Pharmaceutical composition for treating tumors and combination therapeutic agent

Abstract

The present invention provides technology that, in a metastatic cancer patient having a peritoneal metastasis and having a separate tumor from the peritoneal metastasis, treats the separate tumor. Provided is a pharmaceutical composition for treating, in a metastatic cancer patient having a peritoneal metastasis and a first tumor at a site other than the peritoneum, the first tumor. The pharmaceutical composition comprises astatine-211 conjugated to a targeting moiety for a tumor-associated antigen expressed on peritoneal metastasis cell surfaces, is administered intraperitoneally, and is administered in combination with an immune checkpoint inhibitor.

Description

Pharmaceutical compositions and combination therapies for tumor treatment

[0001] The present disclosure relates to the treatment of tumors.

[0002] The abdominal cavity is the space in the bodies of mammals, including humans, that houses organs such as the digestive and urinary systems, and the organs within the abdominal cavity are covered by the peritoneum. Peritoneal dissemination occurs when cancer cells originating in an organ within the abdominal cavity leak out of the organ, metastasize to the peritoneum, and form a tumor scattered across the peritoneal surface, resembling seeds. Peritoneal dissemination can originate from gastrointestinal cancers such as gastric cancer, pancreatic cancer, and colorectal cancer, as well as ovarian cancer and ureteral cancer. In particular, peritoneal dissemination is one of the most common types of metastasis, along with lymph node metastasis and liver metastasis, in gastric cancer.

[0003] Currently, there are few effective treatments for peritoneal dissemination. Complete removal of peritoneal dissemination through surgery is difficult, and even if it appears to have been removed macroscopically after surgery, recurrence is common. For this reason, for example, gastric resection is not typically performed for peritoneal dissemination of gastric cancer; instead, systemic chemotherapy using intravenous or oral anticancer drugs is often used. Regardless of the specific treatment, cancer with peritoneal dissemination generally poses a serious prognosis. Peritoneal dissemination itself can lead to life-threatening conditions such as intestinal obstruction, ascites, hydronephrosis, and peritoneal carcinomatosis, along with symptoms such as significant abdominal distension, nausea, and vomiting. Furthermore, highly metastatic malignant tumors present simultaneously with peritoneal dissemination also serve as breeding grounds for continued peritoneal dissemination and the development of other metastatic cancers. Naturally, reducing or eliminating these tumors as much as possible is desirable. However, the fact that they coexist with peritoneal dissemination and have already metastasized makes targeted, effective treatment even more difficult.

[0004] Non-Patent Document 1 reports that astatine-211 (α-ray emitting nuclide) was used in a peritoneal metastasis model created by injecting HER2-positive human gastric cancer cells into the peritoneal cavity of immunodeficient mice. 211 described that the anti-HER2 antibody trastuzumab labeled with trastuzumab (At) was able to eliminate peritoneal dissemination.

[0005] Patent Document 1, which does not specifically refer to peritoneal dissemination, describes that the combination of checkpoint protein inhibition and targeted therapy for cancer cell damage may provide a therapy with less toxicity and improved efficacy. Patent Document 1 describes the effects of actinium-225 ( 225 The study demonstrated this effect by combining an anti-IGF-1R antibody labeled with IGF-1R (Ac) with a checkpoint inhibitor. Treatment with the checkpoint inhibitor alone resulted in a modest reduction in tumor volume in the mouse colon cancer. 225 It was reported that treatment with Ac-labeled antibody alone showed a greater reduction in tumor volume, but when the two were administered together, the tumor volume was further reduced significantly.

[0006] Li et al., Cancer Sci 108 (2017) 1648-1656

[0007] International Publication No. 2020 / 115556

[0008] The present inventors have found that intraperitoneal administration of certain alpha-emitting nuclides targeted against tumor-associated antigens expressed on the cell surface of peritoneal dissemination, combined with the use of immune checkpoint inhibitors, not only reduces peritoneal dissemination but also significantly inhibits the growth of other tumors present at sites other than the peritoneum. Thus, embodiments of the present disclosure provide a technique for treating other tumors in metastatic cancer patients who have peritoneal dissemination and a tumor other than the peritoneal dissemination.

[0009] The present disclosure includes the following embodiments: [1] A pharmaceutical composition for treating a first tumor in a metastatic cancer patient with peritoneal dissemination and a first tumor at a site other than the peritoneum, the pharmaceutical composition comprising astatine-211 conjugated to a targeting moiety for a tumor-associated antigen expressed on the cell surface of peritoneal dissemination, the pharmaceutical composition being administered intraperitoneally, and the pharmaceutical composition being administered in combination with an immune checkpoint inhibitor. [2] The targeting moiety comprises an antibody or antigen-binding fragment thereof for the tumor-associated antigen, or the targeting moiety comprises an antibody or antigen-binding fragment thereof for the tumor-associated antigen, and 211The pharmaceutical composition according to [1], wherein the immune checkpoint inhibitor is conjugated to astatine-211 via the group At-C6H4-CONH-. [3] The pharmaceutical composition according to [1] or [2], wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-CTLA-4 antibody, an anti-PD-L1 antibody, or an antigen-binding fragment thereof, or a combination of two or more thereof. [4] The pharmaceutical composition according to any one of [1] to [3], wherein the pharmaceutical composition is for treating the first tumor and the peritoneal dissemination together. [5] The pharmaceutical composition according to any one of [1] to [4], wherein the first tumor is a primary tumor and the peritoneal dissemination is a metastatic tumor from the first tumor, or wherein both the first tumor and the peritoneal dissemination are metastatic tumors from a primary tumor. [6] The pharmaceutical composition according to [5], wherein the primary tumor is gastric cancer. [7] The pharmaceutical composition according to any one of [1] to [6], wherein the tumor-associated antigen is selected from the group consisting of CEACAM5, CEACAM6, CLDN4, CLDN7, EpCAM, CA72-4, CA19-9, and a growth factor receptor. [8] The pharmaceutical composition according to [7], wherein the growth factor receptor is selected from the group consisting of EGFR, HER2, EGFR3, EGFR4, c-MET, FGFR1, FGFR2, FGFR3, FGFR4, a VEGF receptor, an insulin receptor, and a PDGF receptor. [9] The pharmaceutical composition of [8], wherein the tumor-associated antigen is FGFR4, and the targeting moiety is (i) an anti-FGFR4 antibody or an antigen-binding fragment thereof, comprising a light chain variable region comprising an amino acid sequence having 95% or more sequence identity to SEQ ID NO: 1 and a heavy chain variable region comprising an amino acid sequence having 95% or more sequence identity to SEQ ID NO: 2, or (ii) an anti-FGFR4 antibody comprising a light chain comprising an amino acid sequence having 95% or more sequence identity to SEQ ID NO: 4 and a heavy chain comprising an amino acid sequence having 95% or more sequence identity to SEQ ID NO: 5.

[10] A combination therapy for treating a first tumor in a patient with metastatic cancer who has peritoneal dissemination and a first tumor at a site other than the peritoneum, the combination therapy comprising astatine-211 in combination with an immune checkpoint inhibitor selected from the group consisting of an anti-PD-1 antibody, an anti-CTLA-4 antibody, an anti-PD-L1 antibody, and antigen-binding fragments thereof, and combinations of two or more thereof, wherein the astatine-211 is conjugated to a targeting moiety against a tumor-associated antigen expressed on the cell surface of peritoneal dissemination and is administered intraperitoneally, and the immune checkpoint inhibitor is administered intraperitoneally or intravenously.

[11] The targeting moiety comprises an antibody or antigen-binding fragment thereof against the tumor-associated antigen, or the targeting moiety comprises an antibody or antigen-binding fragment thereof against the tumor-associated antigen, and 211The combination therapeutic agent according to

[10] , which is conjugated to astatine-211 via the group At-C6H4-CONH-.

[12] The combination therapeutic agent according to

[10] or

[11] , which is intended for use in treating the first tumor and the peritoneal dissemination together.

[13] The combination therapeutic agent according to any one of

[10] to

[12] , wherein the first tumor is a primary tumor and the peritoneal dissemination is a metastatic tumor from the first tumor, or wherein the first tumor and the peritoneal dissemination are both metastatic tumors from a primary tumor.

[14] The combination therapeutic agent according to

[13] , wherein the primary tumor is gastric cancer.

[15] The combination therapeutic agent according to any one of

[10] to

[14] , wherein the tumor-associated antigen is selected from the group consisting of CEACAM5, CEACAM6, CLDN4, CLDN7, EpCAM, CA72-4, CA19-9, and a growth factor receptor.

[16] The combination therapy of

[15] , wherein the growth factor receptor is selected from the group consisting of EGFR, HER2, EGFR3, EGFR4, c-MET, FGFR1, FGFR2, FGFR3, FGFR4, VEGF receptor, insulin receptor, and PDGF receptor.

[17] The combination therapy of

[16] , wherein the tumor-associated antigen is FGFR4, and the targeting moiety is (i) an anti-FGFR4 antibody or an antigen-binding fragment thereof, comprising a light chain variable region comprising an amino acid sequence having 95% or more sequence identity to SEQ ID NO: 1 and a heavy chain variable region comprising an amino acid sequence having 95% or more sequence identity to SEQ ID NO: 2, or (ii) an anti-FGFR4 antibody comprising a light chain comprising an amino acid sequence having 95% or more sequence identity to SEQ ID NO: 4 and a heavy chain comprising an amino acid sequence having 95% or more sequence identity to SEQ ID NO: 5.

[0010] Figure 1 shows the FGFR4 antibody conjugated to the FGFR4 antibody. 211 Figure 2 illustrates the progression of primary tumor size in mice treated with At, immune checkpoint inhibitors, or a combination thereof, compared to untreated control tumor size. Figure 2 illustrates an experiment similar to Figure 1, but using only one immune checkpoint inhibitor instead of two. Figure 3 illustrates the progression of primary tumor size in mice treated with At, immune checkpoint inhibitors, or a combination thereof, compared to untreated control tumor size.211 By intraperitoneal (ip) administration of At-antiFGFR4 211 Peritoneal accumulation of unconjugated At 211 The quantitative results are shown in comparison with At administration, intravenous administration (iv), and intragastric administration (ig). 211 Figure 5 shows photographs of abdominal incision showing the state of peritoneal dissemination on day 10 after At administration. 211 The images show hematoxylin and eosin staining (top row), anti-phosphorylated histone H2A.X staining (middle row), and anti-HMGB1 staining (bottom row) of peritoneal dissemination tissue on day 7 after At administration.

[0011] In one aspect, the present disclosure provides a pharmaceutical composition for treating a first tumor in a metastatic cancer patient with peritoneal dissemination and a first tumor at a site other than the peritoneum. The pharmaceutical composition comprises astatine-211 conjugated to a targeting moiety for a tumor-associated antigen expressed on the cell surface of peritoneal dissemination. The pharmaceutical composition is also characterized by being administered intraperitoneally and in combination with an immune checkpoint inhibitor. The tumor in this embodiment is a malignant tumor capable of metastasis. The patient is a mammalian patient, and a human patient is particularly preferred.

[0012] In the present disclosure, tumor-associated antigens (commonly abbreviated as TAA in the art) refer to cell surface antigens whose expression is enhanced in tumor peritoneal dissemination cells compared to non-tumor cells, as understood by those skilled in the art. In the present disclosure, tumor-associated antigens also encompass tumor-specific antigens that are detected in tumor cells but not in non-tumor cells. Tumor-associated antigens may also be tumor-preferential antigens that are expressed in non-tumor cells but at significantly higher levels in tumor cells. Those skilled in the art can confirm tumor-associated antigens using techniques such as immunostaining and immunoblotting. Because tumor-associated antigens are expressed at higher levels on the surface of tumor peritoneal dissemination cells than non-tumor peritoneal cells, it is understood that astatine-211 conjugated to a targeting moiety (described below) and administered intraperitoneally will selectively accumulate in peritoneal disseminations.

[0013] Specific examples of tumor-associated antigens that can be used in this embodiment include CEACAM5, CEACAM6, CLDN4, CLDN7, EpCAM, CA72-4, CA19-9, and growth factor receptors. Specific examples of growth factor receptors include EGFR, HER2, EGFR3, EGFR4, c-MET, FGFR1, FGFR2, FGFR3, FGFR4, VEGF receptor, insulin receptor, and PDGF receptor. EGFR stands for epidermal growth factor receptor, FGFR stands for fibroblast growth factor receptor, VEGF stands for vascular endothelial growth factor, and PDGF stands for platelet-derived growth factor. FGFR4 is a particularly suitable tumor-associated antigen, particularly preferred for peritoneal dissemination when the primary lesion is gastric cancer. While the surface antigens that are particularly highly expressed in peritoneal dissemination may vary from patient to patient, it is believed that most peritoneal disseminations exhibit elevated expression of one of the antigens listed in this paragraph. For example, Toh et al., BMJ Open Gastro 2020;7:e000452, reported that in most gastric cancers, the cell surface antigens CEACAM5, CEACAM6, CLDN4, CLDN7, EpCAM, CA72-4, and CA19-9 serve as markers for distinguishing them from normal cells. In another example, Inokuchi et al., World Journal of Surgical Oncology (2017) 15:2, reported that FGFR4 expression was elevated in over 80% of gastric cancers, especially those with peritoneal dissemination. Surface markers from the primary tumor are typically maintained in metastatic tumors.

[0014] In certain embodiments, before the pharmaceutical composition or combination therapeutic agent of the present disclosure is administered, a procedure may be performed to confirm that any of the above-mentioned tumor-associated antigens are expressed at increased levels in the patient's peritoneal dissemination compared to non-tumor cells. Furthermore, it is also possible to identify tumor-associated antigens other than those specifically mentioned above present in the peritoneal dissemination of an individual patient without undue trial and error, for example, by performing a comprehensive test according to the method described in Toh et al., supra, or Yamamoto et al., supra, or by using a cell surface protein gene microarray, which can detect gene locus amplification or elevated mRNA expression, and other methods well known to those skilled in the art.

[0015] A targeting moiety for a tumor-associated antigen is a molecular entity that selectively binds to the tumor-associated antigen, preferably comprising (or consisting of) an antibody or antigen-binding fragment thereof against the tumor-associated antigen. Antibodies against the aforementioned cell surface antigens are often commercially available, and preparing such antibodies or antigen-binding fragments is within the skill of those in the art. Antigen-binding fragments, including single-chain Fv (scFv), Fab, (Fab')2, and the like, are also well known to those skilled in the art. Preferably, the antibody is a humanized or fully human antibody. The targeting moiety may also be a nucleic acid aptamer. Nucleic acid aptamers can provide high binding specificity and affinity comparable to antibodies.

[0016] In a specific preferred embodiment, the tumor-associated antigen is FGFR4, and the targeting moiety can be an anti-FGFR4 antibody or an antigen-binding fragment thereof. In the present disclosure, terms such as "anti-(antigen) antibody," "antibody against (antigen)," and the like refer to an antibody that specifically binds to that antigen, as understood by those skilled in the art. The anti-FGFR4 antibody or antigen-binding fragment thereof preferably comprises a light chain variable region comprising an amino acid sequence having 95% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO:1, and a heavy chain variable region comprising an amino acid sequence having 95% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO:2. The 100% sequence identity among these corresponds to the light and heavy chain variable regions of the hLD1.v22 monoclonal antibody described in Bumbaca et al., mAbs 2011 Vol. 3 Issue 4 Pages 376-386. The anti-FGFR4 antibody can further preferably comprise a constant region corresponding to the constant region of human IgG1. The anti-FGFR4 antibody preferably comprises a light chain comprising an amino acid sequence having 95% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO:4, and a heavy chain comprising an amino acid sequence having 95% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO:5. The anti-FGFR4 antibody or antigen-binding fragment thereof preferably comprises a light chain CDR1 comprising the amino acid sequence of RTSQDISNFLN (SEQ ID NO:8), a light chain CDR2 comprising the amino acid sequence of YTSRLHS (SEQ ID NO:9), and a light chain CDR3 comprising the amino acid sequence of QQGNALPYT (SEQ ID NO:10), and a heavy chain CDR1 comprising the amino acid sequence of NHWMN (SEQ ID NO:11), a heavy chain CDR2 comprising the amino acid sequence of MILPVDSETTLEQKFKD (SEQ ID NO:12), and a heavy chain CDR3 comprising the amino acid sequence of GDISLFDY (SEQ ID NO:13). These are complementarity-determining regions (CDRs) according to the Kabat definition.

[0017] Antibodies or antigen-binding fragments thereof or other targeting moieties of the present disclosure, including the anti-FGFR4 antibodies or antigen-binding fragments thereof described above, can be used, for example, in: 211Astatine-211 ( 211 The linker structure -CH-CONH- can be obtained by a conjugation reaction using N-succinimidyl 3-trimethylstannyl benzoate, as described below. 211 At (or the precursor trimethylstannyl group) can be attached, for example, to the meta position of the benzene ring (-CH-). It is understood that -CONH- represents an amide group, and that the nitrogen atom (N) can come from an amino group on an antibody or antigen-binding fragment thereof.

[0018] Astatine-211 is an alpha-emitting nuclide with a half-life of 7.2 hours. The half-life is related to the rate of cell-damaging energy release. The benefits of the presently disclosed embodiments are believed to be due to the properties of astatine-211, which can effectively damage tumor cells with its moderately short half-life and the short range inherent in alpha rays. In a preferred embodiment, astatine-211 is conjugated to a targeting moiety via a 3-trimethylstannyl group linked to the targeting moiety. Specifically, as described in Lindegren et al. J Nucl Med 2008; 49:1537-1545, a 3-trimethylstannyl group linked to an antibody can be reacted with astatine-211 to replace the 3-trimethylstannyl substituent with an astatine-211 substituent. Linking the 3-trimethylstannyl group to an antibody can be preferably achieved, for example, by reacting the NHS ester group of N-succinimidyl 3-trimethylstannyl benzoate with an amino group on the antibody to form an amide bond, as understood by those skilled in the art. However, the above is merely a preferred example of astatine-211 conjugation method, and it will be understood by those skilled in the art that possible conjugation methods are not limited thereto. For example, Green et al., Blood. 2015 Mar 26; 125(13): 2111-2119, describes astatine-211 conjugation method that does not involve either stannyl groups or NHS ester groups. These same conjugation reactions can also be applied to targeting moieties other than antibodies.

[0019] In the present disclosure, the modifier "first" in the term "first tumor" is used for the convenience of distinguishing a tumor located at a site other than the peritoneum from peritoneal dissemination. In an individual patient with metastatic cancer, the first tumor and peritoneal dissemination typically originate from a common primary tumor. For example, the first tumor may be located in the primary tumor itself, and the peritoneal dissemination may be a metastatic tumor from the first tumor. Alternatively, both the first tumor and the peritoneal dissemination may be metastatic tumors from a common primary tumor. In this case, for example, the first tumor may be formed by metastasis from the primary tumor, and the peritoneal dissemination may be formed by metastasis from the first tumor. Those skilled in the art can clearly recognize multiple tumors originating from a common primary tumor based on the diagnostic history of an individual patient and / or tumor biological testing (e.g., genomic analysis and / or antigen analysis). The primary tumor is typically a cancer of the digestive tract, such as the stomach, pancreas, or colon, or of an organ in the abdominal cavity, such as the ovary or ureter, and the most typical primary tumor is gastric cancer.

[0020] The pharmaceutical composition of this embodiment is administered in combination with an immune checkpoint inhibitor (ICI). Administration in combination with an ICI means that the pharmaceutical composition and the ICI are administered to a patient separately, with one drug acting under the influence of the other. Therefore, the pharmaceutical composition and the ICI do not necessarily need to be administered at exactly the same time, and embodiments in which the pharmaceutical composition and the ICI are administered simultaneously, for example, in the same formulation, are not excluded. For example, the pharmaceutical composition containing astatine-211 may be administered intraperitoneally, while the ICI may be administered intraperitoneally or intravenously. Preferably, the ICI is also administered intraperitoneally. The ICI may be administered before, after, or simultaneously with astatine-211 administration, or any combination thereof. Preferably, the ICI is administered within two weeks before or within one week after astatine-211 administration. Even in situations where administration of the immune checkpoint inhibitor alone or the astatine-211 conjugate alone does not significantly suppress the growth of the first tumor, the combination of the two can synergistically suppress tumor growth.

[0021] ICIs and their uses are well known to those skilled in the art. The ICI used in this embodiment can be, for example, an anti-PD-1 antibody, an anti-CTLA-4 antibody, an anti-PD-L1 antibody, or an antigen-binding fragment thereof, or a combination of two or more thereof. A combination of an anti-PD-1 antibody or its antigen-binding fragment and an anti-CTLA-4 antibody or its antigen-binding fragment, or a combination of an anti-PD-L1 antibody or its antigen-binding fragment and an anti-CTLA-4 antibody or its antigen-binding fragment is more preferred.

[0022] The pharmaceutical composition of this embodiment containing radioactive astatine-211 is administered intraperitoneally. Therefore, it is understood that the pharmaceutical composition of the present disclosure provides internal radiation therapy. The dose of radioactive astatine-211 can be varied as appropriate by those skilled in the art. For example, a pharmaceutical composition containing 0.1 MBq to 2 GBq, 0.4 MBq to 1 GBq, or 1 MBq to 600 MBq of astatine-211 can be administered intraperitoneally (e.g., to humans). It is understood that the dose corresponding to the same level of effect may differ between species, for example, because the abdominal surface area and body weight of mice and humans differ. The radiation dose can be adjusted appropriately based on this disclosure and the knowledge of those skilled in the art. In a specific embodiment, a pharmaceutical composition containing 20 MBq to 1 GBq of astatine-211 can be administered intraperitoneally to a human. In a specific embodiment, a pharmaceutical composition containing 0.1 to 10 MBq / kg body weight of astatine-211 can be administered intraperitoneally to a human.

[0023] Peritoneal dissemination presents a unique situation in which multiple small tumors cluster on a single surface, enabling simultaneous treatment. Because intraperitoneally administered alpha-emitting astatine-211 targets and accumulates in peritoneal dissemination via the targeting moiety, the pharmaceutical composition can also effectively reduce, eliminate, or treat peritoneal dissemination itself. However, this embodiment is characterized by using the unique tumors known as peritoneal dissemination as a springboard, or by using the peritoneum containing peritoneal dissemination as an active breeding ground for the induction or amplification of tumor-specific immunity, to treat a primary tumor located away from the peritoneum. It is believed that the effect on the primary tumor located away from the peritoneum involves cellular immunity induced in the peritoneal dissemination and then circulating throughout the body. As described above, the serious condition of peritoneal dissemination and other malignant tumors outside the peritoneum coexisting in the same body makes it even more difficult to perform targeted and effective treatment. However, embodiments of the present disclosure can be characterized by their ability to systematically treat complex cancers by utilizing the unique pathology of peritoneal dissemination. Tumor treatment can include reducing the tumor growth rate, reducing tumor volume, or eliminating the tumor. The pharmaceutical compositions and combined therapeutic agents of the present disclosure can also be provided for use in treating peritoneal dissemination together with the first tumor.

[0024] The pharmaceutical composition of the present embodiments may include a pharmaceutically acceptable carrier or excipient in addition to the astatine-211 conjugate. For example, the astatine-211 conjugate and / or ICI may be provided and / or administered in the form of an aqueous solution. The aqueous solution may include one or more pharmaceutically acceptable salts, buffering agents, preservatives, etc. Embodiments of the present disclosure may also be described as a method of treating the first tumor, or an astatine-211 conjugate for use in treating the first tumor, or use of an astatine-211 conjugate in the manufacture of a medicament for treating the first tumor.

[0025] In one aspect, the present disclosure provides a combination therapy for treating a first tumor in a patient with metastatic cancer who has peritoneal dissemination and a first tumor at a site other than the peritoneum. The combination therapy comprises astatine-211 conjugated to a targeting moiety for a tumor-associated antigen expressed on the cell surface of peritoneal dissemination, in combination with an ICI selected from the group consisting of an anti-PD-1 antibody, an anti-CTLA-4 antibody, an anti-PD-L1 antibody, and antigen-binding fragments thereof, and combinations of two or more thereof. In this combination therapy, the astatine-211 is administered intraperitoneally, and the ICI is administered intraperitoneally or intravenously. It will be understood by those skilled in the art that the elements of this embodiment of the combination therapy correspond to the elements of the embodiment of the pharmaceutical composition described above, and therefore, the explanations provided for the pharmaceutical composition described above also apply to the combination therapy.

[0026] The two agents of the combined therapy, i.e., the astatine-211 conjugate and the ICI, are administered in combination with each other. It is not necessary for the astatine-211 conjugate and the ICI to be administered at exactly the same time, and embodiments in which the astatine-211 conjugate and the ICI are administered simultaneously, e.g., in the same formulation, are not excluded. For example, the astatine-211 conjugate may be administered intraperitoneally, while the ICI may be administered intraperitoneally or intravenously. Preferably, the ICI is also administered intraperitoneally. The ICI may be administered before, after, or simultaneously with the administration of astatine-211, or any combination thereof. Preferably, the ICI is administered within two weeks before or within one week of the administration of astatine-211.

[0027] When multiple numerical ranges are described herein, the upper or lower limit of a given numerical range can be combined with the upper or lower limit of another given numerical range, and it is understood that the resulting combined numerical ranges are also disclosed in this application. In this specification, the phrase "comprising A" or "having A" means, unless the context requires otherwise, that an object that includes not only A but also other elements, and is understood to also include a disclosure of an object that includes only A and not other elements, i.e., an object consisting of A. Unless the context requires otherwise, a reference to a singular object is understood to also include a disclosure of an embodiment in which multiple objects are used.

[0028] The following examples will explain the embodiments of the present disclosure in more detail, but the present invention is not limited to the specific experimental conditions exemplified in these examples. In particular, the examples show experiments using anti-FGFR4 antibodies for the convenience of the model animals used, but it will be understood by those skilled in the art that embodiments of the present invention can also use tumor-associated antigen targeting moieties other than anti-FGFR4 antibodies based on the same principles.

[0029] [Preparation of Astatine-211-Labeled Anti-FGFR4 Antibody] 1. Preparation of Anti-FGFR4 Antibody Expression Vector For use in experiments using immune-competent peritoneal dissemination model mice, an anti-FGFR4 monoclonal antibody was prepared containing the light chain variable region (SEQ ID NO: 1) and heavy chain variable region (SEQ ID NO: 2) of the hLD1.v22 monoclonal antibody described in Bumbaca et al., mAbs 2011 Vol. 3 Issue 4, Pages 376-386. This antibody can recognize both human and mouse FGFR4. The constant region sequence used was that of human IgG1. The signal sequence for secretory expression was that described in WO 2008 / 148519 (SEQ ID NO: 3).

[0030] Based on the amino acid sequences of the light chain (SEQ ID NO: 4) and heavy chain (SEQ ID NO: 5) of the above anti-FGFR4 antibody, DNA containing gene sequences (SEQ ID NO: 6 for the light chain and SEQ ID NO: 7 for the heavy chain) that were codon-optimized for expression in CHO (Chinese hamster ovary) cells was artificially synthesized (Eurofins Genomics). The artificially synthesized light and heavy chain gene sequences were amplified by PCR and inserted into the plasmid vector pcDNA3.4 (#A14697, Thermo Fisher Scientific) to clone the light and heavy chain expression plasmid DNA. This plasmid was used to transform Escherichia coli competent cells DH5-α. The plasmid was extracted and purified from the isolated strain, and integration of the target gene at the intended location was confirmed by nucleic acid sequencing.

[0031] 2. Expression of anti-FGFR4 antibody expression vector and antibody purification. CHO-S cells were transfected with the light and heavy chain plasmid DNAs using the ExpiCHO Expression System Kit (A29133, Thermo Fisher Scientific). The weight ratio of the light chain and heavy chain plasmids cotransfected was 1:1. Other dosages were determined according to the kit manual. CHO-S cells were cultured in sterile Erlenmeyer flasks (SEF125V, SEF250V, or SEF1000V, Violamo) with vented filter caps in a high-humidity shaker (CS-LR, TAITEC) in a CO2 incubator (Prescyto, MG-71C, TAITEC). After transfection with the above plasmid DNA and incubation for 7–10 days, the culture supernatant was collected and the target antibody protein was purified using rProtein A Sepharose Fast Flow (#17127901, Cytiva). PBS (phosphate-buffered saline) was used as the binding and washing buffer, and 0.1 M sodium citrate, pH 3.0, was used as the elution buffer. 1 mL of elution was performed and collected in a tube. 40 μL of 1 M Tris-HCl, pH 9.0, was added to each mL of collected sample for neutralization. After measuring the protein concentration, the necessary fractions were pooled and concentrated using an ultrafiltration membrane to a concentration of 6–10 mg / mL. The concentrated sample was filter-sterilized and stored in a -80°C freezer.

[0032] 3. m-MeATE Conjugation of Antibodies. Antibody conjugation with N-succinimidyl 3-trimethylstannylbenzoate (#S692025, Toronto Research Chemicals) (hereafter referred to as "m-MeATE") was performed following the procedure described by Lindegren et al. J Nucl Med 2008; 49:1537-1545. Specifically, the antibody buffer was first replaced with 0.2 M sodium carbonate, pH 8.5, using a PD MidiTrap™ G-25 (#28918008, Cytiva). The antibody concentration was then adjusted to 4 mg / mL with the same buffer. Next, 10 μL of 25 μmol / mL m-MeATE in DMSO was added to 0.5-1.0 mL of antibody solution (4 mg / mL concentration) with stirring, and the mixture was incubated at 25°C for 30 minutes with gentle stirring. The unbound m-MeATE was then removed by buffer exchange with PBS using a PD MidiTrap™ G-25. The eluate contained anti-FGFR4 antibody linked to a 3-trimethylstannyl group ("anti-FGFR4-ATE"). The solution was filter-sterilized and stored at -80°C.

[0033] 4. Antibody labeling with astatine-211. Based on Lindegren et al., we labeled anti-FGFR4-ATE with astatine-211 ( 211 Anti-FGFR4-ATE (At) labeling was performed. 200 μg of anti-FGFR4-ATE was added with 0.2 M acetate buffer (pH 5.5) to adjust the protein concentration to 200 μg / 200 μL, and the pH was confirmed to be 5.5 using pH test paper. 211 Add 15 μL of astatine activation solution (20 μg / mL N-iodosuccinimide (NIS) / methanol / 1% acetic acid) to At. 211The FGFR4-ATE solution was added to the astatine solution and incubated for 1 minute. Next, 3 μL of NIS (1 mg / mL) / methanol / 1% acetic acid was added and incubated for 1 minute. Then, 5 μL of 0.25 M ascorbic acid was added. Finally, the buffer was replaced with PBS using a PD SpinTrapG25 (#28918004, Cytiva) to remove free astatine. 211 At-conjugated anti-FGFR4 antibody ( 211 A PBS solution of At-antiFGFR4 was administered to animals as described below.

[0034] [Tumor-bearing mice and their treatment] 1 x 10 cells of the mouse gastric cancer cell line YTN16 (Yamamoto et al., Cancer Science. 2018;109:1480-1492) were cultured in a 2000-well plate. 7 The cells were suspended in 500 μl of Hank's balanced salt solution and intraperitoneally injected into immunocompetent 6-week-old female C57BL / 6 mice to form peritoneal dissemination. Yamamoto et al. confirmed that YTN16 expresses FGFR4 highly. In some experiments, 5 x 10 cells were injected intraperitoneally. 6 YTN16 cells were suspended in 100 μl of Hank's balanced salt solution and injected subcutaneously into the back of the same mice at the same time as the intraperitoneal administration to form subcutaneous tumors. Subcutaneous tumors represent primary tumors separate from peritoneal dissemination. Starting one week after injection, subcutaneous tumor size was measured twice weekly.

[0035] In an experiment to examine the combined effect with ICI, 13 days after tumor cell inoculation, 200 μg of anti-mouse PD-1 antibody and 100 μg of anti-mouse CTLA-4 antibody were dissolved in 500 μl of PBS and administered intraperitoneally. 14 days after tumor inoculation, the same as above was administered. 211 At-antiFGFR4 solution ( 211 The radioactivity of At was 0.6 MBq. 211 Instead of At-antiFGFR4 211Experiments were also conducted in which At alone was administered, and in which these antibodies were administered intravenously or intragastrically. 20 and 27 days after tumor implantation, 200 μg of anti-mouse PD-1 antibody and 100 μg of anti-mouse CTLA-4 antibody were similarly administered intraperitoneally. Subsequent subcutaneous tumor size measurements were performed twice weekly.

[0036] [Results] Figure 1 shows the size (average volume, unit: mm ) of the subcutaneous tumor, which was the primary tumor separate from the peritoneal dissemination. 3 In Figure 1, the horizontal axis shows the time course of measurements taken twice a week starting from day 7 after tumor cell inoculation. The first ICIs administration was performed on day 13 after tumor inoculation. 211 At-antiFGFR4 administration was given on day 14. ICIs were also administered on days 20 and 27. n=10 for each treatment group. 211 The group receiving At-antiFGFR4 alone intraperitoneally did not show significant suppression of primary tumor growth rate compared to the control group. 211 The ICIs-treated group (without At-antiFGFR4) did not show significant suppression of primary tumor growth rate compared to the untreated control group. Although the average tumor size of the ICIs-treated group appears to have decreased at the last measurement time point in Figure 1, this is because several individuals in the group with particularly large tumors died before this time point.

[0037] in contrast, 211 At-antiFGFR4 was administered in combination with ICIs. 211 The growth of the primary extraperitoneal tumor was significantly suppressed in the At-antiFGFR4 + ICIs treatment group compared to the control group, indicating that the combined administration of the two therapeutic agents produced synergistic effects that could not be achieved by either of the two therapeutic agents alone.

[0038] The experiment in Figure 1 used two ICIs at a time. Figure 2 shows essentially the same experiment but with only one ICI or its combination. 211The results are shown in the figure when administered in combination with At-antiFGFR4. The first ICI administration was performed on day 13 after tumor inoculation. 211 At-antiFGFR4 administration was performed on day 14. The same ICI was also administered on days 20 and 27. In this experiment, administration of ICI alone did not suppress the growth of the primary tumor compared to the control, but ICI 211 It can be seen that when combined with At-antiFGFR4, tumor growth is significantly suppressed.

[0039] Figure 3 shows 211 By intraperitoneal (ip) administration of At-antiFGFR4 211 Peritoneal accumulation of unconjugated At 211 Quantitative results are shown for At administration compared with intravenous (iv) and intragastric (ig) administration. a shows the percentage of the input dose detected per gram of peritoneal tissue at the indicated time (h) after administration. b shows the radiation dose per gram of peritoneal tissue in counts per minute. 211 Intraperitoneal administration of At-antiFGFR4 was particularly effective 211 It was found that At accumulates in the abdominal cavity, and 211 The decline in the At signal can be largely explained by its half-life.

[0040] Figure 4 shows 211 Photographs of abdominal incision showing the state of peritoneal dissemination on day 10 after At administration. 211 In the mice in the At intraperitoneal (ip) administration group (b), significant peritoneal dissemination was observed as luminous granular lesions. 211 In the mice (c) that received intraperitoneal administration of At-antiFGFR4, most of the peritoneal dissemination disappeared, resulting in a smooth peritoneal surface. 211In mice receiving intravenous administration of At-antiFGFR4 (d), the extent of peritoneal dissemination was slightly reduced compared to the control group. Figure 5 shows hematoxylin and eosin staining (top row), anti-phosphorylated histone H2A.X staining (middle row), and anti-HMGB1 staining (bottom row) of peritoneal dissemination tissue corresponding to 3 days before Figure 4. In all samples, cell nuclei are stained blue with hematoxylin. Phosphorylated histone H2A.X and HMGB1 are markers that indicate DNA damage and immunogenicity, respectively, and are stained with brown dye. In particular, 211 It was shown that significant cancer cell DNA damage and immunogenicity were induced in the At-antiFGFR4 intraperitoneal administration group. 211 This experiment analyzed the effect of internal irradiation with At alone, and mice were not administered ICI. However, when combined with ICI, as shown in Figures 1 and 2, immunogenic induction in peritoneal dissemination was very effectively utilized, resulting in a systematic and visible suppression of peritoneal dissemination and tumors at other sites.

[0041] This application claims the benefit of priority based on Japanese Patent Application No. 2023-182607, filed on October 24, 2023, the entire contents of which are incorporated herein by reference.

Claims

1. A pharmaceutical composition for treating a first tumor in a metastatic cancer patient with peritoneal dissemination and the first tumor at a site separate from the peritoneum, the pharmaceutical composition comprising astatine-211, the astatine-211 being conjugated to a targeting moiety for a tumor-associated antigen expressed on the cell surface of the peritoneal dissemination, the pharmaceutical composition being administered intraperitoneally, and the pharmaceutical composition being administered in combination with an immune checkpoint inhibitor.

2. The targeting moiety comprises an antibody or an antigen-binding fragment thereof against the tumor-associated antigen, or the targeting moiety comprises an antibody or an antigen-binding fragment thereof against the tumor-associated antigen, and 211 2. The pharmaceutical composition of claim 1, which is conjugated to astatine-211 via the group At-C6H4-CONH-.

3. The pharmaceutical composition of claim 1, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-CTLA-4 antibody, an anti-PD-L1 antibody, or an antigen-binding fragment thereof, or a combination of two or more thereof.

4. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is intended to treat the peritoneal dissemination together with the first tumor.

5. A pharmaceutical composition described in any one of claims 1 to 4, wherein the first tumor is in a primary tumor and the peritoneal seeding is a metastatic tumor from the first tumor, or the first tumor and the peritoneal seeding are both metastatic tumors from a primary tumor.

6. The pharmaceutical composition according to claim 5, wherein the primary lesion is gastric cancer.

7. The pharmaceutical composition according to any one of claims 1 to 4, wherein the tumor-associated antigen is selected from the group consisting of CEACAM5, CEACAM6, CLDN4, CLDN7, EpCAM, CA72-4, CA19-9, and growth factor receptors.

8. The pharmaceutical composition of claim 7, wherein the growth factor receptor is selected from the group consisting of EGFR, HER2, EGFR3, EGFR4, c-MET, FGFR1, FGFR2, FGFR3, FGFR4, VEGF receptor, insulin receptor, and PDGF receptor.

9. The pharmaceutical composition of claim 8, wherein the tumor associated antigen is FGFR4 and the targeting moiety is (i) an anti-FGFR4 antibody or an antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence having 95% or more sequence identity to SEQ ID NO:1 and a heavy chain variable region comprising an amino acid sequence having 95% or more sequence identity to SEQ ID NO:2, or (ii) an anti-FGFR4 antibody comprising a light chain comprising an amino acid sequence having 95% or more sequence identity to SEQ ID NO:4 and a heavy chain comprising an amino acid sequence having 95% or more sequence identity to SEQ ID NO:

5.

10. A combination therapy for treating a first tumor in a metastatic cancer patient with peritoneal dissemination and the first tumor at a site separate from the peritoneum, the combination therapy comprising astatine-211 in combination with an immune checkpoint inhibitor selected from the group consisting of anti-PD-1 antibodies, anti-CTLA-4 antibodies, anti-PD-L1 antibodies, and antigen-binding fragments thereof, and combinations of two or more thereof, wherein the astatine-211 is conjugated to a targeting moiety against a tumor-associated antigen expressed on the cell surface of the peritoneal dissemination and is administered intraperitoneally, and the immune checkpoint inhibitor is administered intraperitoneally or intravenously.

11. The targeting moiety comprises an antibody or an antigen-binding fragment thereof against the tumor-associated antigen, or the targeting moiety comprises an antibody or an antigen-binding fragment thereof against the tumor-associated antigen, and 211 The combination therapy of claim 10, which is conjugated to astatine-211 via the group At-C6H4-CONH-.

12. The combination therapy of claim 10, wherein the combination therapy is for use in treating the peritoneal dissemination in addition to the first tumor.

13. The combination therapeutic agent according to any one of claims 10 to 12, wherein the first tumor is a primary tumor and the peritoneal seeding is a metastatic tumor from the first tumor, or the first tumor and the peritoneal seeding are both metastatic tumors from a primary tumor.

14. The combination therapy of claim 13, wherein the primary lesion is gastric cancer.

15. The combination therapy of any one of claims 10 to 12, wherein the tumor-associated antigen is selected from the group consisting of CEACAM5, CEACAM6, CLDN4, CLDN7, EpCAM, CA72-4, CA19-9, and growth factor receptors.

16. The combination therapy of claim 15, wherein the growth factor receptor is selected from the group consisting of EGFR, HER2, EGFR3, EGFR4, c-MET, FGFR1, FGFR2, FGFR3, FGFR4, VEGF receptor, insulin receptor, and PDGF receptor.

17. The combination therapy of claim 16, wherein the tumor associated antigen is FGFR4 and the targeting moiety is (i) an anti-FGFR4 antibody or an antigen-binding fragment thereof, comprising a light chain variable region comprising an amino acid sequence having 95% or more sequence identity to SEQ ID NO:1 and a heavy chain variable region comprising an amino acid sequence having 95% or more sequence identity to SEQ ID NO:2, or (ii) an anti-FGFR4 antibody comprising a light chain comprising an amino acid sequence having 95% or more sequence identity to SEQ ID NO:4 and a heavy chain comprising an amino acid sequence having 95% or more sequence identity to SEQ ID NO:5.

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