FGFR4 as a target for cancer therapy

Abstract

The present invention provides novel therapeutic approaches for the treatment or prevention of FGFR4-expressing cancers, including those resistant to other therapies.

Description

Technical Field

[0001] This invention relates to the field of cancer. Specifically, this invention provides novel treatment methods for managing cancers expressing FGFR4. Background Technology

[0002] FGFR4 is a tyrosine kinase receptor involved in the regulation of various cell phenotypes, such as proliferation, differentiation, and survival. Imbalances in FGFR signaling have been shown to have oncogenic effects in many cancers (Turner and Grose, 2010). Interestingly, the function and kinase domain of FGFR4 differ from other FGF receptors, suggesting it may have unique roles compared to other FGFRs (Beenken and Mohammadi, 2009). However, due to structural differences between FGFR4 and FGFR1–3, most pan-FGFR inhibitors are insufficiently capable of competitively binding to FGFR4.

[0003] Hormone receptor-positive (HR+) tumors account for 75% of all breast cancers, exhibiting clinical and biological heterogeneity and being a leading cause of cancer death. Several clinical trials have demonstrated that endocrine therapy combined with cyclin-dependent kinase 4 and 6 (CDK4 / 6) inhibitors (CDK4 / 6i) in patients with metastatic HR+ breast cancer, using inhibitors such as palbociclib (Cristofanilli et al., 2016; Finn et al., 2016; Turner et al., 2018), ribociclib (Hortobagyi et al., 2018; Im et al., 2019; Slamon et al., 2018; Tripathy et al., 2018), or abeciclib (Goetz et al., 2017; Johnston et al., 2019; Sledge et al., 2017, 2020), improved progression-free survival (PFS) and overall survival (OS). These studies led to the approval of CDK4 / 6i as a therapy in this context. However, due to the development of CDK4 / 6i resistance mechanisms, almost all patients with advanced disease eventually experience disease progression. Therefore, it is urgent to determine the resistance mechanisms of this new standard of care.

[0004] Targeted therapies such as antibody-drug conjugates (ADCs) represent a promising approach to overcoming current treatment resistance and providing new treatment options. To date, many ADCs targeting breast cancer tumors have been approved. However, only one ADC (ENHERTU; delutecan) has been approved for the treatment of a subset of HR+ patients, specifically those with advanced (unresectable or metastatic) low HR+ / HER2 BC (Modi et al., NEJM 2022). Despite this ADC significantly improving survival, patients still ultimately experience disease progression, representing an unmet need. Recent data show that the proportion of patients experiencing progression during delutecan use was 50% in less than 10 months and as high as 80% in less than 24 months (Modi et al., NEJM 2022).

[0005] Given the above, further methods are still needed to effectively manage cancer. Summary of the Invention

[0006] The inventors have discovered that FGFR4 (fibroblast growth factor receptor 4) is expressed in cancer patients with primary or acquired CDK4 / 6i resistance.

[0007] As shown below, the inventors found that in a clinical cohort (hereinafter referred to as the CDK cohort, including baseline samples—primary and metastatic samples, and advanced samples) of patients with metastatic HR+ / HER2-negative breast cancer treated with CDK4 / 6i (including palbociclib, ribociclib, or abeciclib) in combination with endocrine therapy as first-line, second-line, or subsequent lines of treatment at the Hospital Clinic of Barcelona, ​​higher baseline FGFR4 expression was associated with poorer PFS and OS in CDK4 / 6i-treated patients, regardless of prior treatment lines. Figure 1 A). Furthermore, compared to baseline samples, FGFR4 expression was significantly higher in disease progression (PD) samples ( Figure 1 B). These data support the robustness of FGFR4 as an informative biomarker for CDK4 / 6i therapy response, regardless of the specific nature, stage, or prior treatment of the cancer.

[0008] The inventors characterized baseline, day 15, and surgical samples from 49 postmenopausal patients with HR+ / HER2-negative and PAM50 luminal type B early breast cancer who received CDK4 / 6i ribociclib in combination with letrozole (estrogen therapy) in the context of the CORALLEEN clinical trial (Prat et al., 2020). Figure 1Data in C indicate that higher FGFR4 expression was observed in surgical samples from non-responders who had been treated with CDK4 / 6i compared to responders. Therefore, these data suggest high FGFR4 expression in cases where CDK4 / 6i therapy is ineffective.

[0009] The relationship between FGFR4 and CDK4 / 6i therapy resistance / sensitivity was further elucidated by FGFR4 siRNA-mediated knockdown in both CDK4 / 6i (palbociclib) resistant and sensitive cell lines. See below. Figure 2 BD and FGFR4 silencing significantly inhibited the growth of all CDK4 / 6i resistant cell lines. Furthermore, compared to parental cell lines (MCF-7, T47D, ...), ... Figure 2 A) Compared to all CDK4 / 6i resistant cell lines (MCF7-PR, T47D-PR), IC50 was significantly lower. 50 The values ​​all increased significantly.

[0010] These data lead to the conclusion that the lack of response to CDK4 / 6i in cell lines is due to high expression of FGFR4. Therefore, FGFR4 can provide valuable information to predict whether a subject will develop a positive response to CDK4 / 6i-based therapies before initiating a specific treatment regimen: if the FGFR4 expression level is higher than the reference value, it indicates that the subject's tumor is resistant to the therapy.

[0011] In addition to the above, this marker also indicates the degree of drug resistance: the higher the expression relative to the reference value, the stronger the resistance to treatment.

[0012] In summary, this represents a significant advancement in the management of CDK4 / 6i-based therapies, as oncologists can easily determine at an early stage whether a subject will produce a positive response and to what extent by determining the level of FGFR4 in tissue biopsy or biofluid samples.

[0013] Therefore, in a first aspect, the present invention provides a method for determining the response of a subject diagnosed with cancer to a medical regimen comprising one or more CDK4 / 6i, the method comprising the steps of: (a) determining the expression level of FGFR4 in isolated test samples of the subject, and (b) comparing it with a reference value; wherein if FGFR4 is overexpressed, it indicates that the response to the one or more CDK4 / 6i will be attenuated or absent.

[0014] In a second aspect, the present invention provides the use of FGFR4 as a biomarker for predicting or monitoring a subject’s response to a medical regimen comprising one or more CDK4 / 6i.

[0015] In a third aspect, the present invention provides the use of tools for determining FGFR4 expression levels in the method defined in the first aspect of the present invention.

[0016] In a fourth aspect, the present invention provides a CDK4 / 6i in a method for treating or preventing cancer in a subject, wherein the method comprises: (i) determining the expression level of FGFR4 in an isolated test sample of the subject; (ii) comparing it with a reference value; and (ii) administering a therapeutically effective amount of CDK4 / 6i to the subject if the expression level of FGFR4 in the test sample is equal to or lower than the reference value. This aspect may alternatively be expressed as a method for treating or preventing cancer, comprising (a) determining the expression level of FGFR4 in an isolated test sample of the subject; (b) comparing it with a reference value; and (c) administering a therapeutically effective amount of CDK4 / 6i to the subject if the FGFR4 level is equal to or lower than the reference value.

[0017] In a fifth aspect, the present invention relates to a method for treating or preventing cancer (e.g., breast cancer) in a subject (e.g., a person), which is carried out by stopping the administration of CDK4 / 6i to the subject after determining that FGFR4 expression in the cancer cells of the subject is higher than a reference value.

[0018] The relationship between FGFR4 and CDK4 / 6i therapy resistance / sensitivity was further elucidated by FGFR4 siRNA-mediated knockdown in both CDK4 / 6i (palbociclib) resistant and sensitive cell lines. See below. Figure 2 BD and FGFR4 silencing significantly inhibited the growth of all CDK4 / 6i resistant cell lines. Furthermore, compared to parental cell lines (MCF-7, T47D, ...), ... Figure 2 A) Compared to all CDK4 / 6i resistant cell lines (MCF7-PR, T47D-PR), IC50 was significantly lower. 50 The values ​​were all significantly increased. The MDA-MB-453 cell line was used as a positive control, as high FGFR4 levels had been previously reported.

[0019] Using several in vitro and in vivo models provided below, the inventors have demonstrated that FGFR4 can not only provide valuable information about responses to CDK4 / 6i-based therapies, but that FGFR4 itself can serve as a therapeutic target for overcoming CDK4 / 6i resistance and achieving efficacy in cancer treatment.

[0020] As discussed in more detail below, cell lines that have acquired resistance to CDK4 / 6i have been found to be sensitive to FGFR4 knockdown; consistent with this, FGFR4 knockdown resensitized breast cancer cell lines to CDK4 / 6i in vitro. Figure 2Importantly, these effects were demonstrated in an in vivo breast cancer model using MCF7-palbociclib-resistant cells (called MCF7-PR) and FGFR4 knockdown, with or without CDK4 / 6i palbociclib treatment. FGFR4 gene downregulation reduced tumor growth in the CDK4 / 6i-resistant breast cancer model and also resensitized tumors to CDK4 / 6i palbociclib in the in vivo resistant breast cancer model. Figure 3 D). Therefore, FGFR4 inactivation restored the CDK4 / 6i response and inhibited tumor growth.

[0021] Therefore, in a sixth aspect, the present invention provides an FGFR4-binding molecule or FGFR4 inhibitor for reducing resistance to CDK4 / 6i in subjects with cancer by reducing FGFR4 expression. This aspect can alternatively be described as the use of the FGFR4-binding molecule or FGFR4 inhibitor in the preparation of a medicament for reducing resistance to CDK4 / 6i in subjects with cancer. This aspect can alternatively be described as a method for reducing resistance to CDK4 / 6i in subjects with cancer, the method comprising administering a therapeutically effective amount of the FGFR4-binding molecule or FGFR4 inhibitor to a subject in need. In one embodiment, the FGFR4-binding molecule or FGFR4 inhibitor is an anti-FGFR4 antibody or an anti-FGFR4 antibody-drug conjugate.

[0022] In a seventh aspect, the present invention also provides an FGFR4 binding molecule or an FGFR4 inhibitor for treating cancers in subjects resistant to CDK4 / 6i. This aspect can be described as the use of the FGFR4 binding molecule or FGFR4 inhibitor in the preparation of a medicament for treating cancers in subjects resistant to CDK4 / 6i. This aspect can also be described as a method for treating cancers in subjects resistant to CDK4 / 6i, the method comprising administering a therapeutically effective amount of the FGFR4 binding molecule or FGFR4 inhibitor to the subject in need.

[0023] Due to its relevance to cancer, the inventors are also working to design effective treatments for subjects suffering from cancers associated with FGFR4 expression. FGFR4 can then be used as a marker for cancer cells, and thus as a hook to specifically guide and deliver cytotoxic therapies to tumor cells associated with FGFR4 expression, such as breast cancer cells.

[0024] Therefore, the inventors have discovered that antibody-drug conjugates (ADCs) containing selective antibodies against FGFR4 are highly effective in killing several tumor cell lines expressing FGFR4 (Figures 5, 11 and 12).

[0025] Furthermore, the inventors have discovered that anti-FGFR4 ADCs comprising anti-FGFR4 antibodies that do not bind to FGFR1, FGFR2, and FGFR3 maintain high binding affinity to the target (compared to naked antibodies), and exhibit high stability (when PK curves were determined), safety (no significant nonspecific binding to DNA, insulin, and heparin was detected), and efficacy as an anticancer agent in cell lines and in vivo. See Figures 5-12 for examples.

[0026] In other words, conjugating an FGFR4-specific anti-FGFR4 antibody to a drug via a linker results in a highly stable, safe, and effective ADC. This signifies a major advancement in the field of cancer treatment, particularly in the development of effective and safe ADCs for cancer therapy.

[0027] Here, the toxic payload of the ADC is the driving force behind the therapeutic efficacy.

[0028] In view of the above, in an eighth aspect, the present invention provides an anti-FGFR4 antibody-drug conjugate, wherein the anti-FGFR4 antibody:

[0029] - Directly combined with FGFR4, and

[0030] - It is not specific for FGFR1, FGFR2 and FGFR3.

[0031] In a ninth aspect, the present invention provides a pharmaceutical composition comprising an ADC as defined in the eighth aspect of the present invention, and one or more pharmaceutically acceptable excipients and / or carriers.

[0032] In a tenth aspect, the present invention provides an anti-FGFR4 ADC or pharmaceutical composition as defined herein for the treatment or prevention of cancers expressing FGFR4. This aspect can also be described as the use of the anti-FGFR4 ADC or pharmaceutical composition as defined herein in the preparation of a medicament for the treatment or prevention of cancers expressing FGFR4. This aspect can also be described as a method of treating or preventing cancers expressing FGFR4, the method comprising administering a therapeutically effective amount of the anti-FGFR4 ADC or pharmaceutical composition of the present invention.

[0033] In an eleventh aspect, the present invention provides a method for determining or recommending the initiation of administration of an ADC as defined herein for a subject suffering from cancer, the method comprising the step of determining the expression level of FGFR4; wherein, if FGFR4 expression is confirmed, administration of an ADC as defined herein is recommended.

[0034] In a final aspect, the present invention relates to a method of treating cancer in a subject (e.g., a human) of need, the method comprising administering to the subject a therapeutically effective amount of an anti-FGFR4 antibody-drug conjugate, wherein the subject has been previously identified as having FGFR4 expression in cancer cells that is higher than a reference expression value. In one embodiment, the subject has previously failed treatment with a CDK4 / 6 inhibitor. In another embodiment, the subject discontinues CDK4 / 6 inhibitor treatment after being identified as having high FGFR4 expression in cancer cells, and before administering the anti-FGFR4 ADC. Attached Figure Description

[0035] Figure 1 (A) In the CDK clinical cohort, FGFR4 expression was associated with poor PFS and OS response to CDK4 / 6i therapy. (B) FGFR4 gene expression in baseline and PD samples in the CDK cohort. (C) Comparison of FGFR4 gene expression in responsive and non-responsive tumors after treatment with letrozole (estrogen therapy) in combination with ribociclib (CDK4 / 6i). (D) FGFR4 protein expression in primary breast tumors, lymph node metastases, and tumors refractory to CDK4 / 6i therapy.

[0036] Figure 2 (A) Top panel: Dose response of T47D and MCF7 parental cell lines and palbociclib-resistant cell lines (T47D-PR and MCF7-PR) to CDK4 / 6i palbociclib. Bottom panel: Dose response of T47D, MCF7, ZR-75-1, and BT474 cell lines to CDK4 / 6i palbociclib. (B) Growth dynamics of each cell line after FGFR4 silencing. (C) Top panel: Protein validation of FGFR4 depletion by Western blotting after expression of FGFR4 short hairpin constructs (1 or 2) or control shRNA in MCF7 and MCF7-PR cells; Bottom panel: Dose response of designated control and FGFR4-depleted cell lines to palbociclib. (D) Left panel: Western blot validation of FGFR4 depletion in ZR751 cells after expression of FGFR4 short hairpin constructs (1 or 2) or control shRNA; Right panel: Dose response of specified control and FGFR4-depleted cell lines to palbociclib. (E) Left panel: Western blot validation of FGFR4 expression; Right panel: Dose response of parental ZR751, FGFR4 overexpression (wt), and FGFR4 overexpression constitutive active form (Y367C) to palbociclib.

[0037] Figure 3(A) Growth rates of MCF7 control cells (left) and MCF7-PR cells (right) after solvent placebo or CDK4 / 6i treatment. (B) FGFR4 mRNA levels and proliferation scores of xenograft tumors from (A). (C) FGFR4 expression in samples from (A) as detected by immunohistochemistry. (D) Left: Tumor growth in control and FGFR4-depleted MCF7-PR cells with or without CDK4 / 6i treatment. Right: Representative image of the tumor at the end of the experiment.

[0038] Figure 4A -B. Binding selectivity of mAb1, mAb2, mAb3, mAb4, and mAb5 to FGFR4 vs. other FGFRs.

[0039] Figure 5A -M. In the breast cancer cell line MDA-MB-453 ( Figure 5A -B), T47D PR ( Figure 5C-5G ) and ZR-75-1 ( Figure 5H -J) and the liver cancer cell line Huh7 ( Figure 5K In ), FGFR4 antibody-mediated cytotoxicity when incubated with MMAE.

[0040] Figure 5L In the T47D PR breast cancer cell line, FGFR4 antibody-mediated cytotoxicity mediated by incubation with docalomycin.

[0041] Figure 5M In the breast cancer cell line T47D PR, FGFR4 antibody-mediated cytotoxicity mediated by incubation with MMAF.

[0042] Figure 6A -C. The ADC1 illustrated in the following section ( Figure 6A ), ADC2 ( Figure 6B ) and ADC3 ( Figure 6C Size exclusion chromatography.

[0043] Figure 7A -C. Naked mAb1 antibody, ADC1 ( Figure 7A ), ADC2 ( Figure 7B ) and ADC3 ( Figure 7C ) combined with ELISA.

[0044] Figure 8A -B. Naked mAb1 antibody, ADC1 ( Figure 8A ) and ADC2 ( Figure 8B Serum stability.

[0045] Figure 9A-B. Naked mAb1 antibody, ADC1 ( Figure 9A ) and ADC2 ( Figure 9B It binds to DNA, heparin, and insulin.

[0046] Figure 10A -B. Naked mAb1 antibody, ADC1 ( Figure 10A ) and ADC2 ( Figure 10B Pharmacokinetic evaluation of ).

[0047] Figure 11A -I. ADC1, ADC2, and ADC3 in the MDA-MB-453 breast cancer cell line expressing different levels of FGFR4 ( Figure 11A ), MDA-MB-231 ( Figure 11B ), T47D-PR ( Figure 11C ,11G),ZR-75-1 ( Figure 11D ,11H),BT474 ( Figure 11E ,11I) and the liver cancer cell line Huh7 ( Figure 11F ) cytotoxic activity.

[0048] Figure 12A -B. ADC1, ADC2 and / or ADC3 in ZR-75-1, a breast cancer model expressing FGFR4 and intrinsically resistant to CDK4 / 6i ( Figure 12A ) and BT474 ( Figure 12B The body's effects. Detailed Implementation

[0049] Terms not specifically defined herein shall have the meanings that a person skilled in the art would assign to them based on this disclosure and the context. However, in this specification, unless otherwise stated, the following terms shall have the meanings indicated and shall follow the conventions described below.

[0050] In this specification and the appended claims, the word "comprising" and its variations such as "including" and "containing" should be interpreted as inclusive. That is, where the context permits, these words are intended to convey that other elements or integers not specifically listed may be included. The word "comprising" also includes the term "consisting of".

[0051] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context explicitly indicates otherwise. When used in conjunction with the term “including,” the words “a” and “an” may mean “one” in this context, but are also consistent with the meanings of “one or more,” “at least one,” and “one or more.”

[0052] It should also be noted that the term “or” is generally used in its meaning as including “and / or”, unless the content explicitly indicates otherwise.

[0053] In a first aspect, the present invention provides a method for determining the response of a subject diagnosed with cancer to a medical treatment comprising one or more CDK4 / 6i, the method comprising:

[0054] (a) Determine the expression level of FGFR4 in the isolated test samples of this object, and

[0055] (b) Compare with reference values,

[0056] If the FGFR4 expression level is higher than the reference value, it indicates that the response to a medical regimen containing one or more CDK4 / 6i is weakened or absent.

[0057] The term "FGFR4" is a member of the fibroblast growth factor receptor superfamily. Members of the FGFR family differ from each other in ligand affinity and tissue distribution. A full-length representative protein would consist of an extracellular region (composed of three immunoglobulin-like domains), a single hydrophobic transmembrane segment, and a cytoplasmic tyrosine kinase domain. The extracellular portion of this protein interacts with fibroblast growth factors, initiating a downstream signaling cascade that ultimately influences mitosis and differentiation. The FGFR4 genome is organized with 18 exons instead of 19 or 20, compared to members 1-3. While alternative splicing has been observed, there is no evidence that the C-terminal half of the IgIII domain of this protein varies between the three alternative forms as shown in members 1-3. In the context of this invention, "FGFR4" also includes the spliced ​​form. In one embodiment, FGFR4 is human FGFR4. Human FGFR4 sequences (including variants and isotypes) are known in the art and are covered herein. Examples of illustrative, non-restrictive human FGFR4 are available in GeneBank (Gene ID: 2264, updated July 9, 2023) and UniProt (P22455, 2001-04-27 v2).

[0058] In the context of this invention, "determining a response" includes both predicting a response before the start of a medical protocol for the first or subsequent treatment cycles and monitoring the evolution of the subject during treatment, i.e., whether the subject is developing resistance and, if so, the severity of the resistance.

[0059] As used herein, the term "cancer" refers to a malignant growth characterized by disordered or uncontrolled cell growth. In one embodiment of the invention, the cancer is selected from oral squamous cell carcinoma, head and neck cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, kidney cancer, prostate cancer, sarcoma, melanoma, leukemia, lymphoma, duodenal cancer, small bowel cancer, large bowel cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, rhabdomyosarcoma, or bile duct cancer; particularly, the cancer is breast cancer, liver cancer, gastric cancer, or colorectal cancer. In a particular embodiment, the cancer is breast cancer.

[0060] In some implementations, the cancer is HER2- breast cancer. In other implementations, the cancer is HER2+ breast cancer.

[0061] In some embodiments, the cancer is HR+ / HER2- breast cancer. In some embodiments, the cancer is HR- / HER2+ breast cancer. In some embodiments, the cancer is HR- / HER2- breast cancer. In some embodiments, the cancer is HR+ / HER2+ breast cancer. In some embodiments, the cancer is triple-negative breast cancer (TNBC). In some embodiments, the cancer is HER2-enriched breast cancer. In a preferred embodiment, the cancer is HER2-enriched HR+ / HER2- breast cancer. In the context of this invention, a "CDK4 / 6 inhibitor" (also referred to as "CDK4 / 6i") means a molecule that provides CDK4 / 6 inhibition. CDK4 / 6 are serine-threonine kinases that regulate cell cycle progression. They interact with cyclin D, leading to phosphorylation of retinoblastoma (Rb) protein, which in turn causes the cell cycle G1 checkpoint to advance to S phase. ER signaling can upregulate cyclin D1 levels and activate the CDK4 / 6 signaling pathway. Inhibition of CDK4 / 6 stops cell cycle progression and induces cancer cells to exit the cell cycle and enter a senescent or quiescent state. Therefore, in the context of this invention, "CDK4 / 6 inhibitor" refers to any agent that inhibits the activity of CDK4 and CDK6 kinases.

[0062] In some embodiments, CDK4 / 6i may be derived from pyridopyrimidine, pyrrolopyrimidine, or indobenzocarbazole compounds.

[0063] In some embodiments, CDK4 / 6i may be a derivative of palbociclib (PD0332991), LEE-011 (ribociclib), LY2835219 (abeciclib), G1T28-1, SHR6390, or P276-00, or any of these derivatives. In some embodiments, CDK4 / 6i is palbociclib.

[0064] The terms “palbociclib” or “PD-0332991” (trade name or brand name Ibrance) generally refer to selective CDK4 and CDK6 inhibitors.

[0065] The term "LEE-011" or "Ribocini" usually refers to the selective CDK4 and CDK6 inhibitor with CAS number 1211441-98-3.

[0066] The term “LY2835219” or “abexinib” usually refers to the oral selective CDK4 and CDK6 inhibitor with CAS number 1231929-97-7.

[0067] The term “G1T28-1” refers to a selective CDK4 and CDK6 inhibitor developed and tested by G1 Terapeutics Inc.

[0068] The term "SHR6390" refers to a selective CDK4 and CDK6 inhibitor developed and tested by Jiangsu Hengrui Medicine Co., Ltd.

[0069] The term "P276-00" refers to the selective CDK4 and CDK6 inhibitor with CAS number 920113-03-7.

[0070] In some embodiments, CDK4 / 6i may include, or be composed of, antisense oligonucleotides, shRNA molecules, and / or siRNA molecules that specifically inhibit the expression or activity of CDK4 and / or CDK6. A non-limiting example of CDK4 / 6i includes an antisense, shRNA, or siRNA nucleic acid sequence that is substantially identical (i.e., mostly but not identical) to at least a portion of the CDK4 or CDK6 nucleic acid sequence, wherein the identity of that portion relative to the CDK4 or CDK6 sequence is at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, wherein the percentage identity can be determined as is known in the art, for example, using BLAST or FASTA software. Sequence identity can be determined using suitable algorithms for sequence alignment and sequence identity determination, which is known in itself. Exemplary but non-limiting algorithms include those based on the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10), such as the "Blast 2 sequences" algorithm, for example using published default settings or other suitable settings (e.g., for the BLASTN algorithm: gap open penalty = 5, gap extend penalty = 2, mismatch penalty = -2, match reward = 1, gap decrease value = 50, expected value = 10.0, word length = 28; or for the BLASTP algorithm: matrix = Blosum62, gap open penalty = 11, gap extend penalty = 1, expected value = 10.0, word length = 3).

[0071] In some embodiments, the complementary portion may constitute at least 10 nucleotides, or at least 15 nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, or at least 30 nucleotides, and the length of the antisense nucleic acid, shRNA, or siRNA molecule may be at most 15 nucleotides, or at most 20 nucleotides, or at most 25 nucleotides, or at most 30 nucleotides, or at most 35 nucleotides, or at most 40 nucleotides, or at most 45 nucleotides, or at most 50 nucleotides, or at most 75 nucleotides, or at most 100 nucleotides.

[0072] In the context of this invention, the term "expression" or "expression level" is understood to mean the presence of a protein or messenger RNA that can be detected by standard techniques known in the art.

[0073] In the context of this invention, the term "higher expression," also known as "overexpression," is understood to mean the presence of a protein or messenger RNA that can be detected by standard techniques known in the art, at a level higher than a reference value compared to a reference sample or the median of a reference population.

[0074] In the context of this invention, the term "reference sample" is understood to refer to a sample from a healthy subject. In one embodiment, the cancer is breast cancer, and the reference sample is a breast tissue sample from a healthy subject.

[0075] In this invention, the term "reference value" should be understood as a predefined value for a given molecular marker, derived from the level of said molecular marker in a sample or sample set. If the expression level is determined at the protein level, the "reference value" is a predefined value for the protein quantity, and if the expression level is determined at the mRNA level, the "reference value" is a predefined value for the mRNA quantity. Samples are taken from subjects or groups of subjects whose presence, absence, stage, or course of disease has been correctly determined previously. This value is used as a threshold to distinguish between subjects with the condition to be analyzed and those without.

[0076] The objects from which the "reference value" is derived may include objects where the condition does not exist, objects where the condition exists, or both. Those skilled in the art, using ordinary knowledge, can select objects or groups of objects more suitable for obtaining reference values ​​for each method of the present invention. Methods for obtaining reference values ​​from a selected group of objects are well known in the art. In one embodiment, the reference value is determined from a sample or group of samples from healthy subjects.

[0077] In one embodiment of the method of the present invention, the expression level of FGFR4 is determined before the start of a medical regimen. Therefore, FGFR4 can be used as a predictive biomarker to determine whether a subject will respond to therapy, whether because the subject will be given a CDK4 / 6 inhibitor for the first time or because the subject will repeat a CDK4 / 6 treatment regimen already provided at other stages of the disease. In a particular embodiment of the first aspect of the invention, the subject being analyzed will be given a medical regimen comprising one or more CDK4 / 6 inhibitors for the first time. In an alternative embodiment, the subject has been treated with one or more CDK4 / 6 inhibitors before the FGFR4 expression level is determined. In a further alternative embodiment of the first aspect of the invention, the expression level of FGFR4 is determined during the CDK4 / 6 inhibitor treatment regimen to monitor for the development of CDK4 / 6 inhibitor resistance (when an increase in FGFR4 expression is detected).

[0078] In another embodiment of the first aspect of the invention, the medical treatment further includes endocrine therapy.

[0079] In the context of this invention, the term "endocrine therapy" should be understood as any therapy designed to interrupt the signaling produced by estrogen binding to estrogen receptors. Non-limiting examples include treatments that lower estrogen levels and treatments designed to block estrogen receptors, such as the administration of the drug tamoxifen. Non-limiting examples of endocrine therapy include tamoxifen, aromatase inhibitors, goserelin, leuprorelin, fulvestrant, anastrozole, and raloxifene. For example, endocrine therapy includes estrogen receptor antagonists such as fulvestrant, raloxifene, tamoxifen, toremifene, azoxifen, LU 335563 (desmethylazoxifene), LU 335124, LU 326315, CHE-4227, nafoxitin, lasoxifene, 21if Liben, 20Ln106, LN10L20. (A-007), TAδ-108, Badoxifene acetate (1-{4-[2-(azacycloheptane-1-yl)ethoxy]benzyl}-2-(4-hydroxyphenyl)-3-methyl-1H-indole-5-ol acetate), EKA-923, Alfyxifen, (2)-4-hydroxytamoxifen, Enclomiphene, Femmiphene, Acobifen, EM-652, EM-800, Droloxifene, Edocoxifene, OA5638, TAT-59, OA-7603, Sencomycin, Levomeroxifene, 1C1-164384, BB-3040, CH-4893237, 8K16158, 8K16137, Kai-1901, 8EKM 3471 (P8K-3471), NMK 3339, NMK 3656, SS 8490, 11b-fluoro-7-[5-(methyl-3-[(4,4,5,5,5-pentafluoropentyl)thioalkyl]propylamino)pentyl]estryl-1,3,5(10)-trien-3,17b-diol (8H 646), 11b-fluoro-17a-methyl-7a-5-[methyl(8,8,9,9,9-pentafluorononyl)amino]pentylestryl-1,3,5(10)-trien-3,17b-diol or (+)-3-(4-hydroxyphenyl)-2-[4-(2-piperidin-1-ethoxy)phenyl]-4-(trifluoromethyl)-2H-chromene-7-ol, etc.

[0080] In another embodiment of the first aspect of the invention, determining the expression level of FGFR4 includes determining the amount of FGFR4 mRNA.

[0081] The expression level of FGFR4 mRNA can be determined by quantitative real-time PCR (qPCR), reverse transcription-qPCR (RT-qPCR), nucleic acid microarray, digital molecular barcoding (nCounter Nanostring), branched DNA (bDNA) signal amplification (Quantigene), mass spectrometry (Sequenom), or a combination of these methods. Suitable reagents and kits are available commercially, and those skilled in the art only need to follow the manufacturer's instructions. Illustrative examples are provided below.

[0082] Alternatively, the expression level of FGFR4 can be determined at the protein level. In this embodiment, protein biomarkers include, but are not limited to, the natural sequence polypeptide, isotype, chimeric polypeptide, all homologs, fragments, and precursors of the biomarker, including modified forms of the polypeptide and its derivatives.

[0083] In any of the embodiments provided above or below, the expression level can be determined by immunochemistry.

[0084] As used herein, the term "immunochemistry" refers to various techniques for detecting antigens (typically proteins and peptides, and in this invention, any of the aforementioned proteins alone or in combination) in a sample by utilizing the principle of antibody-specific binding to the antigen.

[0085] Visualizing antibody-antigen interactions can be achieved in a variety of ways. In the most common case, the antibody is coupled to an enzyme (such as a peroxidase) that can catalyze a color-producing reaction.

[0086] Alternatively, antibodies can also be labeled with fluorophores, such as fluorescein or rhodamine. Immunochemical techniques can be direct or indirect. Direct methods are one-step staining methods involving the direct reaction of labeled antibodies (e.g., FITC-conjugated antiserum) with the antigen. While this technique is simple and rapid because it uses only one antibody, it is less commonly used due to less signal amplification (e.g., compared to indirect methods) and lower sensitivity. Indirect methods involve an unlabeled primary antibody (first layer, binding to the target antigen in the sample) and a labeled secondary antibody (second layer, reacting with the primary antibody). If the secondary antibody is conjugated to a fluorescent or enzyme reporter gene, each primary antibody binds to multiple secondary antibodies, resulting in signal amplification, making this method more sensitive than direct detection strategies.

[0087] If the secondary antibody is conjugated to several biotin molecules, it can recruit avidin, streptavidin, or neutral avidin-enzyme complexes, thereby achieving further amplification. In addition to higher sensitivity, the indirect method has the advantage of requiring only a relatively small amount of standard-conjugated (labeled) secondary antibody.

[0088] Using the direct method requires labeling each primary antibody for each target antigen. It's important to remember that immunochemistry techniques can also be used to detect certain nucleic acid sequences if the tagged nucleic acid probe (designed to specifically bind to a particular target nucleic acid sequence) can subsequently be detected with a labeled antibody.

[0089] Therefore, the protein can be detected by using a tagged nucleic acid designed to bind to a specific sequence of the target protein RNA, and then by detecting the tagged nucleic acid with a labeled antibody that selectively binds to the tag.

[0090] Suitable immunoassay procedures include enzyme-linked immunosorbent assay (ELISA), enzyme immunospot assay, agglutination assay, antibody-antigen-antibody sandwich assay, antigen-antibody-antigen sandwich assay, immunochromatography, or other immunoassays well known to those skilled in the art.

[0091] In another embodiment of the method of the present invention, an antibody or fragment thereof capable of binding to a target is used to determine the expression level of a protein.

[0092] In another specific embodiment of the method of the present invention, the tools for carrying out the invention are part of a kit. An antibody or fragment thereof for detecting the target protein may be included in the kit. The kit may additionally include tools (additives, solvents) for visualizing antibody-protein interactions.

[0093] In another embodiment of the method of the present invention, the test sample is selected from biological fluid samples (e.g., serum, blood, plasma), tissue samples (e.g., tissue homogenate) or cell lysates.

[0094] In one implementation, the sample is a biopsy. The term "biopsy" generally refers to a sample of cells or tissue removed from a living object for examination. A biopsy can be an excisional biopsy (i.e., when the entire mass or suspicious area is removed), a cut biopsy or core biopsy (i.e., when only a sample of tissue is removed while preserving the histological structure of the tissue cells), or a needle aspiration biopsy (i.e., when a tissue or liquid sample is removed with a needle in a manner that removes cells but does not preserve the histological structure of the tissue cells).

[0095] In any of the embodiments provided above or below, the expression level is determined in a liquid biopsy, including but not limited to circulating tumor cells, circulating tumor DNA, or the like.

[0096] Before determining the expression levels in a sample, various well-known post-collection preparation and storage techniques can be used to prepare the sample (e.g., fixation, storage, freezing, lysis, homogenization, DNA or RNA extraction, ultrafiltration, concentration, evaporation, centrifugation, etc.).

[0097] In one particular embodiment of the invention, the sample may be a formalin-fixed paraffin-embedded (FFPE) sample or a fresh frozen sample. Preferably, the sample is an FFPE sample. The method of the present invention advantageously allows for the determination of the sensitivity of human subjects with breast cancer to CDK4 / 6i in FFPE samples routinely used in clinical practice.

[0098] In one embodiment, the method of the present invention further includes (i) collecting comparison results (as responders or non-responders) and (ii) storing the information on a data carrier. In the context of the present invention, "data carrier" should be understood as any instrument, such as paper, containing meaningful informational data for the diagnosis or prognosis of endometrial cancer. The carrier can also be any entity or device capable of carrying prognostic data. For example, the carrier may include storage media such as ROM (e.g., CD ROM or 5D semiconductor ROM), or magnetic recording media (e.g., floppy disk or hard disk). Furthermore, the carrier can be a transmissible carrier, such as an electrical or optical signal, which can be transmitted via cable or optical fiber or via radio or other means. The carrier may be constituted by such a cable or other device or tool when the prognostic data is embodied in a signal that can be directly transmitted via a cable or other device or tool. Other carriers involve USB devices and computer files. Examples of suitable data carriers are paper, CDs, USB drives, computer files in a PC, or audio recordings containing the same information.

[0099] In a third aspect, the present invention provides the use of a tool for determining FGFR4 expression levels in the method defined in the first aspect of the invention. All embodiments provided under the first aspect of the invention related to the determination of FGFR4 expression are also embodiments of the third aspect of the invention.

[0100] In a fourth aspect, the present invention provides a CDK4 / 6i in a method for treating or preventing cancer in a subject, wherein the method comprises: (i) determining the expression level of FGFR4 in an isolated test sample of the subject; (ii) comparing it with a reference control value; and (ii) if the expression level of FGFR4 in the test sample is equal to or lower than the level of the reference value, administering a therapeutically effective amount of CDK4 / 6i to the subject. All embodiments provided above under the first aspect of the invention are also embodiments of the fourth aspect of the invention.

[0101] In the context of this invention, "equal to reference value" covers situations where there is no statistically significant difference between the expression level of the test sample and the reference value.

[0102] The term "treatment" includes both therapeutic management of an existing disease or condition, such as treatment for existing cancer, and preventative or preventive measures aimed at preventing or reducing the chances of developing adverse conditions, such as preventing the occurrence, development, and progression of cancer. Beneficial or desired clinical outcomes may include, but are not limited to, reducing tumor burden or decreasing the size and number of metastases, alleviating disease severity, stabilizing (i.e., not worsening) the disease state, delaying or slowing disease progression, and improving or alleviating the disease state. "Treatment" can also mean extended survival relative to the expected survival without treatment. "Improvement" refers to a reduction in the number or severity of cancer signs or symptoms.

[0103] As used in this invention, the term "prevention" or "avoidance" refers to the suppression of the complete development of a disease.

[0104] The method provided by this invention allows for the administration of a therapeutically effective amount of CDK4 / 6i to subjects suffering from cancer, particularly breast cancer, including HR(+)- breast cancer, as taught herein, and these subjects will benefit from such treatment.

[0105] In the context of this invention, the term "therapeuticly effective amount" refers to the amount of an active compound that elicits a biological or medical response in a subject being sought by a surgeon, researcher, veterinarian, physician, or other clinician, which may in particular include relief of symptoms of the treated disease or condition. Methods for determining the therapeutically effective dose of a compound (depending on a particular aspect, a CDK4 / 6i or FGFR4 inhibitor) are known in the art. In one embodiment, the therapeutically effective amount is the amount required to eliminate a tumor, reduce its size, or prevent metastasis.

[0106] The appropriate therapeutic dose of CDK4 / 6i, as taught in this article, can be determined by a qualified physician based on the nature of the CDK4 / 6i, the condition and severity of the disease, and the patient's age, body size, and condition.

[0107] Without limitation, the typical dose range of the CDK4 / 6i to be administered (e.g., palbociclib) may be from about 10 mg to about 1000 mg per dose. For example, the dose range of the CDK4 / 6i to be administered (e.g., palbociclib) may be from about 100 mg to about 500 mg or from about 75 mg to about 200 mg per dose. Alternatively, the CDK4 / 6i palbociclib may be administered by intravenous administration (usually a 50 mg intravenous dose) as an infusion (e.g., a 4-hour infusion).

[0108] For example, CDK4 / 6 inhibitors such as ribociclib (LEE011) can be administered orally daily for 3 weeks followed by a 1-week break, in a 28-day cycle (50-600 mg, usually 600 mg). For example, CDK4 / 6 inhibitors such as LY2835219 (usually 150-200 mg) can be administered orally every 12 hours in a 21- or 28-day cycle.

[0109] In a sixth aspect, the present invention provides an FGFR4 binding molecule or FGFR4 inhibitor for reducing resistance to CDK4 / 6i in subjects with cancer.

[0110] In a seventh aspect, the present invention also provides an FGFR4 binding molecule or an FGFR4 inhibitor for treating cancers in subjects resistant to CDK4 / 6i. In an equivalent aspect, the present invention provides an FGFR4 binding molecule or an FGFR4 inhibitor in a method for treating subjects having cancer cells resistant or refractory to CDK4 / 6 inhibitor therapy.

[0111] As used herein, an "FGFR4 inhibitor" is an entity that inhibits FGFR4. Preferably, the FGFR4 inhibitor is a selective FGFR4 inhibitor, and for example, it is a compound that selectively inhibits FGFR4 compared to FGFR1, FGFR2, and FGFR3. The FGFR4 inhibitor can be a small molecule or a large molecule, such as an antibody or an antibody-containing conjugate. In some embodiments, the FGFR4 inhibitor selectively binds to FGFR4. In some embodiments, the FGFR4 inhibitor reduces the mRNA level of FGFR4. In some embodiments, the FGFR4 inhibitor reduces the protein expression level of FGFR4. In some embodiments, the FGFR4 inhibitor reduces the activity of FGFR4. In some embodiments, the FGFR4 inhibitor reduces the activity of downstream targets of FGFR4. In some embodiments, the FGFR4 inhibitor is an ADC comprising an anti-FGFR4 antibody, wherein the FGFR4 inhibitor is characterized by reducing the mRNA level and / or protein expression of FGFR4, reducing the activity of FGFR4, and / or reducing the activity of downstream targets of FGFR4. In some embodiments, the FGFR4 inhibitor is an ADC comprising an anti-FGFR4 antibody that selectively binds to FGFR4, as described in the eighth aspect of the invention.

[0112] As used herein, an "FGFR4 binding molecule" is an entity that binds to FGFR4. Preferably, the FGFR4 binding molecule selectively binds to FGFR4. In some embodiments, the FGFR4 binding molecule is a selective FGFR4 inhibitor. In some embodiments, the FGFR4 binding molecule is a compound that selectively binds to FGFR4 compared to FGFR1, FGFR2, and FGFR3. The FGFR4 binding molecule can be a small molecule or a large molecule, such as an antibody or an antibody-containing conjugate. In some embodiments, the FGFR4 binding molecule selectively inhibits FGFR4. In some embodiments, the FGFR4 binding molecule reduces the mRNA level of FGFR4. In some embodiments, the FGFR4 binding molecule reduces the protein expression level of FGFR4. In some embodiments, the FGFR4 binding molecule reduces the activity of FGFR4. In some embodiments, the FGFR4 binding molecule reduces the activity of downstream targets of FGFR4. In some embodiments, the FGFR4 binding molecule is an ADC comprising an anti-FGFR4 antibody that selectively binds to FGFR4, as described in the eighth aspect of the invention. In some implementations, the FGFR4-binding molecule is an ADC.

[0113] In one embodiment of the sixth or seventh aspect of the present invention, the FGFR4 inhibitor is a small molecule. Many well-known small molecules in the prior art have the ability to inhibit FGFR4, particularly the ability to irreversibly inhibit FGFR4. Illustrative and non-limiting examples of FGFR4 inhibitors are: Fisogatinib (Blu-554; Blueprint Medicines), H3B-6527 (N-[2-[[6-[[[(2,6-dichloro-3,5-dimethoxyphenyl)amino]carbonyl]methylamino]-4-pyrimidinyl]amino]-5-(4-ethyl-1-piperazinyl)phenyl]-2-acrylamide; CAS No. 170225966-2; H3 Biomedicine), Roblitinib (FGF40, CAS No. 1708971-55-4; Novartis), INCB062079 (Ruggeri B. et al., 2017), and U3-1784 (WO 2016 / 023894, Daiichi Sankyo).

[0114] In an alternative embodiment of the sixth aspect of the invention, the FGFR4 inhibitor is a blocking antibody. Illustrative and non-limiting examples of such antibodies are provided in the following patent applications: WO2009 / 009173, WO2007 / 136893, WO2012 / 138975, WO2010 / 026291, WO2008 / 052798, WO2010 / 004204, WO2014 / 105849, WO2014 / 165287 and WO2016 / 023894, WO2014 / 144737, WO2 The contents of WO2014 / 145909, WO2014 / 011900, WO2015 / 057963, WO2015 / 057938, WO2015 / 030021, WO2015 / 107171, WO2015 / 059668, WO2016 / 064960, WO2016 / 134320, WO2016 / 134314, and WO2016 / 134294 are all incorporated herein by reference.

[0115] In the context of this invention, the term "reduction of resistance to CDK4 / 6i" refers to the partial or complete recovery of sensitivity (i.e., response) to CDK4 / 6i once cancer cells have been treated with an FGFR4 inhibitor. Methods for determining reduction of resistance are well known in the art.

[0116] In one embodiment of the sixth or seventh aspect of the invention, the FGFR4 inhibitor is administered to a subject already receiving CDK4 / 6i-based therapy, either simultaneously, sequentially, or separately. In an alternative embodiment of the sixth aspect of the invention, the FGFR4 inhibitor is administered to a patient who has not yet received any CDK4 / 6i therapy.

[0117] In some implementations, the subject has previously received CDK4 / 6 inhibitor therapy.

[0118] In other embodiments, the subject has not previously received CDK4 / 6 inhibitor therapy.

[0119] In some implementations, the subject has previously received endocrine therapy. In other implementations, the subject has not previously received endocrine therapy.

[0120] In some embodiments, the subject has previously received CDK4 / 6 inhibitor therapy and endocrine therapy. In other embodiments, the subject has not previously received CDK4 / 6 inhibitor therapy and has not previously received endocrine therapy. The cancer may be as described above. In some preferred embodiments, the subject has metastatic HR+ / HER2- breast cancer and has previously been treated with a CDK4 / 6 inhibitor in combination with endocrine therapy.

[0121] In an eighth aspect, the present invention provides an ADC comprising an anti-FGFR4 antibody that selectively binds to FGFR4.

[0122] In the context of this invention, the term "antibody" is an immunoglobulin molecule capable of specifically binding to a target (e.g., carbohydrates, polynucleotides, lipids, peptides, etc.) through at least one antigen recognition site located in the variable region of an immunoglobulin molecule. As used herein, unless the context otherwise requires, the term is intended to cover not only complete polyclonal or monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab′, F(ab′)2, Fv) and domain antibodies (including shark and camel antibodies), as well as antibodies comprising antibody moieties, multivalent antibodies (e.g., COVX-BODY). TM Immunoglobulins include multispecific antibodies (e.g., bispecific antibodies, provided they exhibit the desired biological activity), fusion proteins of antibody fragments as described herein, and any other modified conformations of immunoglobulin molecules containing antigen recognition sites. Antibodies include any class of antibodies, such as IgG, IgA, or IgM (or their subclasses), and antibodies do not need to be of any particular class. Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant domains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known. In one embodiment, the immunoglobulin is a human, mouse, monkey, or rabbit immunoglobulin. In another embodiment, "antibody" is a full-length immunoglobulin or a fragment thereof (e.g., Fab, Fab′, F(ab′)2, Fv).

[0123] As used herein, the term "Fab-containing polypeptide" refers to a polypeptide containing a Fab fragment, a Fab' fragment, or a "(Fab')2 fragment." Fab-containing polypeptides may contain part or all of the wild-type hinge sequence (typically located at the C-terminus of the Fab moiety of the polypeptide). Fab-containing polypeptides may be obtained from or derived from any suitable immunoglobulin, such as from at least one of the various IgG1, IgG2, IgG3, or IgG4 subtypes, or from IgA, IgE, IgD, or IgM. Fab-containing polypeptides can be Fab-containing fusion polypeptides, in which one or more polypeptides are linked to a Fab-containing polypeptide. Fab fusion combines an immunoglobulin's Fab polypeptide with a fusion partner, which can typically be any protein, polypeptide, or small molecule. In fact, any protein or small molecule can be linked to a Fab polypeptide to produce a Fab-containing fusion polypeptide. Fab-containing fusion partners may include, but are not limited to, receptors, adhesion molecules, ligands, enzymes, cytokines, chemokines, or other proteins or target-binding regions of protein domains.

[0124] A "Fab fragment" consists of a heavy chain (CH1 and variable region) and a light chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0125] The “Fab′ fragment” contains a portion of a heavy chain and a light chain. The portion of the heavy chain contains the VH domain and the CH1 domain, as well as the region between the CH1 and CH2 domains, which allows interchain disulfide bonds to be formed between the two heavy chains of the two Fab′ fragments to form the F(ab′)2 molecule.

[0126] The “F(ab′)2 segment” comprises two light chains and two heavy chains, wherein the heavy chains contain a portion of the constant region between the CH1 and CH2 domains, allowing interchain disulfide bonds to form between the two heavy chains. Therefore, the F(ab′)2 segment consists of two Fab′ segments held together by disulfide bonds between the two heavy chains.

[0127] The term "antibody fragment" as used herein refers to only a portion of a complete antibody, wherein the portion preferably retains at least one, preferably most or all, of the functions that would normally be associated with the portion when present in the complete antibody.

[0128] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for the possibility of naturally occurring mutations present in trace amounts. Monoclonal antibodies are highly specific, targeting a single antigen. Furthermore, unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen.

[0129] There are well-known methods for preparing and characterizing antibodies in the prior art. Methods for producing polyclonal antibodies are well-known in the art. In short, polyclonal antibodies are prepared by immunizing animals with proteins; then, serum is collected from the immunized animals and the antibodies are isolated. A variety of animal species can be used to produce antiserum. Typically, animals used for producing antiserum can be rabbits, mice, rats, hamsters, guinea pigs, or goats.

[0130] In addition, monoclonal antibodies (MAbs) can be prepared using well-known techniques. Typically, this method involves immunizing a suitable animal with a disease-associated protein. The immunizing composition can be administered in an amount that effectively stimulates antibody-producing cells.

[0131] The preparation of monoclonal antibodies generally follows the same pathway as that for polyclonal antibodies. An immunogen is injected into the animal as an antigen. The antigen can be mixed with an adjuvant, such as complete or incomplete Freund's adjuvant. The animal is immunized again approximately every two weeks with the same antigen.

[0132] In some embodiments, the monoclonal antibodies described herein may specifically include “chimeric” antibodies (where a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass) and fragments of these antibodies, provided they exhibit the desired biological activity (US4816567).

[0133] The “humanized” form of non-human (e.g., mouse) antibodies is a chimeric antibody, containing a minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies), where the hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody), such as mice, rats, rabbits, or non-human primates with the desired specificity, affinity, and ability. In some cases, the framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in either the recipient or donor antibody. These modifications are made to further improve antibody performance. Typically, humanized antibodies will contain substantially all of the following: at least one, and usually two variable domains, where all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all of the FRs are FRs of the human immunoglobulin sequence. Humanized antibodies may also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin.

[0134] "Human antibody" is an antibody whose amino acid sequence corresponds to that of an antibody produced by humans and / or an antibody prepared using any of the techniques disclosed herein for preparing human antibodies. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0135] In the context of the eighth aspect of this invention, the anti-FGFR4 antibody is an antibody that binds to FGFR4 but not to FGFR1, FGFR2, and FGFR3, as described above. That is, the anti-FGFR4 antibody has no statistically significant effect on binding to FGFR1, FGFR2, and FGFR3. Various immunoassay formats can be used to select antibodies that specifically bind to FGFR4. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that can specifically react with proteins (for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see, for example, Hnasko, ELISA: Methods and Protocols (2015) Humana Press, Chapter 6, pp. 61-69).

[0136] In the context of this invention, when referring to anti-FGFR4 antibodies, the expression "binds to FGFR4" means that the antibody or a fragment thereof interacts with (and binds to) its epitope, whether the epitope is linear or conformational. "Does not bind to FGFR1, FGFR2, and FGFR3" means that anti-FGFR4 does not significantly bind to any of these targets.

[0137] In some embodiments, the anti-FGFR4 antibody molecule is conjugated to a drug or therapeutic agent via a linker. In some embodiments, the anti-FGFR4 antibody molecule is conjugated to two or more drugs or therapeutic agents via a linker. In some embodiments, the anti-FGFR4 antibody molecule is conjugated to four or more drugs or therapeutic agents via a linker. In some embodiments, the anti-FGFR4 antibody molecule is conjugated to five or more drugs or therapeutic agents via a linker. In some embodiments, the anti-FGFR4 antibody molecule is conjugated to six or more drugs or therapeutic agents via a linker. In some embodiments, the anti-FGFR4 antibody molecule is conjugated to seven or more drugs or therapeutic agents via a linker. In some embodiments, the anti-FGFR4 antibody molecule is conjugated to eight or more drugs or therapeutic agents via a linker. The number of drugs or therapeutic agents conjugated to each antibody is called the drug-antibody ratio (DAR). Typically, the DAR corresponds to the average DAR. In some embodiments, antibody attachment determines the drug-antibody ratio (DAR). In some embodiments, the DAR is at least 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the DAR is at most 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the DAR is less than 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the DAR is greater than 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the DAR is 1-10, preferably 2-8, more preferably 6-8, for example 4 or 8. In some embodiments, the DAR is 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the DAR is 2, 4, 8, 16, 32, or more.

[0138] In the context of this invention, the term "connector" should be understood as a bifunctional or multifunctional part that can be used to connect one or more drug moieties to an antibody to form an ADC.

[0139] In one embodiment, the ADC provided by the present invention includes a cleavable linker. The cleavable linker is preferably sufficiently stable extracellularly to achieve therapeutic efficacy. Prior to transport or delivery to cells, the ADC is preferably stable and remains intact, i.e., the antibody remains conjugated to the drug moiety. Once the linker is stable extracellularly in the target cell, it can cleave at an efficient rate. Therefore, an efficient linker will: (i) maintain the specific binding properties of the antibody; (ii) allow delivery of the drug moiety, such as intracellular delivery; and (iii) maintain the therapeutic effect of the drug moiety, such as cytotoxicity.

[0140] Illustrative, non-limiting examples of “cleavable linkers” include acid-labile linkers (e.g., containing hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, light-labile linkers, disulfide-containing linkers (US5208020) (which is incorporated herein by reference in its entirety), or glycosidase-sensitive linkers, such as β-glucuronide-based linkers (US8568728B2 or US11103593B2). Cleavable linkers are generally readily cleaved under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers that can be cleaved by intracellular proteases, such as lysosomal proteases or endosomal proteases. In exemplary embodiments, the linker may be a dipeptide linker, such as a valine-citrulline (Val-Cit), valine-alanine (Val-Ala), or phenylalanine-lysine (Phe-Lys) linker.

[0141] In one embodiment, the linker is cleavable under intracellular conditions, such that cleavage of the linker is sufficient to release the drug from the antibody in the intracellular environment to produce a therapeutic effect. In some embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a specific pH value. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-labile linkers that are hydrolyzable in lysosomes (e.g., hydrazones, hemicarbazones, thiohemicarbazones, cis-aconitamide, orthoesters, acetals, ketals, etc.) can be used. (See, for example, U.S. Patents 5,122,368; 5,824,805; 5,622,929; which are incorporated herein by reference in their entirety.) Such linkers are relatively stable under neutral pH conditions (such as the pH conditions in blood) but unstable below pH 5.5 or 5.0 (the approximate pH of lysosomes). In some embodiments, the hydrolyzable connector is a thioether connector (e.g., a thioether linked to the therapeutic agent via an acylhydrazone bond (see, for example, US5622929, which is incorporated herein by reference in its entirety)).

[0142] In other embodiments, the connector is cleavable under reducing conditions (e.g., a disulfide connector). Various disulfide connectors are known in the art, including those formed, for example, using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)toluene), SPDB, and SMPT (US4880935, which is incorporated herein by reference in its entirety).

[0143] In some embodiments, the linker can be cleaved by a lysing agent (e.g., an enzyme) present in the intracellular environment (e.g., lysosomes, endosomes, or pits). The linker can be, for example, a peptide linker that can be cleaved by intracellular peptidases or proteases (including, but not limited to, lysosomal or endosomal proteases). In some embodiments, the peptide linker is at least two or at least three amino acids in length. The lysing agent may include cathepsins B and D, as well as plasmin, all known to hydrolyze dipeptide drug derivatives, thereby releasing the active drug within the target cell. Most commonly, peptide linkers can be cleaved by enzymes present in cells expressing EGFR. Examples of such linkers are described, for example, in US6214345, which is incorporated herein by reference in its entirety for all purposes. In one specific embodiment, the peptide linker that can be cleaved by intracellular proteases is a Val-Cit linker or a Phe-Lys linker (see, for example, US6214345, which, by reference in its entirety, describes the synthesis of doxorubicin with a Val-cit linker). One advantage of using intracellular protein hydrolysis-release therapeutic agents is that the agent is usually weakened during conjugation, and the serum stability of the conjugate is usually very high.

[0144] In an alternative implementation, the linker is a non-cleavable linker. A non-cleavable linker prevents the release of the cytotoxic agent before the ADC is internalized by the target cells. Once inside the lysosome, the antibody is digested by lysosomal proteases, leading to the release of the cytotoxic agent.

[0145] In the context of this invention, an ADC includes one or more drugs. In some embodiments, the drug is a small molecule. In some instances, the drug is a cross-linking agent, an anti-microtubule agent and / or an anti-mitotic agent, or any cytotoxic or growth-inhibiting agent suitable for mediating the killing of tumor cells. Exemplary cytotoxic agents include, but are not limited to, PDB, auristatin, maytansine, dolalastatin, chalcogenide, nemorubicin and its derivatives, PNU-159682, anthracyclines, vinca alkaloids, taxanes, trichothecene, CC1065, camptothecin, essanotecan, delutecan, elinefadroxil, combretastatin, dolalastatin, docamycin, enediyne, geldmycin, indoline-benzodiazepine dimer, puromycin, tubulolysin, hammetrine, spliceostatin, praldidine, and their stereoisomers, isosteres, analogs, and derivatives having cytotoxic activity.

[0146] As used herein, the term "growth inhibitor" refers to a compound or composition that inhibits cell growth in vitro and / or in vivo. Therefore, a growth inhibitor can be an agent that significantly reduces the percentage of cells in S phase. Examples of growth inhibitors include agents that block cell cycle progression (at locations other than S phase), such as those that induce G1 phase arrest and M phase arrest. Classic M phase blockers include vinca derivatives (vincristine and vinblastine), TAXOL®, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Agents that cause G1 phase arrest can also spill over into S phase arrest, such as DNA alkylating agents like tamoxifen, prednisone, dacarbazine, nitrogen mustard, cisplatin, methotrexate, 5-fluorouracil, and cytarabine. More information can be found in The Molecular Basis of Cancer, edited by Mendelsohn and Israe, Chapter 1, Murakami et al., entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" (WB Saunders: Philadelphia, 1995), especially page 13.

[0147] Anti-FGFR4 antibodies can be conjugated to at least one anti-microtubule agent, such as aurestatin. Aurestatin represents a group of doralastatin analogues that have generally been shown to have anticancer activity by interfering with microtubule kinetics and GTP hydrolysis, thereby inhibiting cell division. For example, aurestatin E (US5635483, which is incorporated herein by reference in its entirety) is a synthetic analogue of the marine natural product doralastatin 10, a compound that inhibits microtubule polymerization by binding to the same site on microtubules by the anticancer drug vincristine. Doralastatin 10, aurestatin PE, and aurestatin E are linear peptides with four amino acids, three of which are characteristic of doralastatin compounds. Exemplary embodiments of the aurestatin subclass of mitotic inhibitors include, but are not limited to, monomethylaurestatin D (MMAD or an aurestatin D derivative), monomethylaurestatin E (MMAE or an aurestatin E derivative), monomethylaurestatin F (MMAF or an aurestatin F derivative), aurestatin F-phenylenediamine (AFP), aurestatin EB (AEB), aurestatin EFP (AEFP), and 5-benzoylvaleric acid-AE ester (AEVB). The synthesis and structure of aurestatin derivatives are described, for example, in WO04010957 or WO02088172, which are incorporated herein by reference in their entirety.

[0148] In one implementation, the anti-FGFR4 antibody is conjugated to at least one MMAE (monomethylaurestatin E). Monomethylaurestatin E (MMAE, Vittin) inhibits cell division by blocking microtubule polymerization. Due to its hypertoxicity, it cannot be used as a drug on its own. In recent developments in cancer therapy, it has been linked to a monoclonal antibody (mAb) that recognizes the expression of specific markers in cancer cells and directs MMAEs to the cancer cells.

[0149] In another embodiment, the drug is a topoisomerase I (TOP1) inhibitor. Examples of TOP1 inhibitors are camptothecin and its derivatives, such as topotecan, irinotecan, beloteccan, ethanotecan, and delutecan. In another embodiment, the drug is pyrrolobenzodiazepine (PBD). For example, PBD dimers recognize and bind to specific DNA sequences and have been shown to be usable as cytotoxic agents. PBD dimers have been conjugated with antibodies, and the resulting ADCs have shown anticancer properties (see, for example, US 2010 / 0203007, which is incorporated herein by reference in its entirety). Exemplary linking sites on the PBD dimer include a five-membered pyrrole ring, a tether between PBD units, and an N10-C11 imine group (see WO 2009 / 016516; US 2009 / 304710; US 2010 / 047257; US 2009 / 036431; US ​​2011 / 0256157; and WO 2011 / 130598, which are incorporated herein by reference in their entirety).

[0150] In another embodiment, the drug is a maytansine molecule. Maytansines are derivatives of maytansine and are mitotic inhibitors that work by inhibiting microtubule polymerization.

[0151] In another embodiment, the ADC comprises one or more chachiomycin molecules. The chachiomycin antibiotic family and its analogues are capable of producing double-strand DNA breaks at subpicomolar concentrations (Hinman et al., Cancer Res 53:33363342, 1993). Exemplary methods for preparing ADCs having a chachiomycin pharmaceutical moiety are described in U.S. Patent Nos. 5,712,374; 5,714,586; 5,739,116; and 5,767,285, which are incorporated herein by reference in their entirety.

[0152] In some embodiments, the ADC comprises anthracyclines. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. Anthracyclines are believed to kill cells through a variety of different mechanisms, including embedding drug molecules into the DNA of cells, thereby inhibiting DNA-dependent nucleic acid synthesis; inducing the generation of free radicals, which subsequently react with cellular macromolecules, causing damage to cells; and / or the interaction of drug molecules with cell membranes. Non-limiting exemplary anthracyclines include: doxorubicin, epirubicin, idarubicin, erythromycin, daunorubicin, doxorubicin, epirubicin, nemorubicin, pentorubicin, and mitoxantrone and their derivatives. For example, PNU-159682 is a potent metabolite (or derivative) of nemorubicin (Quintieri et al., Clin Cancer Res 11(4):1608-1617, 2005). Nemorubicin is a semi-synthetic analogue of doxorubicin, which has a 2-methoxymorpholino group on the glycosidic amino group of doxorubicin (Grandi et al., Cancer Treat Rev17:133, 1990; Ripamonti et al., Br J Cancer 65:703-707, 1992).

[0153] In another embodiment, the drug is a radioisotope used to produce a cytotoxic radiopharmaceutical, referred to as a radioimmunoconjugate. Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic purposes include, but are not limited to, iodine-131, indium-111, yttrium-90, and lutetium-177. Methods for preparing radioimmunoconjugates are known in the art. Examples of radioimmunoconjugates are commercially available, including Zevalin® (DEC Pharmaceuticals) and Bexxar® (Corixa Pharmaceuticals), and radioimmunoconjugates can be prepared using the antibodies of the present invention in a similar manner. In some embodiments, the macrocyclic chelating agent is 1,4,7,10-tetraazacyclododecane-N,N',N”,N”’-tetraacetic acid (DOTA), which can be linked to the antibody via a linker molecule. Such linker molecules are well known in the art and are described in Denardo et al., (1998) Clin Cancer Res. 4(10):2483-90; Peterson et al., (1999) Bioconjug.Chem. 10(4):553-7; and Zimmerman et al., (1999) Nucl. Med. Biol. 26(8):943-50, each of which is incorporated herein by reference in its entirety.

[0154] Suitable ADCs in the context of this invention can be prepared according to any method known to those skilled in the art. For example, the cysteine ​​thiol of an antibody can form a bond with a reactive functional group of a linker or drug-linker intermediate to prepare an ADC. In one embodiment, the conjugates of this invention can be prepared by conjugating a pre-formed drug-linker compound with an antibody or a fragment thereof, as described in, for example, US6441163, US2011 / 0003969, and US2008 / 0145374, followed by a purification step.

[0155] In a ninth aspect, the present invention provides a pharmaceutical composition.

[0156] The exact therapeutic dose of the ADC of this invention can depend on several variables. Some of these variables are: route of administration, drug release time (e.g., instantaneous or prolonged release), administration regimen, pain severity, patient condition, etc.

[0157] The pharmaceutical composition can be prepared in liquid, semi-solid or solid dosage forms, such as injection solutions, drops, juices, syrups, sprays, suspensions, tablets, patches, capsules, dressings, suppositories, ointments, creams, lotions, gels, emulsions, aerosols or multi-particulate forms, such as pills or granules, and, where appropriate, compressed into tablets, dispensed into capsules or suspended in a liquid, or administered as is.

[0158] These compositions can be prepared using conventional tools, equipment, methods, or processes known in the art.

[0159] Pharmaceutically acceptable adjuvants, solvents, or excipients that may be used in these compositions are known to those skilled in the art or are commonly used in the preparation of therapeutic compositions and may be selected, for example, from the group consisting of: excipients, fillers, solvents, diluents, surfactants, colorants, preservatives, disintegrants, flow aids, lubricants, flavoring agents, or binders.

[0160] The term "pharmaceutical acceptable" refers to a pharmaceutically acceptable material, composition, or solvent. Each component must be pharmaceutically acceptable, meaning it is compatible with the other ingredients in the pharmaceutical composition. It must also be suitable for contact with human tissues or organs without causing excessive toxicity, irritation, allergic reactions, immunogenicity, or other problems or complications in animals, particularly humans, that are commensurate with a reasonable benefit / risk ratio.

[0161] The choice of physiologically compatible adjuvant or the amount of adjuvant used depends on the form of administration of the pharmaceutical composition, i.e., oral, subcutaneous, parenteral, intravenous, intraperitoneal, intradermal, intramuscular, intranasal, sublingual, rectal, ear, or tympanic cavity. Formulations in the form of tablets, sugar-coated pills, capsules, granules, pellets, drops (especially ear drops), juices, or syrups are preferably suitable for oral administration; solutions, suspensions, readily resoluble dry formulations, or sprays are preferably suitable for parenteral, topical, or inhalation administration. Compounds according to the invention used in pharmaceutical compositions according to the invention (in reservoir, dissolved, or dressing form, or, if appropriate, with the addition of other agents that promote penetration into the skin) are suitable for transdermal administration. Oral or transdermal formulations may also release the corresponding compound of the invention in a delayed manner.

[0162] For example, for oral administration in tablet or capsule form, the active pharmaceutical ingredient can be combined with an orally administered, non-toxic, pharmaceutically acceptable inert carrier such as lactose, starch, sucrose, glucose, methylcellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, etc.; for oral administration in liquid form, the oral pharmaceutical ingredient can be combined with any orally administered, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerin, water, etc. Furthermore, suitable binders, lubricants, disintegrants, and colorants may be incorporated into the mixture when needed or necessary. Suitable binders include starch, gelatin, natural sugars such as glucose or β-lactose, corn sweeteners, natural and synthetic gums such as gum arabic, tragacanth, or sodium alginate, carboxymethyl cellulose, polyethylene glycol, waxes, etc. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, etc. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc.

[0163] Gelatin capsules contain active ingredients and powdered carriers such as lactose, starch, cellulose derivatives, magnesium stearate, and stearic acid. Similar diluents can be used to manufacture compressed tablets. Both tablets and capsules can be formulated as long-acting release products to continuously release the drug over several hours. Compressed tablets can be sugar-coated or film-coated to mask any unpleasant tastes and protect the tablet from atmospheric effects, or enteric-coated for selective disintegration in the gastrointestinal tract.

[0164] Liquid dosage forms for oral administration may contain colorants and flavorings to increase patient acceptance.

[0165] The dosage of the drug composition administered will, of course, vary depending on the intended use and known factors, such as the recipient's age, health condition, and weight; the nature and severity of symptoms; concurrent treatments (if any); the frequency of treatment; and the desired effect. The recipient can be any type of mammal, but is preferably a human.

[0166] In one embodiment of the invention, the pharmaceutical composition is lyophilized.

[0167] In a tenth aspect, the present invention provides the use of the ADC provided herein in any of the above embodiments for treating cancers expressing FGFR4. Illustrative and non-limiting examples of cancers expressing FGFR4 are oral squamous cell carcinoma, head and neck cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, kidney cancer, prostate cancer, sarcoma, melanoma, leukemia, lymphoma, kidney cancer, duodenal cancer, small bowel cancer, large bowel cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, or bile duct cancer; particularly, the cancer is breast cancer, liver cancer, gastric cancer, or colorectal cancer. In one particular embodiment, the cancer expressing FGFR4 is breast cancer. Other embodiments of cancer and breast cancer are as described above. In the context of the present invention, cancer is cancer in which FGFR4 expression is present, regardless of the expression levels of FGFR1, FGFR2, or FGFR3. In the context of the present invention, the therapeutic effect is due to selective action on FGFR4.

[0168] In one implementation, cancer is associated with overexpression of FGFR4 (i.e., FGFR4 expression is higher than in non-cancer individuals). Protocols and tools for determining FGFR4 expression levels in isolated samples of subjects are well known (as described above and below).

[0169] In one embodiment of the sixth, seventh, or eighth aspect of the invention, the subject with cancer is also resistant or intractable to medical treatments containing one or more CDK4 / 6i.

[0170] In some preferred embodiments, the cancer is metastatic HR+ / HER2- breast cancer, and the subject has previously been treated with CDK4 / 6i inhibitors in combination with endocrine therapy.

[0171] In another embodiment of the invention, the ADC is administered in combination with other therapeutic agents, such as other anticancer agents, such as chemotherapeutic agents, including mitotic inhibitors, alkylating agents, antimetabolites, embedded antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, endocrine therapies, immunomodulators, and anti-angiogenic agents; anti-allergic agents; antinausea agents (or antiemetics); analgesics; cytoprotective agents; and combinations thereof. Other anticancer treatments include radiotherapy.

[0172] Commonly considered chemotherapy agents for combination therapy include: anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), liposomal cytarabine injection (DepoCyt®), dacarbazine (DTIC-Dome®), and actinomycin D. D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezatabin, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin, Alkeran®, Purinetol®, Methotrexate, Novantrone®, Mirotta, Taxol®, Yttrium 90 / MX-DTPA, Pentostatin, Gliadel®, Teniposide, Vumon®, Thioguanine, Thiotepa, Tirazone®, Hycamptin®, Velban®, Oncovin®, and Navelbine®.

[0173] Non-limiting examples of alkylating agents include nitrogen mustards (such as nitrogen mustard, cyclophosphamide, melphalan, uracil mustard, or chlorambucil), alkyl sulfonates (such as busulfan), and nitrosoureas (such as carmustine, lomustine, semustine, streptozotocin, or dacarbazine).

[0174] Non-limiting examples of antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs (such as mercaptopurine or thioguanine).

[0175] Non-limiting examples of natural products include vinca alkaloids (such as vincristine, vinblastine or vincristine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as actinomycin D, daunorubicin, doxorubicin, bleomycin, procainomic acid or mitomycin C) and enzymes (such as L-asparaginase).

[0176] Other non-limiting examples of agents include platinum coordination complexes (such as cis-diamine dichloroplatin II, also known as cisplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine), and adrenocortical inhibitors (such as mitotane and ammonia-glutathione).

[0177] Non-limiting examples of hormones and antagonists include adrenocortical hormones (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), estrogens (such as diethylstilbestrol and ethinylestradiol), anti-estrogens (such as tamoxifen), and androgens (such as testosterone propionate and fluoromethesterone). Examples of commonly used chemotherapy drugs include doxorubicin, eczemab, cytarabine, BiCNU, busulfan, CCNU, carboplatin, cisplatin, cyclophosphamide, daunorubicin, DTIC, 5-FU, fludarabine, hydroxyurea, idarubicin, ifosfamide, methotrexate, photomycin, mitomycin, mitoxantrone, nitrogen mustard, paclitaxel (or other taxanes, such as docetaxel), vinblastine, vincristine, and VP-16. Some newer drugs include gemcitabine (Gemzar), Herceptin, irinotecan (Camptosar, CPT-11), leustatin, Navelbine, Rituxan STI-571, Taxotere, topotecan (Hycamtin), capecitabine (capecitabine), zevallin, and calcitriol.

[0178] In one embodiment, the immunomodulator is a PD-1 / PD-L1 immunomodulator. In some embodiments, the PD-1 / PD-L1 immunomodulator is a PD-1 blocker. In some embodiments, the PD-1 / PD-L1 immunomodulator is an anti-PD-1 blocking antibody. In some embodiments, the PD-1 / PD-L1 immunomodulator is an anti-PD-L1 blocking antibody. PD-1 and PD-L1 therapies are well known in the art and include, but are not limited to, cimipril (Libtayo), nivolumab (Opdivo), pembrolizumab (Keytruda), atezolizumab (Tecentriq), avelumab (Bavencio), durvalumab (Imfinzi), pembrolizumab (Keytruda), nivolumab (Opdivo), cimipril (Libtayo), atezolizumab (Tecentriq), retivalimumab (zynyz), and dotalimumab (Jemperli).

[0179] Other non-limiting examples of immunomodulators that can be used in the context of this invention are AS-101 (Wyeth-Ayerst Labs.), brompiridamine (Upjohn), interferon-gamma (Genentech), GM-CSF (granulocyte-macrophage colony-stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immunoglobulin (CutterBiological), IMREG (Imreg from New Orleans, Louisiana), SK&F 106528, and TNF (tumor necrosis factor; Genentech), etc.

[0180] Another common treatment for certain types of cancer is surgery, such as surgically removing the cancer or a portion thereof. Another example of treatment is radiation therapy, such as applying radioactive material or energy (like external beam therapy) to the tumor site to help eradicate the tumor or shrink it before surgical removal.

[0181] In one embodiment, the antibody-drug conjugate of the present invention is combined with a second compound having anticancer properties in a pharmaceutical combination formulation or in a dosing regimen as a combination therapy. The second compound in the pharmaceutical combination formulation or dosing regimen may have activity complementary to the combined immunoconjugate, such that they do not adversely affect each other. For example, the antibody-drug conjugate of the present invention may be administered in combination with chemotherapeutic agents, tyrosine kinase inhibitors, FGF downstream signaling pathway inhibitors, IAP inhibitors, Bcl2 inhibitors, or Mcl1 inhibitors, but is not limited thereto.

[0182] In one embodiment of the invention, the ADC is administered in combination with a CDK4 / 6 inhibitor. In another embodiment of the invention, the ADC is administered in combination with endocrine therapy. In yet another embodiment of the invention, the ADC is administered in combination with both a CDK4 / 6 inhibitor and endocrine therapy.

[0183] As used herein, the term "drug combination" refers to a fixed combination, or non-fixed combination or kit of reagents, for combined administration in the form of a single dose unit, wherein two or more therapeutic agents may be administered independently simultaneously or separately at time intervals.

[0184] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat the therapeutic condition or ailment described in this disclosure. Such administration includes the combined administration of these therapeutic agents in a substantially simultaneous manner, such as administration in a single capsule having a fixed proportion of active ingredients.

[0185] Alternatively, this administration may include the combined application of the individual active ingredients in multiple or separate containers (e.g., capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, this administration also encompasses the sequential use of each type of therapeutic agent at approximately the same time or at different times. In either case, the treatment regimen will provide the beneficial effects of the combination of drugs in treating the condition or ailment described herein.

[0186] The therapeutically effective amount of an ADC or drug composition, alone or in combination with any other anticancer drug, as defined above, is the amount that provides a beneficial therapeutic effect.

[0187] Although the invention has been described in conjunction with its specific embodiments, many alternatives, modifications and variations will be apparent to those skilled in the art in light of the foregoing description.

[0188] The aspects and embodiments of the invention disclosed herein are further supported by the following non-limiting examples.

[0189] Example

[0190] I. The relationship between FGFR4 and CDK4 / 6i resistance

[0191] FGFR4 is a potential driver of CDK4 / 6 inhibitory resistance.

[0192] FGFR4 gene expression was evaluated in a clinical cohort (CDK cohort) of patients (aged over 18 years) with a histological diagnosis of HR+ / HER2-negative metastatic breast cancer treated with CDK4 / 6 inhibitors (including palbociclib, ribociclib, and abeciclib) at Barcelona hospitals and clinics between 2014 and 2021. In this cohort, 134 baseline samples (including 61 primary and 73 metastatic samples) and 28 disease progression (PD) samples were available for molecular characterization. RNA was extracted using the High Pure FFPE RNA Isolation Kit (Roche) according to the manufacturer's protocol, and gross dissection was performed as needed to avoid contamination by normal tissue. At least 100 ng of total RNA was analyzed on the nCounter platform (Nanostring Technologies) using the Breast Cancer 360 Panel (which measures the expression of 771 breast cancer-related genes and 5 housekeeping genes (ACTB, MRPL19, PSMC4, RPLP0, and SF3A1)). In univariate analysis, FGFR4 gene expression in baseline samples was the number one gene most significantly associated with poor progression-free survival (PFS; hazard ratio [HaR] [95% confidence interval (CI)] = 1.46 [1.19–1.78], p < 0.001) and overall survival (OS; HaR [95% CI] = 2.01 [1.49–2.72], p < 0.001). Figure 1 A). PFS was defined as the time from the start of endocrine therapy and CDK4 / 6i treatment to progression. OS was defined as the time from the start of endocrine therapy and CDK4 / 6i treatment to death or last follow-up. Survival estimates were derived from Kaplan-Meier curves, and differences were tested using the log-rank test. Prognostic significance for each variable was tested using univariate and multivariate Cox models. All differences were considered significant at p-values ​​(p) < 0.05. Furthermore, compared to baseline samples, FGFR4 expression was significantly higher in patients with disease progression (PD). Figure 1 B).

[0193] Following the same method, the inventors also evaluated FGFR4 expression in surgical samples from the ribociclib and letrozole groups in the context of the CORALLEEN Phase II trial (NCT03248427; Prat et al. 2020). The inventors characterized baseline, day 15, and surgical samples from 49 postmenopausal women with HR+ / HER2-negative early breast cancer. This was a trial conducted in postmenopausal women with primary, operable HR+ / HER2-negative breast cancer, in which patients were randomized (1:1) to receive: six 28-day cycles of ribociclib (600 mg orally once daily for 3 weeks, followed by a 1-week break) plus daily letrozole (2.5 mg orally daily); or four cycles of doxorubicin (60 mg intravenously every 21 days). 2 ) and cyclophosphamide (600 mg / m² intravenously) 2 ), followed by weekly paclitaxel (80 mg / m² intravenously). 2 Treatment lasted 12 weeks. The total duration of neoadjuvant therapy was 24 weeks, with the primary endpoint being the proportion of disease with a low PAM50 recurrence risk (ROR) at surgery in each group. Higher FGFR4 expression was observed in surgical samples from non-responders compared to CDK4 / 6i responders. Figure 1 C).

[0194] Immunohistochemical analysis (IHC) using the D3B12 antibody (CAT#: 8562, Cell Signaling Technology) was performed to analyze FGFR4 expression in primary breast tumors, lymph node metastases, and CDK4 / 6i-resistant breast tumors. Compared to primary tumors and lymph node metastases, FGFR4 protein expression (quantified by H score) was significantly increased in CDK4 / 6i-resistant tumors. Figure 1D ).

[0195] These results demonstrate positive selection of FGFR4 during treatment and its association with the phenotype of non-responders.

[0196] Functional validation of FGFR4 as a driver of CDK4 / 6 drug resistance

[0197] Next, the inventors created experimental cell line models of acquired palbociclib resistance derived from commercially available breast cancer cell lines MCF7, T47D, ZR75-1, and BT474 (ATCC), and cultured them according to the supplier's recommendations. MCF7 and T47D were sensitive to CDK4 / 6i palbociclib. Figure 2 A, as shown in the image above). The ZR-75-1 cell line is HR+ / HER2-, and the BT474 cell line is HR+ / HER2+. Both cell lines exhibit intrinsic resistance to CDK4 / 6i palbociclib. Figure 2A (see figure below). MCF7-derived palbociclib-resistant (MCF7-PR) and T47D-derived palbociclib-resistant (T47D-PR) cell lines were established by treating MCF7 and T47D cells with palbociclib for 6 months, starting at a low concentration (1 nM) and increasing the concentration at each passage until reaching 1 μM. Both HR+ / HER2- cells were maintained in complete medium containing 500 nM palbociclib. The IC50 of sensitive MCF7 and T47D cells, as well as the derived palbociclib-resistant MCF7-PR and T47D-PR cell lines, was evaluated by seeding 5000 tumor cells per well in 96-well plates and adding an increased dose of palbociclib the following day. 50 Four days later, proliferation was assessed using CyQUANT NF Cell Proliferation (ThermoFisher Scientific) according to the manufacturer's instructions. Fluorescence measurements were performed using an FL800 fluorescence microplate reader at 485nm excitation and 520nm emission detection. The survival fraction relative to untreated control cells was calculated using the following formula: Cell viability % = (treated / untreated) * 100, and IC50 was calculated using a nonlinear regression model with GraphPad PRISM software. 50 value.

[0198] These results indicate that the IC50 of palbociclib-resistant cell lines... 50 The IC50 was higher than that of the corresponding sensitive cell lines. 50 (IC) 50 MCF7 250nM, IC 50 MCF7-PR 2000nM; IC 50 T47D 50nM, IC 50 T47D-PR 1800nM, IC 50 ZR-75-13100nM, IC 50 BT474 ~2.9e16nM) Figure 2 A, Table 1).

[0199] Table 1. IC50 of CDK4 / 6 inhibitor (palbociclib) in breast cancer cell lines 50

[0200]

[0201] To elucidate the potential role of FGFR4 in palbociclib-sensitive and palbociclib-resistant cell line models, the inventors employed FGFR4 siRNA-mediated knockdown. A subset of breast cancer cell lines were transfected with either 20 nmol / L of FGFR4-targeting siGENOME SMARTpool (M-003134-02, FGFR4 siRNA) or Silencer® Select Negative Control #1 (NT siRNA) (ThermoFisher Scientific) according to the manufacturer's instructions using Lipofectamine RNAiMax transfection reagent (Invitrogen). After 6 days of culture, cells were labeled with Hoechst 33342 (Invitrogen), and fluorescence was subsequently measured using a Gen5 microplate reader and Imager Software. Compared to cells transfected with non-targeting (NT) siRNA, FGFR4 silencing significantly attenuated the growth of palbociclib-resistant MCF7-PR and T47D-PR cells, but did not affect the growth of sensitive parental MCF7 and T47D cells. Furthermore, FGFR4 silencing inhibited the growth of intrinsically resistant ZR751 cells and the FGFR4-mutant cell line MDA-MB-453 (used as a positive control). Figure 2 B). MDA-MB-453 cells primarily express a mutant form of FGFR4, namely FGFR4 Y367C, which leads to ligand-independent activation of kinase activity and activation of downstream signaling of FGFR4 (Hagel et al., 2015).

[0202] Next, FGFR4 depletion was induced by stable shRNA expression and confirmed by Western blotting using standard procedures. Specifically, susceptible MCF7 and resistant MCFR7-PR and ZR751 were infected with two independent stable FGFR4 shRNAs (shFGFR4#1 and shFGFR4#2) and a non-targeted disordered shRNA (shControl) as a control. Figure 2 (C-2D). The response to palbociclib was assessed by incubating cells with increased concentrations of palbociclib as described above. FGFR4 knockdown consistently reduced the IC50 of both palbociclib-resistant cell lines. 50 This restored the sensitivity of MCF7-PR cells to palbociclib to the level of MCF7 parental cells (IC50). 50 MCF7-PR shControl 1553 nM, IC 50 MCF7-PR shFGFR4#1 576.4 nM,IC 50MCF7-PR shFGFR4#2 449.3 nM; MCF7-PR shControl VS MCF7-PR shFGFR4#1 p<0.0001; MCF7-PR shControl VS MCF7-PR shFGFR4#2 p<0.0001) ( Figure 2 C), and also restored sensitivity to intrinsically resistant ZR751 (IC). 50 ZR751 shControl 3803 nM, IC 50 ZR751 shFGFR4#1 80.23nM, IC 50 ZR751 shFGFR4#2 124.7 nM; ZR751 shControl VS ZR751 shFGFR4#1 p=0.0009; ZR751 shControl VS ZR751 shFGFR4#2 p=0.001 1) ( Figure 2 D).

[0203] To verify that FGFR4 is a driver of CDK4 / 6i resistance, the inventors developed ZR751 cells overexpressing wild-type FGFR4 (FGFR4wt) or constitutively active FGFR4 (FGFR4 Y367C) (Roidl et al., 2010). Control cells were infected with a backbone vector expressing red fluorescent protein (RFP). Overexpression of FGFR4 in ZR751 cells was confirmed. Figure 2 E, left figure), and led to decreased sensitivity of two FGFR4 overexpressing cell lines to palbociclib (IC). 50 ZR751 RFP353.8 nM, ZR751 FGFR4wt ~4.2e6, ZR751 FGFR4 Y367C ~1.2e16) ( Figure 2 E, right figure).

[0204] In summary, these findings reveal an association between FGFR4 expression and resistance to CDK4 / 6i in breast cancer patients and breast cancer cell lines.

[0205] To test whether CDK4 / 6i resistance in vivo depends on FGFR4, the inventors developed an experimental model to test the systemic effects of CDK4 / 6i and explore experimental FGFR4-targeted therapy. 1x10 6 MCF7 control and palbociclib-resistant (MCF7-PR) breast cancer cells were implanted into the mammary fat pads of female Balb / c nude mice, and tumors were induced to grow in the presence of supplemented estrogen. Prior to inoculation, the cells were cultured as described above. The tumors were examined by palpation (50 mm).3 Afterward, mice were randomly assigned to groups and orally administered either the solvent or palbociclib (50 mg / kg) 5 days a week until the end of the experiment. Tumor volume was measured twice weekly using digital calipers during the experiment. As previously reported, the experimental MCF7 model of HR+ breast cancer responded to CDK4 / 6i systemic therapy and limited tumor growth (solvent vs. palbociclib, p=0.018). Figure 3 A, left figure). Conversely, palbociclib-resistant MCF7 derivatives (MFC7-PR) developed lesions regardless of CDK4 / 6i treatment, confirming that palbociclib resistance was maintained in vivo (solvent vs. palbociclib, p=0.907). Figure 3 A, right figure). After harvesting the tumor, the inventors used the proliferation 60 gene nCounter panel to analyze FGFR4 gene expression and proliferation gene characteristics, and confirmed that palbociclib-resistant tumors expressed high levels of FGFR4 and showed increased proliferation scores. Figure 3 B). These observations were confirmed by immunohistochemical (IHC) analysis of FGFR4. For this purpose, FFPE-embedded tumors were excised into 4 μm pieces, and after standardized dewaxing, antigen retrieval was performed using heated citrate buffer (pH 6.0). Primary anti-FGFR4 antibody (4H2B10B2, Merck) was incubated at a 1:500 dilution and detected using a Dako (Agilent) EnVision Flex system. Figure 3 C).

[0206] Next, the inventors investigated the effect of gene-depleted FGFR4 in palbociclib-resistant MCF7 cells (MCF7-PR) in vivo. Control cells expressing disordered shRNA and FGFR4 knockdown cells expressing shRNA targeting FGFR4 (shFGFR4#1) were cultured and injected into mice using the same method described above. When the tumor reached approximately 50 mm... 3 Mice were randomly assigned to groups and treated orally with either a solvent or palbociclib (50 mg / kg) five days a week until the end of the experiment. Notably, FGFR4 depletion strongly inhibited in vivo tumor growth of MCF7-PR cells. Figure 3 (D) The effect was amplified when mice were treated with CDK4 / 6i palbociclib as a concomitant therapy, indicating a synergistic effect in the latter case. These results suggest that targeting FGFR4 halts tumor growth and restores CDK4 / 6i responses.

[0207] III. Identification of five selective monoclonal antibodies against FGFR4 for delivering cytotoxic payloads.

[0208] MMAE

[0209] In the first step, we evaluated the selectivity of several antibodies for FGFR4. For this purpose, solid-binding assays were performed to evaluate the binding selectivity of mAb1 (CAT#: NS-028CN, Creative Biolabs), mAb2 (CAT#: ARG53740, Arigo Laboratories), mAb3 (U3-1784; Bartz et al 2019; DOI: 10.1158 / 1535-7163; MCT-18-0048), mAb4 (ThermoFisher CAT#: MA5-28550), and mAb5 (US11643467B2) to FGFR4 and other members of the FGFR family (FGFR1, FGFR2, FGFR3). For this purpose, MAXISORP flat-bottom F96 Nunc plates were coated overnight at 4°C with 1 μg / mL of recombinant His-tagged FGFR 1-4 (Sino Biological). After washing and blocking, increased concentrations of mAb1, mAb2, mAb3, mAb4, or mAb5 were added, and the mixture was incubated at room temperature (RT) for 1 hour. Antigen-binding antibodies were detected using HRP-conjugated anti-mouse Fc secondary antibody (Dako), and colorimetric readings were performed at 630 nm using TMB. Results showed that mAb1, mAb2, mAb3, mAb4, and mAb5 were selective binders to FGFR4, while the binding signal to FGFR1-3 was negligible. Figure 4A -B).

[0210] In the second phase, the inventors sought to confirm the ability of these antibodies to deliver cytotoxic payloads to cells expressing FGFR4. To this end, commercial cytotoxic payloads (Moradec, CAT#: AM-202-AE) were delivered to several FGFR4-expressing cancer cell lines using specific anti-FGFR4 mAb1, mAb2, mAb3, mAb4, and mAb5 antibodies.

[0211] MDA-MB-453 and ZR-75-1 breast cancer cell lines were cultured according to the supplier's recommendations (ATCC). The Huh7 liver cancer cell line (CLS) was cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1% penicillin-streptomycin (Gibco). T47D-PR cells were cultured in Dulbecco modified Eagle medium (DMEM) / nutrient mixture F-12 supplemented with 10% v / v heat-inactivated fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA), 1% GlutaMAX (Gibco; Thermo Fisher Scientific Inc.), and 1% penicillin / streptomycin (Sigma-Aldrich, Saint Louis, MO, USA) in a humidified incubator at 37°C and 5% CO2. Palbociclib (500 nM) was also added to the culture medium for T47D-PR cells (Selleckchem, Houston, TX, USA). MDA-MB-453 breast cancer cell line, T47D-PR breast cancer cell line (acquired CDK4 / 6i resistance), ZR-75-1 breast cancer cell line (CDK4 / 6i intrinsic resistance), and Huh7 liver cancer cell line were plated in triplicate and incubated with serially diluted control mouse IgG (hereinafter also referred to as "isotype antibody," Absolute antibody CAT: Ab00102-1.1), mAb1, mAb2, mAb3, mAb4, or mAb5 (at specified concentrations).

[0212] Subsequently, 6.6 nM of cytotoxic payload (containing an anti-Fc antibody conjugated to MMAE) was added and incubated for 6 days. At the end of the experiment, cell number was assessed by adding CellTiter 96 Aqueous One Solution Reagent (Promega) according to the manufacturer's instructions. Finally, absorbance at 490 nm was recorded using a TECAN Infinite MNano+ microplate reader. These results showed a dose-dependent effect of these anti-FGFR4 antibodies, while the isotype control had no effect. These results indicate that FGFR4 is crucial for this effect, and that anti-FGFR4 specific antibodies can be used to deliver cytotoxic drugs to a variety of cancer cells (…). Figure 5A -K).

[0213] MMAF and Docalamycin

[0214] T47D-PR cells were plated in triplicate and incubated with serially diluted control mouse IgG (hereinafter also referred to as "allotype antibody," Absolute antibody CAT: Ab00102-1.1) or FGFR4 mAb1 (at a specified concentration). Subsequently, a cytotoxic payload (containing an anti-Fc antibody conjugated with docalamycin DM (Moradec, CAT#: AM-202-DD) or MMAF with an uncleavable linker (Moradec, CAT#: AM-201-AF)) was added at a 1:6 ratio, and the cells were incubated for 6 days. At the end of the experiment, cell counts were assessed by adding CellTiter 96 Aqueous One Solution Reagent (Promega) according to the manufacturer's instructions. Finally, absorbance at 490 nm was recorded using a TECAN Infinite MNano+ microplate reader. These results demonstrate a dose-dependent effect of these anti-FGFR4 antibodies, while the allotype control showed no effect. These results indicate that FGFR4 is crucial for this effect, and that anti-FGFR4 specific antibodies can be used to deliver cytotoxic drugs to a variety of cancer cells. Figure 5L -M).

[0215] III. Preparation and characterization of the antibody-drug conjugates (ADCs) of the present invention suitable for cancer treatment.

[0216] MMAE

[0217] Production of ADC1 and similar ADCs

[0218] SuO-Val-Cit-PAB-MMAE(I) (13 equivalents, 5 mM in DMSO solution) was added to a phosphate-buffered saline solution of mAb1 and the allotype antibody (5 mg / mL), and the mixture was reacted on an orbital shaker at 25 °C for 2 hours. The average DAR was measured by high-resolution mass spectrometry and found to be 4.3.

[0219]

[0220] The reaction was quenched by adding 50 mM Tris·Cl (pH 7.4), and purification was achieved by repeated washing with phosphate-buffered saline via spin dialysis using a 10 kDa MW stop membrane. Purity was assessed by SDS-PAGE and SEC-HPLC using a MAbPac™ SEC-1 analytical column (ThermoFisher). For SEC-HPLC, 50 μl of sample (1 mg / mL) was injected and run at 0.15 mL / min for 30 min in phosphate-buffered saline (pH 7.4). Protein eluted as a single peak at 280 nm, with no aggregates. Figure 6A ).

[0221] Production of ADC2 and similar ADC2

[0222] Add 5 equivalents of TCEP to a solution of mAb3 and allotype antibody (5 mg / mL) in phosphate buffer (pH 7.0) containing 1 mM EDTA, and incubate the mixture on an orbital shaker at 25 °C for 90 min. Then, add 8 equivalents of Mc-Val-Cit-PAB-MMAE (from 5 mM stock solution in DMSO), and incubate the mixture on an orbital shaker at 25 °C for 45 min. Excess linker-load was then quenched with L-Cys (32 equivalents), and the remaining free load was removed via gel filtration using a Sephadex desalting column. The results were analyzed by SDS-PAGE and SEC-HPLC using MAbPac. TM Purity was assessed using a ThermoFisher SEC-1 analytical column. For SEC-HPLC, 50 μl of sample at 1 mg / mL was injected and run at 0.15 mL / min for 30 min in phosphate-buffered saline (pH 7.4). Proteins eluted as a single peak at 280 nm, with no aggregates. Figure 6B The average DAR was measured by hydrophobic interaction chromatography (HIC), and the DAR was found to be 3.9.

[0223] delutidecan

[0224] Production of ADC3 and similar ADC3

[0225] 100 equivalents of TCEP were added to a solution of mAb1 and allotype antibody (5 mg / mL) in phosphate buffer (pH 7.0) containing 1 mM EDTA, and the mixture was incubated on an orbital shaker at 25 °C for 90 min. Excess TCEP was removed by gel filtration using a Sephadex desalting column. Then, 20 equivalents of Mc-GGFG-Dxd (from a 5 mM stock solution in DMSO) were added, and the mixture was incubated on an orbital shaker at 25 °C for 45 min. Excess linker-load was then quenched with L-Cys (32 equivalents), and the remaining free load was removed by gel filtration using a Sephadex desalting column. The results were analyzed by SDS-PAGE and SEC-HPLC using MAbPac. TM Purity was assessed using a ThermoFisher SEC-1 analytical column. For SEC-HPLC, 50 μl of sample at 1 mg / mL was injected and run at 0.15 mL / min for 30 min in phosphate-buffered saline (pH 7.4). Proteins eluted as a single peak at 280 nm, with no aggregates. Figure 6CThe average DAR was measured by high-resolution mass spectrometry, and the DAR was found to be 7.4.

[0226] Combination of ADC1-3 and FGFR4

[0227] Next, the binding of ADC1 to FGFR4 was verified using the above method via a solid-state binding assay. The results confirmed that once conjugated with the drug, the antibody retained its ability to effectively bind to FGFR4, and its affinity was similar to that of the unconjugated antibody (IC50). 50 mAb1 0.93 nM, IC 50 ADC1 0.82 nM Figure 7A ).

[0228] The binding of ADC2 and ADC3 to FGFR4 was also verified by solid-state binding assays using the same method. Figure 7B -C).

[0229] Serum stability of ADC1-2

[0230] Mouse serum was spiked with 100 μg / mL of naked mAb1 or ADC1 and incubated at 37°C for 7 days. Samples from days 3 and 7 were quantified using the FGFR4 conjugation assay as described above. Results showed that after 7 days, the concentrations of mAb1 and drug-conjugated ADC1 were almost identical, indicating that the ADC was stable and showed no significant difference from the naked antibody. Figure 8A Serum stability of ADC2 was determined using the same method. Figure 8B ).

[0231] Nonspecific binding of ADC1-2

[0232] The inventors evaluated the nonspecific binding of ADC1 and mAb1 to DNA, heparin, and insulin. For this purpose, MAXISORP flat-bottom F96 Nunc plates were coated overnight at 4°C with PBS solution (pH 7.4) containing DNA (10 μg / mL), heparin (10 μg / mL), and insulin (5 μg / mL). The wells were washed with water and blocked with PBS solution containing 2% BSA at room temperature for 1 hour. 100 nM antibody was added to the wells in triplicate and incubated at room temperature for 1 hour. The plates were washed three times with water, and the nonspecific binding antibody was detected using HRP-conjugated anti-human Fc secondary antibody (Dako). The score was calculated as the ratio of the ELISA signal from the 100 nM antibody to the signal from the well containing buffer (instead of the primary antibody). Gantemab was used as a positive control (Ctrl+; Jain et al. 2017). Results showed that an ADC score below 5 indicated specificity of the conjugate to FGFR4 (…). Figure 9A The specificity of ADC2 was determined using the same method. Figure 9B ).

[0233] Pharmacokinetic (PK) study of ADC1 in mice

[0234] The pharmacokinetic properties of ADC1 were assessed by administering a single intravenous injection of 10 mg / kg to Balb / c mice, with plasma collected at 10 min, 6 h, 24 h, 48 h, 7 days, 14 days, and 21 days post-injection using heparin-lithium capillary blood collection. Blood samples were centrifuged at 2000 xg for 5 min at 4°C to obtain anemic platelet-rich plasma, and antibody levels were quantified using the FGFR4 binding assay as described above. The corresponding concentrations were determined using a nonlinear regression 4-parameter standard calibration curve. Results showed that the PK curve of ADC1 was highly similar to that of the carrier antibody (mAb1). Figure 10A ).

[0235] Pharmacokinetic (PK) study of ADC2 in mice

[0236] The pharmacokinetic properties of ADCs were assessed by administering a single intravenous injection of 3 mg / kg ADC1 to Balb / c mice, with plasma collected at 24, 48, 36, and 120 hours post-injection using heparin-lithium capillary blood collection. Platelet-rich plasma was obtained by centrifugation at 2000 xg for 5 minutes at 4°C, and antibody levels were quantified using the FGFR4 conjugate assay as described above. The corresponding concentrations were determined using a nonlinear regression 4-parameter standard calibration curve. The concentrations of total ADC2 and conjugated ADC2 are shown in the figure. Figure 10B middle.

[0237] IV. Biological effects on cancer cells and animal models of cancer

[0238] Cytotoxicity assay using ADC1

[0239] The cytotoxic activity of ADC1 was evaluated in a group of FGFR4-expressing breast and liver cancer cell lines (MDA-MB-453, CDK4 / 6i resistant cell lines ZR751, BT474, and T47D-PR, and Huh7) and FGFR4-negative breast cancer cell line MDA-MB-231 (ProteinAtlas, https: / / www.proteinatlas.org / ENSG00000160867-FGFR4 / cell+line; Turunene et al., 2019). As a control, mouse IgG control antibody (used in the previous section) was conjugated to the same payload (isotype-ADC) according to the procedure described above. Cell lines were cultured as detailed above and seeded in 200 μl of cell culture medium in 96-well plates (triple copies) at a density of 2000 to 12500 cells per well. After overnight incubation, the medium was replaced with serially diluted ADC1 starting at 1 μg / ml. A negative control consisting only of culture medium was also included. On day 6, CellTiter 96 Aqueous One Solution Reagent (Promega) was added to the cells according to the manufacturer's instructions, and absorbance was recorded at 490 nm using a TECAN Infinite MNano+ microplate reader. After subtracting background absorbance, the effect of each replicate of ADC or allotype-ADC on cell proliferation was calculated as follows: Cell viability % = (ADC treatment group / negative control group) * 100. Regardless of cancer type, the ADC targeting FGFR4, rather than the non-targeting allotype-ADC, exhibited cytotoxic activity against FGFR4-expressing cancer cell lines at very low doses, while having no effect on FGFR4-negative cell lines. Figure 11A -F).

[0240] Cytotoxicity assays using ADC2 and ADC3

[0241] The cytotoxic activity of ADC2 and / or ADC3 was evaluated in the acquired CDK4 / 6i resistant breast cancer cell line T47D-PR. As a control, human IgG control antibody (used in the previous section) was conjugated with the same payload (isotype-ADC) according to the procedure described above. The cell lines were cultured as detailed above and seeded in 200 μl of cell culture medium in 96-well plates (triple copies) at a density of 2000 cells per well. After overnight incubation, the medium was replaced with serially diluted ADC starting at 3.5 μg / ml. A negative control of medium only was also set up. On day 6, CellTiter-Glo Reagent (Promega) was added to the cells according to the manufacturer's instructions, and the luminescence values ​​were recorded using a Biotek SYNERGY H1M luminescent microplate reader. After subtracting background absorbance, the effect of each replicate of ADC or isotype-ADC on cell proliferation was calculated as follows: Cell viability % = (ADC treatment group / negative control group) * 100 ( Figure 11G ).

[0242] The cytotoxic activity of ADC2 was evaluated in the intrinsically CDK4 / 6i-resistant breast cancer cell line ZR-75-1. As a control, the human IgG control antibody (used in the previous section) was conjugated with the same payload (isotype-ADC) according to the procedure described above. The cell line was cultured as detailed above and seeded in 200 μl of cell culture medium in 96-well plates (triple copies) at a density of 4000 cells per well. After overnight incubation, the medium was replaced with serially diluted ADC starting at 1.2 or 10.5 μg / ml. A negative control consisting of medium only was also set up. On day 6, CellTiter-Glo Reagent (Promega) was added to the cells according to the manufacturer's instructions, and the luminescence values ​​were recorded using a Biotek SYNERGY H1M luminescent microplate reader. After subtracting background absorbance, the effect of each replicate of ADC or isotype-ADC on cell proliferation was calculated as follows: Cell viability % = (ADC treatment group / negative control group) * 100 ( Figure 11H ).

[0243] The cytotoxic activity of ADC2 was evaluated in the intrinsically CDK4 / 6i resistant breast cancer cell line BT474. As a control, the human IgG control antibody (used in the previous section) was conjugated with the same payload (isotype-ADC) according to the procedure described above. The cell line was cultured according to the detailed method described above and seeded in 96-well plates at a density of 3000 cells per well in 200 μl of cell culture medium (triple). After overnight incubation, the culture medium was replaced with serially diluted ADC starting at 0.4 μg / ml. A negative control of culture medium only was also set up. On day 6, CellTiter-GloReagent (Promega) was added to the cells according to the manufacturer's instructions, and the luminescence values ​​were recorded using a Biotek SYNERGY H1M luminescent microplate reader. After subtracting background absorbance, the effect of each replicate of ADC or isotype-ADC on cell proliferation was calculated as follows: Cell viability % = (ADC treatment group / negative control group) * 100 ( Figure 11I ).

[0244] The above results indicate that ADCs targeting FGFR4 exhibit cytotoxicity in breast cancer cell lines that are intrinsically resistant to or have acquired resistance to CDK4 / 6 inhibitors.

[0245] In vivo antitumor activity of ADC1

[0246] The effect of ADC1 on tumor growth was investigated in 12-week-old female Balb / c nude mice in the presence of supplemented estrogen. All mice received a subcutaneous injection of 1x10 ADC1 on their backs. 6 ZR751 cells were resuspended 1:1 in Matrigel:PBS1X. When the tumor reached 100 mm... 3 Mice were randomly assigned to different treatment groups: solvent, allotype-ADC (as described above), and ADC1. Treatment was administered intravenously once weekly at a dose of 5 mg / kg or the corresponding volume in the case of solvent-treated mice. Tumor volume was determined by caliper measurement until the end of the experiment. Treatment with FGFR4-specific ADC (ADC1) significantly reduced tumor growth compared to allotype-ADC or solvent. Notably, ADC1 treatment led to tumor regression at the end of the experiment (allotype-ADC vs ADC1, p < 0.0001; excipient vs ADC1, p < 0.0001). Figure 12A ).

[0247] In vivo antitumor activity of ADC1, ADC2 and ADC3

[0248] In 7-8 week old female Balb / c nude mice, the effects of ADC1, ADC2, and ADC3 on tumor growth were investigated in the presence of supplemented estrogen. All mice were subcutaneously injected with 1x10 ADC1 on the back. 7BT-474 cells were resuspended 1:1 in Matrigel:PBS1X. When the tumor reached 100 mm... 3 Mice were randomly assigned to different treatment groups: solvent, allotype ADC2, allotype ADC3, ADC1, ADC2, and ADC3. Treatment was administered intravenously at doses of 4 mg / kg for ADC1, ADC2, and allotype ADC2, and 10 mg / kg for ADC3 and allotype ADC3, or the corresponding volume in the case of solvent-treated mice. Tumor volume was determined by caliper measurement until the end of the experiment. Treatment with FGFR4-specific ADCs (ADC1, ADC2, and ADC3) significantly reduced tumor growth compared to the corresponding allotype-ADC or solvent. Notably, all ADC treatments (with different payloads, DAR, and conjugation methods) resulted in tumor regression at the end of the experiment (solvent vs. ADC1, p<0.05, solvent vs. ADC2, p<0.05, solvent vs. ADC3, p<0.05). Tumor volume was shown in [data missing]. Figure 12B middle.

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[0271] US11643467B2

[0272] Terms and Conditions

[0273] For completeness, various aspects of the present invention are set forth in the following numbered clauses:

[0274] Clause 1. A method for determining the response of a subject diagnosed with cancer to a medical regimen comprising one or more CDK4 / 6 inhibitors, the method comprising the steps of:

[0275] (a) Determine the expression level of FGFR4 in isolated test samples of the object, and

[0276] (b) Compare with reference values,

[0277] If FGFR4 is overexpressed, it indicates that the response to one or more CDK4 / 6 inhibitors will be weakened or absent.

[0278] Clause 2. The method according to Clause 1, wherein the expression level of FGFR4 is determined before the start of the medical protocol.

[0279] Clause 3. The method according to Clause 2, wherein the object is the first administration of a medical regimen containing one or more of the CDK4 / 6 inhibitors.

[0280] Clause 4. The method according to any one of the preceding clauses, wherein the subject has been treated with one or more CDK4 / 6 inhibitors prior to determining the FGFR4 expression level.

[0281] Clause 5. The method according to any one of Clauses 1-2, wherein the expression level of FGFR4 is determined during the medical protocol.

[0282] Clause 6. The method according to any one of the preceding clauses, wherein the one or more CDK4 / 6 inhibitors are selected from palbociclib, ribociclib, or abeciclib; in particular, the medical regimen includes administration of palbociclib.

[0283] Clause 7. The method according to any one of the preceding clauses, wherein the medical treatment further includes endocrine therapy.

[0284] Clause 8. The method according to any one of the preceding clauses, wherein determining the expression level of FGFR4 includes determining the amount of mRNA FGFR4.

[0285] Clause 9. The method according to any one of the preceding clauses, wherein the expression level of FGFR4 is determined by PCR.

[0286] Clause 10. The method according to any one of the preceding clauses, wherein the expression level is determined at the protein level.

[0287] Clause 11. The method according to Clause 10, wherein the expression level of said protein is determined using an antibody or a fragment thereof capable of binding to said protein.

[0288] Clause 12. The method according to Clause 11, wherein the antibody or fragment thereof forms part of the kit.

[0289] Clause 13. The method according to any one of the preceding clauses, wherein the test sample is selected from biological fluid samples (e.g., serum, blood, plasma), tissue samples (e.g., tissue homogenate) or cell lysates.

[0290] Clause 14. Use of FGFR4 as a biomarker for predicting or monitoring a subject’s response to a medical regimen containing one or more CDK4 / 6 inhibitors.

[0291] Clause 15. Use of tools for determining FGFR4 expression levels in the methods defined in any of Clauses 1-14.

[0292] Clause 16. The method according to any one of Clauses 1-13 or the use according to any one of Clauses 14-15, wherein the subject has breast cancer, particularly HR(+) breast cancer; particularly HR(+) / HER2 negative breast cancer.

[0293] Clause 17. A CDK4 / 6 inhibitor in a method for treating cancer of a subject, wherein the method comprises:

[0294] (i) Determine the expression level of FGFR4 in isolated test samples of the object.

[0295] (ii) Compare with reference values, and

[0296] (iii) If the expression level of FGFR4 in the test sample is equal to or lower than the reference level, the CDK4 / 6 inhibitor is administered to the subject.

[0297] Clause 18. An anti-FGFR4 antibody-drug conjugate (ADC), wherein the anti-FGFR4 antibody:

[0298] - Directly combined with FGFR4, and

[0299] - It is not specific for FGFR1, FGFR2 and FGFR3.

[0300] Clause 19. The ADC as described in Clause 18, wherein the antibody directly binds to FGFR4.

[0301] Clause 20. An ADC according to any one of Clauses 18-19 above, wherein the drug binds to the antibody via a cleavable linker.

[0302] Clause 21. The ADC according to any one of the preceding clauses, wherein the cleavable linker is a peptide linker that can be cleaved by intracellular proteases, particularly a Val-Cit linker or a Phe-Lys linker.

[0303] Clause 22. The ADC according to any one of the preceding clauses, wherein the drug binds to the Lys residues of the antibody via a cleavable linker.

[0304] Clause 23. The ADC according to any one of the preceding clauses, wherein the antibody is a human antibody or a humanized antibody.

[0305] Clause 24. An ADC according to any one of the preceding clauses, wherein said drug is auristatin, particularly monomethylauuristatin E.

[0306] Clause 25. The ADC as described in Clause 24, wherein the drug-antibody ratio is 2 to 6, particularly 4.

[0307] Clause 26. A pharmaceutical composition comprising any one of Clauses 18-25, an antibody-drug conjugate, and a pharmaceutically acceptable carrier.

[0308] Clause 27. The pharmaceutical composition according to Clause 26, wherein the composition is a lyophilized composition.

[0309] Clause 28. An anti-FGFR4 ADC according to any one of Clauses 18-25 above, or a pharmaceutical composition according to any one of Clauses 26-27, for the treatment of a subject suffering from cancer expressing FGFR4.

[0310] Clause 29. An ADC for the use described in Clause 28, wherein the subject has cancer that overexpresses FGFR4.

[0311] Clause 30. An ADC for the use described in any one of Clauses 28-29 above, wherein the cancer is selected from oral squamous cell carcinoma, head and neck cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, kidney cancer, prostate cancer, sarcoma, melanoma, leukemia, lymphoma, kidney cancer, duodenal cancer, small intestine cancer, liver cancer, pancreatic cancer, bladder cancer, rhabdomyosarcoma, or bile duct cancer; particularly, the cancer is breast cancer, liver cancer, gastric cancer, or colorectal cancer.

[0312] Clause 31. An ADC for use according to any one of Clauses 28-30 above, wherein the subject is also resistant to a medical regimen containing one or more CDK4 / 6 inhibitors.

[0313] Clause 32. An ADC for use according to any one of Clauses 28-31 above, wherein the ADC or composition is administered in combination with one or more therapeutic agents selected from the group consisting of: anticancer agents, antiallergic agents, antinausea agents (or antiemetics), analgesics, cytoprotective agents, and combinations thereof.

[0314] Clause 33. An FGFR4 inhibitor for reducing resistance to CDK4 / 6 inhibitors in subjects with cancer.

[0315] Clause 34. An FGFR4 inhibitor for the treatment of cancer in subjects resistant to CDK4 / 6 inhibitors.

[0316] Clause 35. An FGFR4 inhibitor for use according to any one of Clauses 33-34, selected from small molecules, anti-FGFR4 antibodies, or anti-FGFR4 antibody-drug conjugates.

[0317] Clause 36. An FGFR4 inhibitor for the use described in Clause 35, wherein the FGFR4 inhibitor is an anti-FGFR4 antibody.

[0318] Clause 37. An FGFR4 inhibitor for the use described in Clause 36, wherein the antibody does not bind to FGFR1, FGFR2, or FGFR3.

[0319] Clause 38. An FGFR4 inhibitor for use under any one of Clauses 33-37, which is an anti-FGFR4 antibody-drug conjugate as defined in any one of Clauses 19-25.

[0320] Clause 39. An FGFR4 inhibitor for use according to any one of Clauses 33-38, wherein the subject has breast cancer.

[0321] Clause 40. An FGFR4 inhibitor for any of the purposes described in any of Clauses 33-39, administered in combination with one or more anticancer agents, antiallergic agents, antinausea agents (or antiemetics), analgesics, cytoprotective agents, and combinations thereof.

[0322] Clause 41. A method for determining or recommending the initiation of administration of an ADC as defined herein for a subject having or suspected of having cancer, the method comprising the step of determining the expression level of FGFR4; and, if FGFR4 expression is confirmed, recommending the administration of the ADC of the present invention.

Claims

1. An FGFR4 binding molecule in a method for treating a cancer of a target, wherein the target has cancer cells that are resistant or refractory to CDK4 / 6 inhibitor therapy.

2. The FGFR4 binding molecule for use according to claim 1, wherein the subject has previously received CDK4 / 6 inhibitor therapy.

3. The FGFR4 binding molecule for use according to claim 1, wherein the subject has not previously received CDK4 / 6 inhibitor therapy.

4. The FGFR4 binding molecule for use according to any one of claims 1 to 3, wherein the determination is based at least in part on a sample isolated from said object, such as a tissue biopsy or a biofluid sample, said object having been identified as cancer cells resistant to CDK4 / 6 inhibitor therapy.

5. The FGFR4 binding molecule for use according to any one of claims 1 to 4, wherein the object is identified as having cancer cells resistant to CDK4 / 6 inhibitor therapy by means of a method comprising determining whether the object has cancer cells with elevated FGFR4 expression levels compared to a reference value.

6. The FGFR4 binding molecule for the use of claim 5, wherein the reference value is the FGFR4 level of a healthy subject.

7. The FGFR4 binding molecule for use according to any one of claims 1 to 4, wherein the object is identified as having cancer cells resistant to CDK4 / 6 inhibitor therapy by means of a method comprising determining whether the object has cancer cells with elevated FGFR4 expression levels compared to an object having cancer cells that respond to CDK4 / 6 inhibitor therapy.

8. The FGFR4 binding molecule for use according to claim 6 or 7, wherein the assay is an in vitro assay.

9. The FGFR4 binding molecule for use according to claim 8, wherein the in vitro assay is selected from one or more of the following: gene sequencing, immunohistochemistry, qPCR, and flow cytometry.

10. The FGFR4 binding molecule for use according to any one of claims 1 to 9, wherein the cancer cells are HR+.

11. The FGFR4 binding molecule for use according to any one of claims 1 to 9, wherein the cancer cell is HR-.

12. The FGFR4 binding molecule for use according to any one of claims 1 to 9, wherein the cancer cell is HER2+.

13. The FGFR4 binding molecule for use according to any one of claims 1 to 9, wherein the cancer cell is HER2-.

14. The FGFR4 binding molecule for use according to any one of claims 1 to 9, wherein the cancer cells are HR+ / HER2- or HR+ / HER2+.

15. The FGFR4 binding molecule for use according to any one of claims 1 to 14, wherein the FGFR4 binding molecule binds to FGFR4 with an EC50 of less than 1 nM, less than 0.5 nM, less than 0.1 nM, or less than 0.01 nM.

16. The FGFR4-binding molecule for the use of claim 15, wherein the EC50 is determined by solid-binding assay.

17. The FGFR4 binding molecule for use according to any one of claims 1 to 1, wherein the IC50 of the CDK4 / 6 inhibitor therapy is... 50 The values ​​are 300nM, 400nM, 500nM, and 600nM.

18. The FGFR4-binding molecule for the use of claim 17, wherein the IC 50 The value was determined by cell proliferation assay.

19. The FGFR4 binding molecule for use according to any one of claims 1 to 18, wherein the FGFR4 binding molecule does not bind to FGFR1, FGFR2 or FGFR3.

20. The FGFR4 binding molecule for use according to any one of claims 1 to 19, wherein the FGFR4 binding molecule is a small molecule.

21. The FGFR4 binding molecule for use according to any one of claims 1 to 19, wherein the FGFR4 binding molecule is an antibody or an antigen-binding fragment thereof.

22. The FGFR4 binding molecule for use according to claim 21, wherein the antibody or its antigen-binding fragment is Fab, F(ab')2, Fv, single-chain Fv (scFv), biantibody, single-domain antibody or camel antibody.

23. The method according to any one of claims 1 to 22, wherein the FGFR4 binding molecule inhibits the FGFR4 signaling pathway.

24. The FGFR4 binding molecule for use according to any one of claims 1 to 22, wherein the FGFR4 binding molecule reduces FGFR4 mRNA levels.

25. The FGFR4 binding molecule for use according to any one of claims 1 to 22, wherein the FGFR4 binding molecule reduces FGFR4 protein levels.

26. The FGFR4 binding molecule for use according to any one of claims 1 to 25, wherein the FGFR4 binding molecule is linked to a cytotoxic agent.

27. The FGFR4 binding molecule for use according to claim 26, wherein the cytotoxic agent is selected from one or more of the following: PDB, antimicrotubule agents (e.g., auristatin), maytansine, dolalastatin, chalcogenine, nemorubicin and its derivatives, PNU-159682, anthracyclines, vinca alkaloids, taxanes, trichothecene, CC1065, camptothecin, elinefadroxil, combroretin, dolalastatin, docamycin, enediyne, gerdemycin, indoline-benzodiazepine dimer, puromycin, tubulolysin, hammetrine, spliceostatin, praldidine, and their stereoisomers, isosteres, analogs and derivatives.

28. The FGFR4 binding molecule for use according to claim 27, wherein the antimicrotubule agent is auristatin, preferably selected from monomethyl auristatin D (MMAD or auristatin D derivative), monomethyl auristatin E (MMAE or auristatin E derivative), monomethyl auristatin F (MMAF or auristatin F derivative), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), and 5-benzoylvaleric acid-AE ester (AEVB).

29. The FGFR4-binding molecule for the use of claim 28, wherein the auristatin is MMAE or MMAF.

30. The FGFR4 binding molecule for use according to claim 27, wherein the cytotoxic agent is a topoisomerase I inhibitor, such as delutecan or esanotecan; or a DNA alkylating agent, such as docalomycin.

31. The FGFR4 binding molecule for use according to any one of claims 2426 to 30, wherein the FGFR4 binding molecule is linked to the cytotoxic agent via a connector.

32. The FGFR4 binding molecule for use according to claim 31, wherein the linker is a non-cleavable linker.

33. The FGFR4 binding molecule for use according to claim 32, wherein the connector is a cleavable connector.

34. The FGFR4 binding molecule for use according to claim 33, wherein the cleavable linker is a peptide linker capable of being cleaved by intracellular proteases.

35. The FGFR4 binding molecule for use according to claim 33 or 34, wherein the cleavable linker is a valine-citrulline linker or a Phe-Lys linker.

36. The FGFR4 binding molecule for use according to claim 35, wherein the linker is valine-citrulline-para-aminocarbamate (VC-PABC).

37. The FGFR4 binding molecule for use according to any one of claims 32 to 36, wherein the linker binds to one or more Lys residues.

38. The FGFR4 binding molecule for use according to any one of claims 32 to 36, wherein the linker binds to one or more Cys residues.

39. The FGFR4 binding molecule for use according to any one of claims 1 to 38, wherein the object has cancer cells that are resistant or refractory to endocrine therapy.

40. The FGFR4 binding molecule for use according to any one of claims 1 to 39, wherein the subject has previously received endocrine therapy.

41. The FGFR4 binding molecule for use according to claim 40, wherein the subject has previously received CDK4 / 6 inhibitor therapy.

42. The FGFR4 binding molecule for use according to any one of claims 1 to 4039, wherein the subject has not previously received endocrine therapy.

43. The FGFR4 binding molecule for use according to claim 39, wherein the subject has not previously received CDK4 / 6 inhibitor therapy.

44. The FGFR4 binding molecule for use according to any one of claims 1 to 43, wherein the cancer is selected from one or more of the following: oral squamous cell carcinoma, head and neck cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, kidney cancer, prostate cancer, sarcoma, melanoma, leukemia, lymphoma, kidney cancer, duodenal cancer, small intestine cancer, liver cancer, pancreatic cancer, bladder cancer, rhabdomyosarcoma, and bile duct cancer.

45. The FGFR4 binding molecule for use according to claim 4344, wherein the cancer is breast cancer, and wherein the breast cancer is HR+ breast cancer, HER2- breast cancer, HER2+ breast cancer, HR+ / HER2- breast cancer, HR+ / HER2+ breast cancer, or triple-negative breast cancer.

46. ​​The FGFR4 binding molecule for use according to any one of claims 1 to 45, wherein the cancer is a metastatic cancer, preferably wherein the cancer is metastatic HR+ / HER2- breast cancer, and the subject has previously been treated with a CDK4 / 6i inhibitor in combination with endocrine therapy.

47. The FGFR4 binding molecule for use according to any one of claims 1 to 46, wherein the CDK4 / 6 inhibitor therapy is palbociclib, ribociclib, or abeciclib.

48. An anti-FGFR4 antibody-drug conjugate (ADC) for the treatment or prevention of cancers expressing FGFR4, wherein the anti-FGFR4 antibody: - Directly combined with FGFR4, and - It is not specific for FGFR1, FGFR2 and FGFR3.

49. The ADC for use according to claim 48, wherein the drug binds to the antibody via a connector, the connector being a cleavable connector.

50. An ADC for use according to any one of the preceding claims, wherein the cleavable linker is a peptide linker capable of being cleaved by intracellular proteases, particularly a valine-citrulline linker or a Phe-Lys linker; in particular, the linker is a valine-citrulline-para-aminocarbamate (VC-PABC).

51. An ADC for use according to any one of the preceding claims, wherein the drug binds to one or more Lys or Cys residues of the antibody via the cleavable linker.

52. An ADC for use according to any one of the preceding claims, wherein the drug is selected from: (I) an antimicrotubule agent, preferably auristatin, more preferably monomethylauribatin E (MMAE) or monomethylauribatin F (MMAF); (II) a topoisomerase I inhibitor, preferably delutecan or esanotecan; or (III) a DNA alkylating agent, such as docalamycin.

53. The ADC for use according to claim 52, wherein the drug-antibody ratio is 1 to 10, preferably 2 to 8, more preferably 4 to 8.

54. The ADC for use according to claim 53, wherein (I) the drug is an antimicrotubule agent, preferably auristatin, more preferably monomethylauribatin E (MMAE) or monomethylauribatin F (MMAF), and the drug-antibody ratio is 4, or (II) the drug is a topoisomerase I inhibitor, preferably delutecan or esanotecan, and the drug-antibody ratio is 4 or 8.

55. An ADC for use according to any one of the preceding claims, administered in the form of a pharmaceutical composition together with one or more pharmaceutically acceptable carriers and / or excipients.

56. An ADC for use according to any one of the preceding claims, wherein the subject suffers from cancer associated with FGFR4 overexpression.

57. An ADC for use according to any one of the preceding claims, wherein the subject is also resistant to medical regimens comprising one or more CDK4 / 6 inhibitors, and optionally also resistant to other anticancer treatment regimens.

58. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 48 to 57 and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is a lyophilized composition.

59. The ADC for use according to any one of claims 48 to 57 or the pharmaceutical composition according to claim 58, for treating a subject suffering from cancer expressing FGFR4.

60. The ADC of claim 59, wherein the subject suffers from cancer that overexpresses FGFR4.

61. The ADC for use according to any one of claims 59 to 60, wherein the cancer is selected from oral squamous cell carcinoma, head and neck cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, kidney cancer, prostate cancer, sarcoma, melanoma, leukemia, lymphoma, kidney cancer, duodenal cancer, small bowel cancer, liver cancer, pancreatic cancer, bladder cancer, rhabdomyosarcoma, or bile duct cancer; particularly, the cancer is breast cancer, liver cancer, gastric cancer, or colorectal cancer; more particularly, the cancer is breast cancer, preferably wherein the cancer is metastatic HR+ / HER2- breast cancer, and the subject has previously been treated with a CDK4 / 6 inhibitor in combination with endocrine therapy.

62. The ADC for use according to any one of claims 58 to 61, wherein the subject is also resistant to medical regimens comprising one or more CDK4 / 6 inhibitors.

63. The ADC for use according to any one of claims 58 to 61, wherein the ADC or the pharmaceutical composition is administered in combination with one or more therapeutic agents selected from the group consisting of: anticancer agents, antiallergic agents, antinausea agents (or antiemetics), analgesics, cytoprotective agents, and combinations thereof.

64. An FGFR4 inhibitor for reducing resistance to CDK4 / 6 inhibitors in subjects with cancer.

65. An FGFR4 inhibitor in a method for treating or preventing cancer in a subject resistant to a CDK4 / 6 inhibitor.

66. The FGFR4 inhibitor for use according to claim 65, wherein the FGFR4 inhibitor does not bind to FGFR1, FGFR2, or FGFR3.

67. The FGFR4 inhibitor for use according to any one of claims 64 to 66, wherein the inhibitor is selected from small molecules, anti-FGFR4 antibodies or anti-FGFR4 antibody-drug conjugates; particularly, the FGFR4 inhibitor is an anti-FGFR4 antibody-drug conjugate.

68. An FGFR4 inhibitor for use according to any one of claims 64 to 66, wherein it is an anti-FGFR4 antibody-drug conjugate as defined in any one of claims 1 to 6.

69. An FGFR4 inhibitor for use according to any one of claims 64 to 6866, wherein the subject suffers from cancer selected from: oral squamous cell carcinoma, head and neck cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, kidney cancer, prostate cancer, sarcoma, melanoma, leukemia, lymphoma, kidney cancer, duodenal cancer, small bowel cancer, liver cancer, pancreatic cancer, bladder cancer, rhabdomyosarcoma, or bile duct cancer; particularly, said cancer is breast cancer, liver cancer, gastric cancer, or colorectal cancer; more particularly, said cancer is breast cancer.

70. An FGFR4 inhibitor for use according to any one of claims 64 to 69, wherein it is administered in combination with one or more anticancer agents, antiallergic agents, antinausea agents (or antiemetics), analgesics, cytoprotective agents, and combinations thereof.

71. A method for determining or recommending the initiation of administration of an ADC as defined herein for a subject having or suspected of having cancer, the method comprising the step of determining the expression level of FGFR4; and, if FGFR4 expression is confirmed, recommending the administration of the ADC of the present invention.

72. A method for determining the response of a subject diagnosed with cancer to a medical regimen comprising one or more CDK4 / 6 inhibitors, the method comprising the steps of: (a) Determine the expression level of FGFR4 in isolated test samples of the subject, and (b) Compare with reference values, If FGFR4 is overexpressed, it indicates that the response to one or more CDK4 / 6 inhibitors will be weakened or absent.

73. The method of claim 72, wherein the expression level of FGFR4 is determined before the medical protocol is initiated.

74. The method of claim 73, wherein the object is the first administration of a medical regimen comprising one or more of the CDK4 / 6 inhibitors.

75. The method according to any one of claims 72 to 74, wherein the subject has been treated with one or more CDK4 / 6 inhibitors prior to determining the FGFR4 expression level.

76. The method according to any one of claims 72 to 75, wherein the expression level of FGFR4 is determined during the medical protocol.

77. The method according to any one of claims 72 to 76, wherein the one or more CDK4 / 6 inhibitors are selected from palbociclib, ribociclib, or abeciclib; particularly, the medical regimen includes administration of palbociclib.

78. The method according to any one of claims 72 to 77, wherein the medical regimen further comprises endocrine therapy.

79. The method according to any one of claims 72 to 78, wherein determining the expression level of FGFR4 comprises determining the amount of mRNA FGFR4.

80. The method according to any one of claims 72 to 79, wherein the expression level of FGFR4 is determined by PCR.

81. The method according to any one of claims 72 to 80, wherein the expression level is determined at the protein level.

82. The method of claim 81, wherein the expression level of the protein is determined using an antibody or a fragment thereof capable of binding to the protein.

83. The method of claim 82, wherein the antibody or a fragment thereof forms part of the kit.

84. The method according to any one of claims 72 to 83, wherein the test sample is selected from biological fluid samples (e.g., serum, blood, plasma), tissue samples (e.g., tissue homogenate) or cell lysates.

85. Use of FGFR4 as a biomarker for predicting or monitoring a subject’s response to a medical regimen containing one or more CDK4 / 6 inhibitors.

86. Use of the tool for determining FGFR4 expression levels in the method defined in any one of claims 71 to 84.

87. The method according to any one of claims 71 to 84 or the use according to claim 85 or 86, wherein the subject has breast cancer, particularly HR(+) breast cancer; particularly HR(+) / HER2 negative breast cancer.

88. A CDK4 / 6 inhibitor in a method for treating cancer of a subject, wherein the method comprises: (i) Determine the expression level of FGFR4 in isolated test samples of the object. (ii) Compare with reference values, and (ii) If the expression level of FGFR4 in the test sample is equal to or lower than the reference level, the CDK4 / 6 inhibitor is administered to the subject.

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