets, capsules, powders, granules, ointments, liquids, emulsions, etc. solid, semi-solid, liquid, or gaseous forms, such as steroids, injectables, inhalants, and aerosols The preparation can be formulated into:

[0082] Pharmaceutical formulations suitable for administration to an individual (e.g., suitable for human administration) are generally sterile. In addition, there are detectable pyrogens or conditions that contraindicate administration to patients via the selected route of administration. It may be free from certain other contaminants.

[0083] In pharmaceutical dosage forms, the drug may be present alone or in combination with a second pharmaceutically active compound, e.g. 2 anticancer drugs (including but not limited to small molecule anticancer drugs) in appropriate combination or The following methods and carriers / excipients are merely examples and should not be construed as limiting the scope of the present invention. It is not intended to be limiting.

[0084] For oral preparations, the drug may be administered alone or in the form of tablets, powders, granules or capsules. and suitable additives for making the sugars, such as lactose, mannitol, corn denat. The conventional additives, such as potato starch or cellulose, derivatives, acacia, corn starch, or gelatin, with a binder Corn starch, potato starch, or sodium carboxymethylcellulose a disintegrant such as talc or magnesium stearate, and a lubricant such as talc or magnesium stearate, if necessary. For example, it may be used in combination with diluents, buffers, humectants, preservatives, and flavoring agents. do.

[0085] Drugs may be administered parenterally (e.g., intravenously, intratumorally, intraarterially, intraosseously, intramuscularly, intracerebrally, intraventricularly, In certain embodiments, the agent can be formulated for administration (intrathecal, subcutaneous, etc.). , vegetable oils or other similar oils, synthetic fatty glycerides, esters of higher fatty acids, or The complex is dissolved, suspended, or dissolved in an aqueous or non-aqueous solvent, such as propylene glycol. By emulsifying, and if necessary, adding solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizing agents The composition can be formulated for injection together with conventional additives such as antiseptics and antiseptics.

[0086] Pharmaceutical compositions containing a drug may be prepared by dissolving the drug in any physiologically acceptable carrier, with the drug having the desired purity. by mixing with the carrier, excipient, stabilizer, surfactant, buffer, and / or isotonicity agent. Acceptable carriers, excipients and / or stabilizers may be used. The dosage and concentration are non-toxic to recipients and contain no buffers, e.g., phosphate, citrate, or the like. acid salts, and other organic acids; ascorbic acid, glutathione, cysteine, methionine and Antioxidants, including citric acid, preservatives (e.g., ethanol, benzyl alcohol, phenol ethanol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, or a combination thereof), arginine, glycine, ornithine leucine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, Alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline , and combinations thereof, such as amino acids, monosaccharides, disaccharides, and other sugars, low molecular weight ( polypeptides (less than about 10 residues), proteins such as gelatin or serum albumin, E Chelating agents such as DTA, trehalose, sucrose, lactose, glucose, mannose , maltose, galactose, fructose, sorbose, raffinose, glucosamine sugars such as N-methylglucosamine, galactosamine, and neuraminic acid, and / or Alternatively, use Tween, Brij Pluronic, Triton-X, or polyethylene. Nonionic surfactants such as polyethylene glycol (PEG) are included.

[0087] The pharmaceutical composition may be in liquid form, lyophilized form, or a liquid form reconstituted from a lyophilized form. The lyophilized preparation may be in the form of a liquid preparation, in which case the lyophilized preparation will be reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is to add a certain amount of purified water (typically the lyophilized The amount of the solution added back is equal to the volume removed during drying, but the pharmaceutical composition for parenteral administration A solution containing an antimicrobial agent may be used for the manufacture of the article.

[0088] The aqueous formulation of the drug may have a pH in the range of, for example, about 4.0 to about 7.0 or about 5.0 to about 6.0, Alternatively, it can be prepared in a pH buffer at a pH of about 5.5. Examples of buffers suitable for the pH include phosphate buffers, histidine buffers, citrate buffers, These include succinate buffers, acetate buffers, and other organic acid buffers. The buffer concentration can be, for example, For example, about 1 mM to about 100 mM, or about 5 mM, depending on the buffer and the desired tonicity of the formulation. M can be about 50 mM.

[0089] To adjust the tonicity of the formulation, a tonicity agent can be included in the formulation. The agents include sodium chloride, potassium chloride, glycerin, and any of the amino acids. In some embodiments, water may be used. The formulation is isotonic, although hypertonic or hypotonic solutions may also be suitable. The term "solution" is the same as any other solution to which it is compared, such as saline or serum. The tonicity agent is used in an amount of about 5 mM to about 350 mM, for example, 1 It can be used in an amount of 00 mM to 350 mM.

[0090] To reduce aggregation and / or minimize particulate formation in the formulation Surfactants may be added to the formulation to reduce adsorption and / or surface active agents. Examples of the antibacterial agent include polyoxyethylene sorbitan fatty acid esters (Tween), poly Oxyethylene alkyl ether (Brij), alkylphenyl polyoxyethylene ether ether (Triton-X), polyoxyethylene-polyoxypropylene copolymer ( Poloxamer, Pluronic) and sodium dodecyl sulfate (SDS) are examples. Examples of suitable polyoxyethylene sorbitan fatty acid esters include polysorbates 20 (sold under the trademark Tween 20™), and Polysorbate 80 (Tween A suitable polyethylene-polypropylene copolymer is sold under the trademark n 80™. Examples of such compounds are Pluronic® F68 or Poloxamer 188 (trademark). Suitable polyoxyethylene alkyl ethers are sold under the trade name An example is that sold under the trademark Brij™. Examples of surfactant concentrations are: It can range from about 0.001% to about 1% w / v.

[0091] Cryoprotectants are added to protect the drug from destabilizing conditions during the freeze-drying process. For example, known cryoprotectants include sugars (including glucose and sucrose), polysaccharides, and the like. nucleotides (including mannitol, sorbitol and glycerol), and amino acids (containing alanine, glycine, and glutamic acid). Cryoprotectants are used in approximately 10 ml It may be included in an amount of M to 500 nM.

[0092] In some embodiments, the pharmaceutical composition comprises a drug and an ingredient identified above, e.g. , surfactants, buffers, stabilizers, isotonicity agents), and Dimethyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or or propylparaben, benzalkonium chloride, and combinations thereof. In other embodiments, the formulation is essentially free of preservatives, e.g. It is contained at a concentration ranging from about 0.001 to about 2% (w / v).

[0093] kit As summarized above, the present disclosure provides kits. Such kits find use, for example, in practicing the methods of the present disclosure. For example, the kit of the present disclosure includes an agent that inhibits CLCF1-CNTFR signaling, and and instructions for administering the agent to an individual identified as having a KRAS mutant cancer.

[0094] The kits of the present disclosure contain the CLCF1-CNTFR signal transduction vector described in the methods section above. The present invention may include any of the agents that inhibit neural transmission, the description of which is incorporated herein for brevity. By way of example, the subject kit may be the same as that described in the Methods section above. The present invention also includes any of the engineered ligands, soluble CNTFR polypeptides, etc. It can be seen.

[0095] In certain embodiments, the instructions for the kit of the present disclosure include instructions for treating a patient with KRAS mutant lung cancer. The drug then includes instructions for administering the drug to the identified individual. For example, the instructions may include instructions for administering the drug to the identified individual. To administer the drug to individuals identified as having S-mutant non-small cell lung cancer (NSCLC) The kit may include instructions for administering to an individual identified as having KRAS mutant NSCLC. If instructions for administering the drug are included, the instructions should include information about KRAS-mutated lung adenocarcinoma (L The drug may include instructions for administering the drug to an individual identified as having UAD.

[0096] According to some embodiments, the kit of the present disclosure comprises an amino acid substitution at position 12 of human KRAS. instructions for administering the drug to an individual identified as having a KRAS mutant cancer, including wherein the numbering is the same as SEQ ID NO: 1. In certain embodiments, such Such kits are from the group consisting of G12A, G12C, G12D, G12S, and G12V. The drug is administered to individuals identified as having KRAS mutant cancers containing amino acid substitutions selected from the Instructions for administration may be included.

[0097] The subject kits include CLCs present in unit dose (e.g., ampoules) or multi-dose forms. an amount of an agent (e.g., a pharmaceutically acceptable salt thereof) that inhibits F1-CNTFR signaling; (e.g., in a pharmaceutical composition comprising a carrier containing the compound). In some embodiments, the kit may include one or more (e.g., two or more) unit doses of a composition comprising an agent. The term "unit dose" is used herein to mean a single unit dose (e.g., an ampoule). When used, the formulation shall be in physically separate units suitable as single doses for human and animal subjects. Each unit contains a predetermined amount of a composition calculated to be sufficient to produce the desired effect. The amount of the unit dose will depend on the individual, the particular drug used, and the effect to be achieved. In yet another embodiment, the effect of the drug on the vasopressin receptor agonist (vasopressin receptor agonist) is dependent on various factors, including the efficacy and safety of the drug, and the pharmacodynamics associated with the drug. Alternatively, the kit may contain a single multiple dose of the composition.

[0098] The components of the kit may be present in separate containers, or multiple components may be present in a single container. Suitable containers include single tubes (e.g., vials), ampoules, plates, etc. one or more wells of a plate (e.g., 96-well plate, 384-well plate, etc.) Includes:

[0099] Any instructions (e.g., instructions for use (IFU)) included with the kit The instructions may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. The instructions can thus be printed within the kit, for example as a package insert, or or on the label of the component's container (i.e., associated with the packaging or sub-packaging) In other embodiments, the instructions are provided on a portable flash drive, DVD, CD, or - electronic storage residing on a suitable computer-readable storage medium such as a ROM, diskette, etc. In yet another embodiment, the actual instructions are present in the kit. obtain instructions from a remote source, e.g., via the Internet, but not in the An example of this embodiment is a method for reading instructions and / or or a kit containing a web address where the instructions can be downloaded. Similarly, the means for obtaining the instructions are recorded on a suitable substrate.

[0100] The following examples are offered by way of illustration and not by way of limitation.

[0101] [experiment] Example 1—Expression of CLCF1 and CNTFR, and CLCF1 in human LUAD -Oncogenic effects of CNTFR signaling Analysis of public gene expression data showed that CLCF1 was significantly increased in lung adenocarcinoma (LUA) compared with normal lung. D) was significantly upregulated in the CLCF1 gene (data not shown). The present study was associated with decreased survival in patients with KRAS mutations [Cox hazard ratio: 2. 53 (95% CI 1.43-4.48), p-value: 0.001) compared with patients without KRAS mutations There was no association in patients [Cox hazard ratio: 0.86 (95% CI 0.51 to 1.00)]. 4), p-value: 0.56]. This result indicates that KRAS-mediated carcinogenesis These findings suggest a specific role for CLCF1 signaling in KRAS mutant LUAD. FR expression did not show the same pattern [Cox hazard ratio: 1.36 (95% CI 0.65-2.82), p-value: 0.41]. Previous studies have shown that in mouse lung tumors, Cancer-associated fibroblasts (CAFs) have been shown to be the major source of CLCF1. To determine whether human lung CAFs also provide a source of CLCF1, we cultured CAFs from human lungs. CLCF1 was isolated from lung cancer patients and matched normal lung fibroblasts (NLFs). The present study demonstrated significant improvements in six of eight human CAFs compared with patient-matched NLFs. However, the LUAD cell lines tested also secreted CLCF1, which The presence of both paracrine and autocrine signaling of this cytokine in human LUAD suggests the presence.

[0102] We next assessed the functional role of CLCF1 in cell lines exposed to recombinant CLCF1. Ligand induced increased proliferation in all LUAD cell lines tested (Figure 1, Panel A). When binds to the CNTFR / LIFR / gp130 complex, gp130 is phosphorylated, Downstream signals including STAT3 and ERK are activated. LCF1 induced STAT3 phosphorylation (Figure 1, panels B–D). To further investigate the functional significance of CLCF1-CNTFR signaling in RN Two different shRNAs were used to reduce the amount of CNTFR on the cell surface. The knockdown used significantly reduced the viability of all five LUAD cell lines tested. CNTFR knockdown also significantly reduced the CL of LUAD cell lines (Figure 1, panels E–G). It also inhibited laminogenic growth (Figure 1, panels H and I), reducing sphere size and proliferation in 3D culture. This resulted in a reduction in the number of CNTFR receptors (Figure 1, panels J and K). The aim of this study was to determine whether the downregulation of IFN-γ affected tumor growth in vivo. CNTFR knockdown in all LUAD cell lines reduced xenograft formation (Figure 1, panels L and M). Furthermore, from LUAD cells with CNTFR knockdown The tumors formed had a lower proliferation index and higher apoptosis compared to control tumors. (Figure 1, panels N and O). To test whether CLCF1 is secreted, we knocked down CLCF1 in H2009 and The cells were transplanted as xenografts. Similar effective tumor growth reduction was observed in both cutaneous and subcutaneous xenograft tumors. In this study, it was suggested that the source of CLFC1 is primarily autocrine from the tumor cells themselves. It was.

[0103] To determine the mechanism of action of CNTFR blockade in LUAD, we investigated MAPK, AKT, and The effects of knockdown on the STAT3 and STAT4 signaling pathways were evaluated, all of which Previously identified as activated downstream of gp130: ERK and S6 Phosphorylation of α, β, and β decreased in tumors after CNTFR knockdown, and these were mediated by MAPK / ERK, respectively. The results indicated an effect on the AKT and IL-1 pathways. Decreased phosphorylation of STAT3 was also observed. Taken together, these results suggest that CLCF1-CNTFR signaling is essential for LUAD. It is active in CNTFR and plays a role in promoting carcinogenesis. The mechanism of CNTFR inhibition involves the activation of STAT3 and ERK. and attenuation of the activity of several signaling cascades, including AKT signaling. Indicates that it is included.

[0104] Example 2 - Soluble receptor decoys for inhibiting the CLCF1-CNTFR signaling axis Preparation of The above functional studies suggest that inhibition of CLCF1-CNTFR signaling may offer therapeutic opportunities for lung cancer. This supports the possibility that effective strategies to target this pathway may be needed. CNTFR is a C-terminal peptide that is synthesized following proteolytic cleavage of the C-terminal propeptide. On the cell surface via glycosylphosphatidylinositol (GPI) linkages formed by When bound to CLCF1, CNTFR binds to gp13 0 and LIFR (Figure 2, panel A, ii). CNTFR is secreted from the membrane but still binds to CLCF1 and expresses CNTFR. It can activate downstream signaling even in cells that do not express it (Figure 2, Panel A , iii). Therefore, effective blocking of CLCF1 requires binding of the decoy to CLCF1. Both an increase in IL-1 and a decrease in IL-1 binding to gp130 and LIFR are required (Fig. 2, Panel B). Le A, iv).

[0105] Directed evolution was used to identify mutants with higher affinity for CLCF1, gp130, and LI We have designed soluble CNTFR mutants that lack binding to the FR. It functions as a selective ligand trap and blocks CLCF1-mediated oncogenic signaling. To develop a high-affinity receptor decoy, we hypothesized that CNTFR The DNA encoding the extracellular domain of the The corresponding protein library (approximately 10 8 transformants) as fusion The mutants with increased CLCF1 binding were enriched for the CLCF1-displayed mutants (Figure 2, Panel B). To identify the 3 Lau strains, the library was screened using flow cytometry. After screening of the strains, T174P and S237F emerged as consensus mutations, Considerable variation was observed at other amino acid positions. To do this, 20 different clones were randomly selected from the sorted population and analyzed using S Using the taggered Extension Process (StEP) method A second library was generated by shuffling. This library was sorted and screened using a combination of The screening stringency was increased (Figure 2, Panel C). After cloning, a combination of four mutations (R110Q, T174P, S237F, and I287F) was detected. Quantitative yeast display binding studies showed that each of these mutations increased the expression of CLCF1 The combination of all four mutations contributed to a higher binding affinity for β-glucan (Figure 2, panel D). The apparent K d This CNTFR mutant (mutant 4) ) was carried forward for further optimization.

[0106] CLCF1-CNTFR binding is mediated through heterodimerization of LIFR and gp130 Reduce the formation of this complex while sequestering CLCF1 to activate downstream signaling. Modifying CNTFR to reduce or prevent this is beneficial for its inhibitory activity. The mother-displayed CNTFR interacts with gp130 and LIFR in a CLCF1-dependent manner. Therefore, CNTFR mutant 4 was confirmed to form a complex with the co-receptor The nucleotide sequence was further engineered to reduce its binding to ribosomal RNA. Dam mutations were introduced into CNTFR mutant 4, and the resulting library was incubated with CLCF1. The mutants with reduced LIFR binding signals were identified by flow cytometry. Two concepts that reduce binding to LIFR were investigated (Figure 2, Panel E). Mutations (Y177H and K178N) were identified (Figure 2, panel F). Two mutations conferred high-affinity CLCF1 binding, and four mutations conferred low-affinity binding to gp130. Two additional alanine substitutions (T268A and D269A) were incorporated that were shown to attenuate Combined, the final mutant, eCNTFR, was created.

[0107] Example 3 - Characterization of soluble eCNTFR Because structural information for the full-length CNTFR is not available, we used the Phyre 2 server to Modeling wtCNTFR and eCNTFR revealed that the tertiary mutations in eCNTFR Three of the four mutations identified by affinity maturation (T174P , S237F, and S287F) have been shown to be important for cytokine binding. The aromatic cluster (F172, F199, and F238) and conserved residues (E The soluble eCNTFR was located proximal to the C with a terminal hexahistidine tag (eCNTFR-His), or antibody Fc domain It was recombinantly expressed as an N-terminal fusion to CLCF1 (eCNTFR-Fc) and showed affinity to CLCF1. The activity was measured using a microtiter plate-based assay. Both His and eCNTFR-Fc have picomolar binding affinities for CLCF1. In contrast, the soluble wild-type CNTFR construct (wtC The binding affinity of CLCF1 was weaker for the wtCNTFR-His and wtCNTFR-Fc. Using a similar approach, we investigated the correlation between gp130 and LIFR. In these experiments, the binding interactions between wtCNTFR and both receptors were characterized. In contrast to the construct, the eCNTFR construct did not have detectable binding to gp130 and LIFR. The protein was sized to avoid glomerular filtration. Increasing the size significantly increases serum half-life, and the Fc domain promotes FcRn-mediated cleavage. The half-life can be further extended by recycling. R-Fc fusions were used to further characterize the effects of eCNTFR in animal models of LUAD. Evaluated.

[0108] CNTF is another ligand for CNTFR, and signaling through CNTF is important for neuronal survival. eCNTFR is directed to CLCF1 rather than CNTF Manipulating the binding selectivity of -Fc can potentially reduce side effects from inhibition of CNTF signaling. Furthermore, CLCF1 acts only via CNTFR. CNTF also binds to the IL-6 receptor (IL-6R) and induces CL CF1 and CNTF have distinct functional roles in regulating signaling pathways Although wtCNTFR-Fc exhibited binding to recombinantly produced CNTF, However, eCNTFR-Fc did not (Figure 3, panel D). Consistent with the yeast display binding data for FR and eCNTFR, CLC Affinity maturation of CNTFR against F1 increased its specificity for CLCF1 and CNT In addition, eCNTFR-Fc resulted in decreased binding to wtCN Compared with TFR-Fc, it binds to mouse CLCF1 with higher affinity, and CLCF1 binds to mouse CLCF1 with higher affinity. This demonstrates the usefulness of in vivo experiments when derived from a mammalian cell line (Figure 3, Panel E). Mouse CLCF1 can activate CNTFR in human cells.

[0109] eCNTFR-Fc effectively sequesters CLCF1 and blocks the formation of the receptor complex. To assess whether the receptor complex can be expressed in a specific manner, we investigated the effect of the wtCNTFR on the function of the other subunits of the receptor complex. A competitive assay was performed to measure the effect of eCNTFR-Fc on the interaction between each of the units. A combined assay was designed to detect eCN in wells coated with wtCNTFR-His. Incubation with TFR-Fc inhibited the expression of CLCF1, LIFR, and gp130 constructs. was prevented from interacting with wtCNTFR-His (Figure 3, panel F). TFR-Fc effectively neutralizes CLCF1 and inhibits gp130 signaling. in the presence and absence of a soluble CNTFR construct to determine whether LUAD cells were stimulated with CLCF1. wtCNTFR-Fc inhibited STAT3 (Tyr7 05), whereas eCNTFR-Fc increased phosphorylation of ribosomal protein 1 (rRNA) in both cell lines tested. Furthermore, the interaction with eCNTFR-Fc was reduced (Figure 3, panels G and H). Incubation inhibited CLCF1-mediated survival (Fig. 3, panels I and J).

[0110] Example 4 - eCNTFR-Fc inhibits Ras-GTP loading by reducing Ras-GTP loading Selectively inhibits KRAS mutant cells The above demonstrates that CLCF1 expression is mediated by oncogenic KRAS. Currently, KRAS mutations are associated with a specific prognosis for survival in patients with LUAD. Few tumor treatment options exist, making it challenging to develop new therapies for this subset. This is particularly clinically important. To identify these, we collected a panel of LUAD cell lines with diverse genotypes and analyzed their cell viability. The effect of eCNTFR-Fc on these cell lines was evaluated (Figure 4, Panel A). The sensitivity varied, with the least sensitivity (no effect) being in normal lung cells (NL20). The most sensitive was the LUAD cell line A549. All cell lines were KRAS mutant. The cell lines exhibited moderate sensitivity. In contrast, H1755 and H1395 (both BRAF G469A ) cells were completely insensitive to CNTFR blockade. F G469A The mutation is a "class 2" mutation that signals as a constitutively active dimer and is predicted to be independent of upstream KRAS signaling. Two KRAS mutant cell lines carrying the H mutation (Figure 4, panel A) expressed eCNTFR-Fc The Q61H mutant KRAS was completely insensitive to blockade of the intrinsic GTPase lacking activity, it is unable to regulate upstream signals that regulate GTPase-activating proteins (GAPs). GAP is expected to be insensitive to GTPase hydrolysis. The amount of GTP-bound KRAS was measured in both KRAS mutant and wild-type KRAS cells. Taken together, these results suggest that CLCF1-CNTFR regulates the expression of CLCF1 through gp130. Transduces signals to activate GAP, which then regulates KRAS GTP binding, thus This is consistent with a model in which the ATP-dependent ATP synthase regulates downstream signals.

[0111] After binding to a ligand, CNTFR activates gp130, which in turn activates SHP2. Second, SHP2 activates oncogenic KRs through regulation of GTP loading. It functions as a major upstream regulator of both AS and wild-type KRAS. In both H23 LUAD cell lines, serum stimulation, as expected, increased the expression of P-SHP2 as well as P- Increased phosphorylation of STAT3 and P-ERK (Figure 4, panels B and C). Stimulation of cell lines with recombinant CLCF1 in the absence of SHP2, STAT3, and The phosphorylation level of ERK was also increased, indicating that the upstream signaling of CLCF1 is via SHP. Treatment with eCNTFR-Fc increased the activity of CLCF2. 1, but was less effective in inhibiting the action of whole serum. This was because serum has other effects independent of the CLCF1-eCNTFR axis. This is expected. To establish the mechanistic relevance more directly, we investigated the effect of CLCF1 on the expression of CLCF1 in cells treated with recombinant CLCF1. directly measure the levels of Ras-GTP in the presence or absence of eCNTFR-Fc The Ras-GTP level increased after CLCF1 treatment, and this The effect was attenuated by eCNTFR-Fc. CLCF1 inhibition indicates a link between FR signaling and oncogenic KRAS is more effective in some KRAS genotypes but not others Taken together, these studies suggest that CLCF1 inhibition may be beneficial in preventing and treating inflammatory bowel disease, as discussed further below. As discussed, it has been suggested that this treatment may be particularly effective in KRAS-mutant tumors.

[0112] Example 5 - eCNTFR-Fc sequestered CLCF1 and inhibited tumor growth in vivo Next, we evaluated the role of eCNTFR-Fc as an antitumor therapeutic agent in vivo. To determine whether NTFR-Fc can effectively sequester mouse CLCF1 To investigate this, non-tumor-bearing mice were treated with a single dose of eCNTFR-Fc. Serum levels of CLCF1 rapidly increased with a concomitant decrease in unbound CLCF1, which persisted for up to 72 hours. These results suggest that eCNTFR- Fc effectively binds to mouse CLCF1 and reduces its availability in serum This shows that it is possible.

[0113] To test the therapeutic efficacy of eCNTFR-Fc, two LUAD cell lines were immunodeficient. The tumors engrafted in mice grew to an average volume of 100 mm 3 When the Treatment resulted in dose-dependent tumor inhibition in both xenograft models. (Figure 5, panels B to D), whereas wtCNTFR-Fc had no effect. The results showed that eCNTFR-Fc inhibits the expression of PDTX in a panel of patient-derived xenograft tumors. Treatment with eCNTFR-Fc significantly reduced the incidence of pulmonary fibrosis in five LUAD PDTX models. Three of these resulted in significant tumor growth inhibition (Figure 5, panels E–G). A significant decrease in IL-1 and an increase in apoptosis were observed in both cell line xenografts and PDTX models. The results were consistent with those observed in the three PDTX models in response to eCNTFR-Fc treatment (Figure 5, panels H-K). The genotypes of the patients were KRAS G12C, KRAS G12V, and EGFR mutant / KRAS wild-type (wt), whereas non-responders had KRAS and EGFR wt. Furthermore, CAFs with the highest CLCF1 expression were associated with eCNTFR-Fc signaling. It was noted that the results were obtained from tumors with genotypes predicted to be most dependent on .

[0114] As observed with CNTFR knockdown, treatment with eCNTFR-Fc also Activation of ERK (Figure 5, panels L-O) and S6 kinase (Figure 5, panels L and M) To assess the time-dependent effects on signaling pathways, tumor-bearing mice were A short-term study was conducted in which mice were treated with eCNTFR-Fc and euthanized at different time points. These results suggest that eCNTFR-Fc first induces the inhibition of STAT3, and then inhibits ER. This suggests that a delayed inhibition of K and S6 signaling follows.

[0115] These studies were then applied to genetically engineered autochthonous, high-grade LUAD. The results were expanded to a mouse (GEM) model. G12D / Trp53 f / f Mice experienced a reduction in tumor burden compared to vehicle-treated controls. Treatment with eCNTFR-Fc also resulted in decreased proliferation, apoptosis, and vasoconstriction (Figure 6, panels A-E). Increased apoptosis and decreased activation of ERK, S6, and STAT3 signaling (Figure 6, panels F-H). Next, eCNTFR-Fc treatment and cisplatin A survival assay was performed to compare the platinum compounds with those from standard chemotherapy regimens. This was chosen for comparison because it is the primary human LUAD therapy (Figure 6, panel I). Both cisplatin and eCNTFR-Fc treatment improved survival. While mice treated with cisplatin had significant weight loss at the end of the study, However, mice treated with eCNTFR-Fc did not lose weight. Extensive evaluation of post-mortem mouse tissues from mice treated with Fc did not reveal any abnormalities. However, platinum chemotherapy has been shown to induce significant adverse effects, including nephrotoxicity. These results strongly support the therapeutic efficacy of eCNTFR-Fc in LUAD. can.

[0116] Further development of eCNTFR-Fc as a bona fide therapeutic agent will require further investigation into the activity of this pathway. This will be particularly strengthened by identifying appropriate biomarkers for CLCF1 expression. There was a slight positive correlation between the expression of β-glucan and decreased survival after treatment with eCNTFR-Fc. The data presented here suggest that certain genotypes may be more sensitive to eCNTFR-Fc. The next step was to examine plasma CLCF1 levels. The question was whether this could serve as an indicator of the activity of this pathway in individual patients. A method for detecting CLCF1 by ELISA was developed (eCNTFR-Fc was captured). This compound acted as a therapeutic agent and was used to measure the levels of CLCF1 in the plasma of cancer patients. A trend toward higher levels of CLCF1 was observed in LUAD patients compared with healthy controls. Furthermore, those with a genotype susceptible to eCNTFR-Fc (the "variant of interest") Patients with the mutation had significantly higher levels of CLC than patients without the mutation. F1 (Figure 6, Panel J). Logistic regression (logit) was used to analyze the data. Further analysis revealed that tumors were specifically targeted by specific mutations (KRAS G12C, KRAS G12V, or KRAS wt / EGFR mutant type) by measuring blood CLCF1 We demonstrated that the odds ratio (OR) of 8.35 (CI 95% 6.3) can predict whether or not 6-10.33), p-value: 0.04]. Taken together, these results suggest that tumor genotyping CLCF1 plasma concentration combined with analysis may provide therapeutic benefit from eCNTFR-Fc. These findings suggest that the CRPC-10001 gene may serve as a useful biomarker for selecting patients with the highest risk of developing CRPC-1001.

[0117] [method] Lung adenocarcinoma mouse model Lox-stop-Lox-Kras G12D (129Sv / Jae), Trp53 f l / fl (FVB), ​​and Rosa26-LSL-tdRFP (C57BL / 6J) mice Mice were maintained in a virus-free environment. At 8–10 weeks of age, mice were transfected with Cre-expressing mice. 5×10 6Mice were infected intranasally with pfu adenovirus (University of Iowa). Starting 8 weeks after the infection, eCNTFR-Fc was administered by intraperitoneal injection three times a week for 4 weeks. (10 mg / kg) or PBS (vehicle). The weight of the mice was measured periodically.

[0118] Survival and gene expression analysis of human LUAD CLCF1 TPM log2 expression for the cohorts (LUAD; LUSC) was analyzed using a In the R programming language using the FirebrowseR package (1.1.35), Downloaded directly from the Broad Institute. TP (primary tumor) or N Only expression data classified as either T (normal) or T (normal) were used. The RSEM Level 3 exam is available on the FIREHOSE Broad GDAC website. The somatic mutations in the LUAD dataset were downloaded directly from UCSC Xena Obtained from a public repository. Samples with non-silent KRAS mutation(s) Only the samples with KRAS silent mutations were associated with the KRAS mutation group. The KRAS wild-type group was not included and was excluded from the analysis. Clinical data for the LUAD survival analysis, including data from published clinical summaries of the TCGA dataset, are available. Survival analysis curves and multivariate Cox hazard regressions were obtained using survmin in R using er (0.4.3.999) and survival packages (2.44-1.1) Cox regression analysis adjusted for age at diagnosis, sex, and cancer stage. Samples were grouped based on corresponding gene expression quantiles (normal vs. high), and normal expression was assessed. Low expression is below the 75th percentile, and high expression is above the 75th percentile.

[0119] Quantitative reverse transcriptase PCR RNA was isolated using TRIzol reagent (Invitrogen) and analyzed by Qiage The cDNA was further purified using a miniRNA column (Qiagen). cDNA was generated using the yNAmo cDNA synthesis kit (New England Biolabs). , quantitative reverse transcriptase PCR (qRT-PCR) was performed using SYBRGreen (Applied Biosystems, see Supplementary Table 5 for primer sequences) qRT-PCR was performed as follows: 10 min at 95°C, 15 s at 95°C for 35 min. cycle, and 60°C for 1 min.

[0120] Generation of patient-derived tumor xenografts (PDTX) Fresh patient samples were cut into 1 x 1 mm pieces and either implanted fresh or later. For use, the tumor fragments were frozen in 90% FBS, 10% DMSO. Corning Matrigel #356234) and NOD scid gamma The tumors were transplanted into the kidney capsule of non-steroidal anti-inflammatory (NSG) mice. Successfully transplanted tumors were identified when they reached approximately 1-2 cm. A section was kept for histology, and the remainder was treated with collagenase at 37°C for 45 minutes. The DNA was digested for 10 min and filtered through a 70 μm filter. Mouse stroma was depleted of cells (Te (Using antibodies against r119, CD45, CD31, and mouse MHC class I). For later passages and drug studies, cells were cultured in 100 µL of α-MEM and 20 µL of The tumors were implanted subcutaneously in Matrigel (Corning) into the flanks of NSG mice (1 per flank). 5×10 5 Xenograft tumor specimens were stored at -80°C until use.

[0121] The cells were passed through 100 μm and 40 μm cell strainers and incubated at 1,200 rpm for 8 min. The cells were incubated in RBC lysis buffer and resuspended in 6 ml of medium. Remove cell debris by spinning through 0.5 ml of serum placed at the bottom of the tube. Biotin-conjugated anti-mouse CD45, CD31, and Ter119 (eBiosciences) to deplete lineage-positive cells from the cells. The mixture was then depleted on a MACS LS column (Miltenyi Biotec). x10 5 Single cells were mixed with Matrigel (BD Biosciences) and incubated for 6 h. Female NSG mice, aged 8 weeks, were injected into the flank. Tumor volumes were measured at the indicated times and ovals were placed in the flank. Body size [0.5 (length x width 2 )] was used to calculate the

[0122] Serum analysis and toxicity studies Blood samples from individual mice are collected at the end of the experiment under terminal anesthesia using cardiac puncture. Serum was separated from the blood within 1 hour by centrifugation at 500 g for 10 minutes. The samples were aliquoted and stored at -80°C for further testing. MP) and complete blood count (CBC) were performed at the Stanford Veterinary School Animal Diagnostic Laborator at rvice Center Toxicity studies, including autopsy and comprehensive histopathological analysis of each organ, were performed by performed by a veterinary pathologist.

[0123] Treatment of mice with eCNTFR-Fc The tumors were 100 mm 3 When the average tumor size reached 100, the average tumor size per group was Mice were stratified into treatment groups based on tumor size. -Fc (10 mg / kg) or PBS (vehicle) intraperitoneally 3 times a week for 2 to 4 weeks. Mice were weighed at the start of the study and periodically during drug treatment. Tumor volumes were measured with digital calipers 3–4 times a week.

[0124] Knockdown studies in xenografts pLKO shRNA constructs were obtained from Thermo Fisher Scientific Lentivirus for each construct was purchased from Eppendorf Biotech. Generated by transfecting 293 cells, 1 day after transfection The viral supernatants were collected on days 1 and 2 and pooled on day 2. The viral supernatants were then diluted to 0. The plates were filtered through a 45 μM PES filter. Approximately 500 μL of fresh medium was used to The virus pellet was resuspended on a thermorocker for 2 hours. ma) Using digestion buffer, dissociate the cells into a single cell suspension and filter on a 70 μM filter. The resulting cell suspension was filtered through a filter and lineage-depleted on a MACS column (as described above). The suspension was then diluted to approximately 5 x 10 per well of a 6-well plate. 6 Plated with cells, Polybrene (Sigma) and virus in the medium were incubated at 1500 rpm for 3 min at room temperature. Spin infect for 10 minutes (Sorvall XRT centrifuge) and then incubate at 37°C. After selection with puromycin (2 μg / mL), cells were trypsinized. The cells were then filtered and viable cells were counted, keeping the viable cell count constant between study groups. The remaining cells were retained for confirmation of gene knockdown. .

[0125] Cell extracts and Western blot analysis For whole cell extracts, cells were lysed in NP-40 lysis buffer (20 mM Tris-HCl l, pH 8.0, 137 mM NaCl, 10% glycerol, 1% NP-40, dH2 0, 1x protease inhibitor (Sigma P8349-1ML), and 1x phosphatase inhibitor (Sigma P8349-1ML). lysis using a tase inhibitor cocktail (Sigma P5726-1ML) for 15 min. The tumor was thawed and placed on ice in Bio-Genomics prior to lysis. Mechanically using a PRO200 homogenizer (PRO Scientific) Protein concentration was determined by BCA assay (Thermo Fisher). Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and purified by Bio The analysis was performed using an ad Chemi Doc instrument. The antibodies used were as follows: P- AKT(#4060, Cell Signaling, 1:1000), T-AKT(# 75692, Cell Signaling, 1:1000), P-ERK1 / 2(#4 370, Cell Signaling, 1:1000), T-ERK1 / 2(#469 5, Cell Signaling, 1:1000), P-STAT3(#9145, C ell Signaling, 1:1000), T-STAT3(#9139, Cell Signaling, 1:1000), GAPDH(#9485, Abcam, 1:1 000).

[0126] Histology and immunohistochemistry Tissue specimens were fixed in 10% buffered formalin for 24 h and then in 70% ethanol until paraffin embedding. Five-micrometer sections were stained with hematoxylin and eosin (HE) or stored in a cool, dry place. Immunohistochemistry was performed using formalin-fixed, paraffin- fixed, and paraffin-treated tissue. This was performed using the biotin-avidin method on polystyrene-embedded mouse and human tissue sections. The following antibodies were used (at the indicated dilutions): P-Akt (#4060, Cell S ignaling, 1:100), P-ERK1 / 2(#4370, Cell Sign aling, 1:400), P-Histone H3(#9701, Cell Sig naling, 1:200), Cleaved Caspase 3(#9661, Ce ll Signaling, 1:200), CNTFR(#175387, Abcam, 1:50). Sections were developed with DAB and counterstained with hematoxylin. Analysis of tumor area and IHC analysis was performed using ImageJ software to measure pixel-by-pixel. This was done by.

[0127] Cell assay Cell viability: In a total volume of 100 μL of medium containing 10% bovine growth serum (BGS), Cells were plated in 96-well plates at 2,000 cells per well (optimal density for growth). After 24 hours of incubation, cell viability was assessed using AlamarBlue ( The assay was performed using the Thermo Fisher Scientific (Thermo Fisher Scientific) according to the manufacturer's instructions for 7 days. The evaluation was carried out.

[0128] Colony formation assay: For long-term colony formation assays, 10,000 cells per well were used. 00–50,000 cells were seeded in a 6-well plate. After 12 days, the cells were incubated in methanol. The tissue was fixed in PBS, stained with crystal violet, photographed, and quantified.

[0129] 3D spheroid methylcellulose assay: For anchorage-independent sphere growth, The cells were plated in 2 mL of complete medium supplemented with 0.5% methylcellulose in a 24-well ultra-low attachment plate. Plates were seeded (20,000 viable cells per well) for 9–20 days. Spheres were then formed (varies depending on the cell line). The spheres were then visualized under a Leica Dmi8 microscope ( The size and number of spheres were quantified using ImageJ.

[0130] Analysis of Ras-GTP levels The level of activated Ras-GTPase was measured using a previously published method similar to that used in the production Ras GTPase ELISA kit (Abcam 134640) was prepared according to the original instructions. Briefly, 10% bovine serum albumin (BSA) was used to determine the cellular markers of IL-16 in 10 cm tissue culture dishes. 1 x 10 cells in RPMI medium supplemented with growth serum and 1% penicillin / streptomycin. 6 Seed cells and incubate at 37°C in 5% CO2 until cells reach 60% confluence. The cells were then resuspended in RPMI and 1% penicillin / streptomycin. The cells were serum-starved for 24 hours with CLCF1 (10 nM) and eCNT. Incubated in FR-Fc (2.5 μM) at 37°C in 5% CO for 20 min The medium was then removed, and the cells were washed once with ice-cold PBS and processed according to the manufacturer's protocol. did.

[0131] statistics Kaplan-Meier survival curves were calculated using the survival time of each mouse from all litter groups. The log-rank test was used to test for significant differences between groups. For gene expression analysis, statistical significance was determined using Prism GraphPad software. Analysis was performed by Student's t-test using the (two-tailed unpaired test depending on experimental variance) A single or paired t-test combined with Dunnett's multiple correction test was used depending on the experiment. Systematically examined using F-tests for both one-way and two-way ANOVA. *P<0.05, **P<0.01, ***P<0.001. Data are from in vivo studies. as mean ± SD for in vitro experiments and mean ± SEM for in vivo experiments In the box plot, the boxes represent the 25th and 75th percentiles. The midline indicates the median, and the whiskers extend to the lowest / highest values ​​within 1.5 times the interquartile range. Drink.

[0132] Logistic regression model Table taken from Stargazer v.5 by Marek Hlavac of Harvard University. 2.2. The model included only blood CLCF1 levels (pg / mL). No other covariates were used.

[0133] Recombinant CLCF1 generation cDNA encoding CLCF1 without the signal peptide sequence (28-225) was , using BsaI and XhoI restriction sites to construct pE The plasmid was cloned into T28b and amplified in DH10B cells. The plasmid was transformed into Rosetta gami cells. The inclusion bodies were incubated with 5 mM D Dissolved in 60% ddH2O, 40% acetonitrile, 0.1% TFA containing TT CLCF1 was purified using reversed-phase high-performance liquid chromatography (RP-HPLC). Protein purity was further analyzed using SDS-PAGE and Nanodrop Protein was quantified using the Thermo Scientific 2000. The extinction coefficient for quantifying the concentration is 39,549 M -1 cm -1 The value of was used.

[0134] Generation of soluble CNTFR, LIFR, and gp130 CNTFR(1-342), LIFR(1-534), and gp130(1-619 ) into the pAdd2 plasmid and incubated with DH1 The purified plasmid was amplified in 0B cells. For expression, the purified plasmid was transfected with PEI (#23966- 2, Polysciences) into human HEK293 cells. Briefly, PEI was dissolved in dH2O to a concentration of 1 g / L. For a transfection volume of 1 L, use 0.5 mg of purified DNA and 1 mL of Each of the PEIs was added to 10 mL of OptiPro serum-free medium (#12309-019, Ther (Fisher Scientific) and mixed immediately. After 15 minutes, The solution was added dropwise to 500 mL of cells. The cells were incubated in a humidified incubator at 37°C and 5% CO2. The cells were incubated on a rotary shaker at 120 RPM in a centrifuge. Protein A (#101142, Fisher Scientific) affinity chromatography was performed. Proteins containing a hexahistidine tag were purified using a Ni-N column. The product was purified using a TA (#30210, Qiagen) affinity column. The protein was further purified using fractionation chromatography. The following extinction coefficients were used: CNTFR mutant: 70,275 M -1 cm -1 , CNTs FR-Fc mutant: 206,410M -1 cm -1 , gp130:130,470M -1 cm -1 , gp130-Fc: 326,800M -1 cm -1 , LIFR: 98,610 M -1 cm -1 , and LIFR-Fc: 263,080M -1 cm -1 .

[0135] Generation and screening of a CNTFR library created via error-prone PCR ng CNTFR was expressed in yeast as a gene fusion to the agglutinin mating protein Aga2p. The cDNA encoding the human CNTFR extracellular domain (residues 18-342) was isolated from Nh Use the eI and BamHI restriction sites to clone the pCTCON2 yeast display plasmid The CNTFR extracellular domain was used as a template to identify the error promoter. Create a loan library and use Taq polymerase (#50-811-694, Fisher Scientific) Mutations were introduced using 5 mM NaCl (R Scientific) and 55 mM MgCl2. Different concentrations of MnCl2 (0, 0.01, 0.05, 0.1, and 0.15 mM) were used. The products from these reactions were analyzed using gel electrophoresis. The purified mutant cDNA and linearized plasmid were then transformed with EBY100 enzyme. The resulting chromosomes are electroporated into the mother, where they assemble in vivo through homologous recombination. The library size was determined by dilution plating and colony counting. 8.1 x 10 7 It was estimated that:

[0136] BD Aria II flow cytometer (Stanford FACS Core) High affinity fluorescently labeled cells were analyzed using fluorescence-activated cell sorting (FACS) using the Yeast expressing CNTFR mutants were isolated and analyzed using a BD FACSCalibur. The screening was performed using a 1 mg / mL BSA containing CLCF1 concentration: Equilibrium binding was achieved by incubating yeast at room temperature in phosphate-buffered saline (PBSA). The conditions were: 20 nM CLCF1 for 3 hours for selection 1, and 20 nM CLCF1 for selection 2. For selection 1, 2 nM CLCF1 was used for 6 hours, and for selection 2, 0.5 nM CLCF1 was used for 12 hours. After incubation with CLCF1, yeast were pelleted, washed, and diluted with CLCF1 at a 1:500 ratio. in PBSA containing chicken anti-c-Myc (#A21281, Invitrogen) The yeast were then washed, pelleted, and incubated at 4°C for 30 minutes with goat anti- Chicken PE (#sc-3730, Santa Cruz Biotech) and mouse PE (#sc-3730, Santa Cruz Biotech) were used. Anti-HIS Hilyte Fluor 488 (#61250-H488, Anasp Secondary labeling was performed on ice for 30 minutes using PBSA containing ec).

[0137] Selected clones were expanded and subjected to further rounds of FACS. After the final round of screening, a Zymoprep kit (#50-444-107, Zymo Plasmid DNA was collected using a cloning system (Denver Research Corp.) and transformed into DH10B elastomers. Transform into microcompetent cells and isolate using a plasmid miniprep kit. Sequencing was performed by Molecular Cloning Laboratories. The samples were analyzed using a FACSCalibur (BD Biosciences). and analyzed the data using FlowJo software (Treestar Inc.). Ta.

[0138] Created via the Staggered Extension Process (StEP) Generation and screening of the CNTFR library The StEP method was performed as previously described, and the resulting library was displayed on yeast. Briefly, isolated from the final round of selection of an error-prone PCR library Twenty unique sequences were randomly selected from the yeast population. 1 ng of each template was used for the synthesis. Combine a total of 20 ng of template and add each to a final concentration of 0.15 μM in sterile dH2O. Primers, 1x PCR buffer, 200 µM dNTP mix, 1.5 mM MgCl 2 and 2.5 U of Taq polymerase, and the total volume was 50 μL. The protocol was run for 100 cycles using the following parameters: 94°C for 30 seconds (denaturation); ) and 55°C for 10 seconds. The products from these reactions were purified using gel electrophoresis. The purified mutant cDNA and linearized plasmid were transformed into EBY100 yeast. Electroporation, where they assemble in vivo through homologous recombination. The library size was determined to be 7.9 × 10 by dilution plating. 7 It was estimated that:

[0139] The screen uses one equilibrium binding selection followed by two kinetic off-rate selections. For dynamic off-rate selection, yeast were incubated with 2 nM CLCF1 at room temperature for 2 h. After incubation, cells were washed twice to remove excess unbound CLCF1 and incubated with 20 nM w Resuspend in PBSA containing tCNTFR-Fc to prevent rebinding of dissociated CLCF1. Regarding the length of the unbinding step, 10 hours were used for sort 2 and 24 hours for sort 3. The library was stained as described above to detect CLCF1 binding and c-myc expression. The clones with the highest CLCF1 binding / c-Myc expression ratio (0.5-1%) were selected by FACS. Select the clones with the highest binding to CLCF1. The library was enriched and plasmid DNA was isolated and sequenced as described above.

[0140] Library generation and screening of CNTFR mutants that do not bind to LIFR To generate CNTFR mutants with reduced binding to LIFR, we used error-prone PC Using R, introduce random mutations into CNTFR mutant 4, approximately 1 x 10 8 Estimated number of individuals A library containing a diverse group of transformants was created. The resulting library was The fusion protein was displayed on the surface of the mouse, and the fusion protein bound to LIFR-Fc in the presence of CLCF1. Screening was performed to isolate the population with reduced signal. To maintain affinity, positive selection was performed against 0.5 nM CLCF1. Alternating with negative selection against increasing concentrations of LIFR-Fc After six rounds of selection, two consensus mutations emerged. (Y177H and K178N). These mutations contribute additively to the reduction of LIFR binding. did.

[0141] Yeast display CNTFR binding assay Yeast displaying the CNTFR construct were incubated with various concentrations of CLCF1 at room temperature for 12 hours. Incubation was allowed to reach equilibrium binding, followed by washing with PBSA and 1:5 The cells were resuspended in PBSA containing chicken anti-c-Myc antibody at a ratio of 1:100 for 30 minutes at 4°C. Next, the yeast were washed, pelleted, and incubated with a 1:100 dilution of goat anti-chicken PE antibody and mouse anti-PE antibody. Secondary standard was prepared using PBSA containing anti-HIS Hilyte Fluor 488 antibody. Identification was carried out on ice for 30 minutes. Samples were then washed and analyzed by BD Accuri flow cytometry. Samples were analyzed by flow cytometry using a BD Bioscience The data were analyzed using FlowJo software (Treestar Informatics). c) was used for the analysis.

[0142] For the assay to detect binding to β receptors, 10 nM CLCF1 was used. Various concentrations of the LIFR construct and / or gp130 construct were added to the yeast-displayed For the His-tagged constructs, mouse anti-HIS Hilyte was added. Binding was detected using Fluor 488 antibody. For detection of Fc fusion constructs, anti-mouse antibody was used. Fc Alexa 488 antibody (#A11029, Thermo Fisher Scientific) was used. Used.

[0143] Cell-free binding assay 96-well plates were coated overnight with 10 μg / mL of anti-HIS antibody or anti-mouse Fc antibody. The plates were then coated and blocked with 5% milk for 1 hour. The plates were then washed twice with PBSA. Various concentrations of soluble CNTFR-HIS or CNTFR-Fc fusion constructs were added to the The cells were incubated with 2 nM CLCF1 in PBSA at room temperature for 12 hours. The mixture was plated on a 96-well plate coated with anti-HIS antibody or anti-mouse Fc antibody, respectively. Plates were then washed twice with PBS. 00 diluted anti-CLCF1 rabbit antibody (#ab26125, Abcam) at room temperature. Incubated for 2 hours, then washed 4 times with PBS. HRP-conjugated anti-rabbit antibody (#111-035-144, Jackson Im Incubate with munoResearch) for 2 hours at room temperature and wash 4 times with PBS For reading, 1-Step Ultra TMB ELISA (#34029, The rmo Fisher Scientific) was used.

[0144] Phosphorylation assay A549 or H23 cells were cultured in 6-well plates at 50% confluence. Cells were grown until CLCF1 (10 nM) and CNTFR constructs (10 nM) were detected. After incubation in PBS at 37°C and 5% CO for 20 minutes, protease inhibitor (#P8 340, Sigma Aldrich) and phosphatase inhibitor (#P5726, S Equal amounts of lysate were collected in NP-40 buffer containing PEG-100 (Aldrich). The gel was loaded onto a 100-Tris gel and transferred to a nitrocellulose membrane. Stan blot analysis was performed using a ChemiDoc XRS system (Bio Detection was performed using NP-40 buffer (20 mM Tris pH 8.0). , 137 mM NaCl, 10% glycerol, and 1% IGEPAL / NP40 It was done.

[0145] CLCF1 cell proliferation assay 5×10 3 A549 and H23 cells were seeded and grown for 24 hours, then incubated in 0.1% Cells were serum starved by incubation in DMEM containing BSA for 24 hours. CLCF1 and CNTFR constructs were then added and incubated at 37°C / 5% CO2 for 72 hours. Next, AlamarBlue reagent (#DAL1025, Fisher Scientific) was added. Scientific) to each well and incubate at 37°C / 5% CO2 for 1 hour. Cellular metabolic activity was measured by measuring fluorescence using 560EX nm / 590EM nm. Error bars represent the standard deviation of triplicate wells. Measured against a negative control using medium alone.

[0146] Analysis of in vivo CLCF1 sequestration of eCNTFR-Fc Non-tumor-bearing NSG mice were intraperitoneally injected with 10 mg / kg body weight of eCNTFR-Fc A single dose of 100 mg / kg of 1000 mg ... The baseline CLCF1 levels were determined using untreated mice. by cardiac puncture at 12, 24, 36, 48, and 72 hours A terminal blood sample was taken at the time of euthanasia, and serum was isolated for analysis. This assay measured free, unbound CLCF1 To ensure detection of the eCNTFR-Fc, eCNTFR-Fc was used as a capture agent. Plates were coated with 10 μg / mL eCNTFR-Fc overnight at room temperature and then incubated with 5% The coated plates were washed twice with PBSA and then collected. The plates were incubated with serum for 2 hours at room temperature. The plates were then washed twice with PBS. After purification, CLCF1 was detected using polyclonal anti-CLCF1 antibody and anti-rabbit HRP. The plate was washed four times with PBS and then 1-Step Ultra TMB The ELISA was developed using ELISA.

[0147] Methods, data, agents (e.g., engineered CNTFR ligands) disclosed herein , including but not limited to reagents, Kim et al. (2019) Nature re Medicine 25:1783-1795, which is incorporated herein by reference. No. 6,299,499, filed on Dec. 1, 2003, which is incorporated herein in its entirety for all purposes.

[0148] Thus, the foregoing merely illustrates the principles of the present disclosure. Although not explicitly described or illustrated herein, it embodies the principles of the present invention and is intended to provide the spirit and scope of the present invention. It will be appreciated that various configurations may be devised that fall within the scope of the present invention. All examples and conditional language recited in the specification are based primarily on the principles of the present invention and the inventors' It is intended to aid the reader in understanding the concepts contributed to the advancement of the art by The examples and conditions are for illustrative purposes only and should not be construed as being limiting to such specifically listed examples and conditions. It should also be noted that the present invention is not limited to the above embodiments, and may be modified in various ways without departing from the spirit and scope of the present invention. All statements herein that cite the term "compound" encompass both structural and functional equivalents thereof. Additionally, such equivalents include both currently known equivalents and those developed in the future. Both the developed equivalents, i.e., any developed product that performs the same function regardless of structure, Accordingly, the scope of the present invention is intended to encompass all elements shown and described herein. It is not intended to be limited to the exemplary embodiments described.

Claims

[Claim 1] The invention described in the specification and drawings of this application.

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