Photoimmunotherapy

Abstract

Provided is a novel antibody-light absorber conjugate for PIT. Provided is an antibody-light absorber conjugate for repeated administration in optimal photoimmunotherapy (PIT) for humans.

Description

Photoimmunotherapy

[0001] The present invention relates to photoimmunotherapy (PIT), molecules used in PIT, and their use in the treatment of disease.

[0002] Currently, the number of cancer deaths worldwide is increasing, with the majority of these cases being solid malignant tumors. Standard treatments for solid malignant tumors include surgical resection and radiochemotherapy, but postoperative complications and reduced quality of life (QOL) are problematic. Therefore, the development of effective treatments with better organ preservation, fewer invasiveness and complications than conventional standard treatments is necessary. Photoimmunotherapy (PIT), developed by the National Cancer Institute in the United States, is a novel cancer treatment method that utilizes light irradiation and cancer cell membrane antigen-specific antibodies conjugated with a light absorber (IRDye700DX (IR-700)). This is a groundbreaking treatment that efficiently delivers the light absorber-conjugated antibody to the target cancer cell membrane antigen and selectively treats only the cancer cells to which the light absorber-conjugated antibody is bound by 690 nm near-infrared light (Non-Patent Document 1). Due to its high cell selectivity, photoimmunotherapy is expected to cause minimal damage to normal cells and minimal invasiveness or complications, making it potentially applicable to patients who are refractory to existing standard treatments. PIT using anti-epidermal growth factor receptor (EGFR) mAb has been approved for recurrent head and neck cancer in Japan, and preclinical studies using other mAbs have also reported efficacy against multiple cancers. The only antibody used clinically in photoimmunotherapy is ASP-1929, an antibody drug consisting of a chemical conjugate of the EGFR antibody cetuximab and IR-700. A phase III international collaborative trial using ASP-1929 is currently underway for patients with advanced or recurrent head and neck cancer, and conditional approval was granted in Japan in October 2020.

[0003] Mitsunaga M. et al. Nat Med. ,2011

[0004] The only antibody drug available for photoimmunotherapy is ASP-1929. However, because the expression frequency of its target molecule, EGFR, is low and heterogeneous, ASP-1929 is not expected to be effective against many types of cancer. Additionally, the number of IR-700 conjugates per antibody is heterogeneous, potentially limiting its therapeutic efficacy. EpCAM-IR-700 is an excellent antibody drug for photoimmunotherapy because it expresses its target antigen uniformly and highly. However, EpCAM-IR-700 has been reported to have nonspecific therapeutic effects in vivo (Isoda Y. et al. Oncotarget. 2018), raising questions about its usefulness.

[0005] An object of the present invention is to provide a novel antibody-light absorber conjugate for PIT that solves these problems.

[0006] As a result of intensive research to solve the above problems, the present inventors selected a cancer antigen that shows high and uniform expression regardless of the type of cancer, and discovered an optimal method for conjugating an antibody that specifically recognizes the selected antigen with a light absorber. Furthermore, the present inventors found that the obtained antibody-light absorber conjugate brings about a specific therapeutic effect in vivo that differs from conventional therapeutic methods, and thus completed the present invention.

[0007] That is, the present invention relates to the following, but is not limited thereto. [1] An antibody-light absorber conjugate for repeated administration in optimal photoimmunotherapy (PIT) to humans, wherein the antibody is an antibody against EpCAM or an EpCAM mutant, and the light absorber is a phthalocyanine derivative. [2] The antibody-light absorber conjugate according to [1], wherein the antibody is adecatumumab and the light absorber is IR-700. [3] The antibody-light absorber conjugate according to [1] or [2], wherein the number of IR-700 bound per adecatumumab molecule (DAR) is 2 to 3. [4] The antibody-light absorber conjugate according to any one of [1] to [3], wherein the PIT treatment consists of one administration of the antibody-light absorber conjugate followed by two separate administrations of light irradiation, and the treatment is repeated once or multiple times every three or four weeks. [5] A pharmaceutical composition for treating a tumor expressing EpCAM, comprising the antibody-light absorber conjugate according to any one of [1] to [4] and a pharmaceutically acceptable carrier. [6] The pharmaceutical composition according to [5], wherein the tumor is an epithelial cancer. [7] The pharmaceutical composition according to [6], wherein the epithelial cancer is a cancer selected from the group consisting of colorectal cancer, gastric cancer, esophageal cancer, prostate cancer, breast cancer, lung cancer, gallbladder cancer, pancreatic cancer, and endometrial cancer. [8] A method for producing an antibody-light absorber conjugate for repeated administration in optimal photoimmunotherapy (PIT) to humans, comprising a step of reacting an antibody against EpCAM or an EpCAM mutant with a phthalocyanine derivative light absorber under neutral conditions at 4°C for 24 hours or at 20°C for 24 hours. [9] A method for treating a tumor expressing EpCAM in a subject in need thereof, the method comprising administering to the subject the antibody-light absorber conjugate described in any one of [1] to [4] or the pharmaceutical composition described in any one of [5] to [7].

[0008] The antibody-light absorber conjugate of the present invention provides a novel antibody-light absorber conjugate for PIT.

[0009] Figure 1A shows the IHC staining intensity of EGFR. Figure 1B shows the IHC staining intensity of HER2. Figure 1C shows the IHC staining intensity of EpCAM. Figure 2 is a graph showing the uniformity of EGFR, HER2, and EpCAM. Figures 3A and 3B show representative endoscopic images. Figures 3C(1) and (2) show the expression of HE, EGFR, HER2, and EpCAM. Figure 4 is a flow cytometry image showing binding to OE-19 cells. Figure 5A is a schematic diagram of an antibody-photoabsorber conjugate, an adecatumumab-IR-700 conjugate. Figure 5B is a graph showing the drug / antibody ratio and the amount of IR-700 used for conjugation. Figure 5C is an SDS-PAGE result showing the weights of adecatumumab and the adecatumumab-IR-700 conjugate. Figure 5D is a flow cytometry image showing the binding of adecatumumab to the adecatumumab-IR-700 conjugate. Figure 6 is a time-lapse fluorescence microscopy image of OE-19 cells after administration of adecatumumab-IR-700 compared to the IR-700 control. Figure 7 is an image showing the localization of the adecatumumab-IR-700 conjugate at the OE-19 tumor site. Figure 8 is an image showing the tumor-to-background ratio of different adecatumumab-IR-700 conjugates. Figure 9A is a schematic diagram showing the analysis procedure for the fluorescence intensity of the adecatumumab-IR-700 conjugate. Figure 9B is an image showing the fluorescence intensity of the adecatumumab-IR-700 conjugate. Figure 9C is a graph showing the fluorescence intensity of the adecatumumab-IR-700 conjugate. Figure 9D is a graph showing the fluorescence intensity of the adecatumumab-IR-700 conjugate. Figure 10A is a graph showing the tumor volume over time in the control group and the adecatumumab-IR-700 conjugate group. Figure 10B is a graph showing the change in body weight over time in the control group and the adecatumumab-IR-700 conjugate group. Figure 10C is an image showing the occurrence of edema in the control group and the adecatumumab-IR-700 conjugate group. Figure 10D is a graph showing the edema rate in the control group and the adecatumumab-IR-700 conjugate group. Figure 10E is an image showing histological changes in the control group and the adecatumumab-IR-700 conjugate group.Figure 10F is an image showing histological changes in the control group and the adecatumumab-IR-700 conjugate group. Figures 11A and 11B are images showing HE pathology specimens of Barrett's esophageal cancer. Figure 12 is a graph showing the change in HT29 tumor volume over time in the control group and the adecatumumab-IR-700 conjugate group. Figure 13 is a graph showing the change in HT29 tumor volume over time in the control group and the adecatumumab-IR-700 conjugate group. Figure 14 is a graph showing the change in HT29 tumor volume over time in the control group and the adecatumumab-IR-700 conjugate group.

[0010] <Terminology> The definitions of terms used in this specification are explained below.

[0011] Antibody: An antibody is a polypeptide ligand that specifically recognizes an antigen and contains a heavy chain immunoglobulin variable region and a light chain immunoglobulin variable region. Antibodies of the present invention include monoclonal antibodies (mAbs) that can bind to antigens, as well as antigen-binding fragments of the mAbs, such as Fab and F(ab')2, single-chain antibodies, single-chain variable fragments (scFv), and scFabs. mAbs and antibody portions can be produced using techniques known to those skilled in the art.

[0012] Light absorber: A light absorber is a substance that can specifically absorb light having a specific wavelength and become photoexcited.

[0013] Sequence identity: In this specification, the percent identity of two amino acid sequences can be determined by visual inspection and mathematical calculation. Percent identity can also be determined using a computer program. Examples of such computer programs include BLAST and ClustalW. In particular, the various conditions (parameters) for identity searches using the BLAST program are described in Altschul et al. (Nucl. Acids. Res., 25, pp. 3389-3402, 1997) and are publicly available from the websites of NCBI and the DNA Data Bank of Japan (DDBJ) (BLAST Manual, Altschul et al., NCB / NLM / NIH, Bethesda, MD 20894; Altschul et al.). Furthermore, genetic information processing software GENETYX Ver. It can also be determined using programs such as DNASIS Pro (Hitachi Software), Vector NTI (Infomax), etc. Sequence identity of nucleotide sequences can also be determined in a similar manner.

[0014] Tumor: As used herein, the term "tumor" refers to the progressive proliferation of cells in a living organism. Tumors, particularly those in which abnormal cells spread to the surrounding area or metastasize to other organs or tissues, are called "malignant tumors" or "cancers." Among these, those derived from epithelial cells are called "cancers." However, in this specification, "tumor," "cancer," and "cancer" may be used interchangeably.

[0015] <Antibody-Photoabsorber Conjugate> According to one aspect of the present invention, there is provided an antibody-photoabsorber conjugate for repeated administration in photoimmunotherapy (PIT) to humans.

[0016] In the antibody-light absorber conjugate of the present invention, a preferred antibody is an antibody against EpCAM or an EpCAM variant, and more preferably an antibody against human EpCAM or a human EpCAM variant. EpCAM (epithelial cell adhesion molecule) is a transmembrane glycoprotein of approximately 40 kDa involved in cell adhesion, cell proliferation, and tumor progression, and examples thereof include human EpCAM having the amino acid sequence shown in Genbank Accession No. AAH14785. An EpCAM variant refers to a polypeptide consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence shown in Genbank Accession No. AAH14785, and which has the function of EpCAM. An EpCAM variant is an EpCAM variant having the function of Genbank Accession No. AAH14785. Preferably, the polypeptide has an amino acid sequence that has 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more sequence identity with the amino acid sequence shown in AAH14785 and has the function of EpCAM.

[0017] A more preferred antibody in the antibody-photoabsorber conjugate of the present invention is adecatumumab, a recombinant human monoclonal antibody of the IgG1 subclass that has binding specificity for EpCAM and is commercially available.

[0018] The light absorber in the antibody-light absorber conjugate of the present invention may be any substance that has strong light absorption in the near-infrared region and can change its shape after photoexcitation to cause physicochemical damage to the cell membrane to which the light absorber is bound. Preferably, the light absorber in the antibody-light absorber conjugate of the present invention is a phthalocyanine derivative such as IR-700.

[0019] The adecatumumab-IR-700 conjugate may be referred to herein simply as adecatumumab-IR-700.

[0020] The antibody-photoabsorber conjugate of the present invention preferably has a binding rate (DAR) of IR-700 per antibody molecule of 2 to 3. When the DAR is within this range, the antibody-photoabsorber conjugate of the present invention can be repeatedly administered with reduced side effects in photoimmunotherapy (PIT) to humans, and can bring about a higher therapeutic effect.

[0021] When the antibody-light absorber conjugate of the present invention is repeatedly administered to a human in photoimmunotherapy (PIT), the administration regimen can be appropriately determined taking into consideration the age, sex, weight, severity of disease, etc. of the subject. For example, one administration of the antibody-light absorber conjugate followed by two separate administrations of light irradiation constitutes one treatment, and the treatment can be repeated once or multiple times every three or four weeks.

[0022] The antibody-light absorber conjugate of the present invention can be produced using the production method described below.

[0023] <Pharmaceutical Composition> One aspect of the present invention relates to a pharmaceutical composition for treating a tumor that expresses EpCAM.

[0024] The pharmaceutical composition of the present invention comprises the antibody-light absorber conjugate and a pharmaceutically acceptable carrier. In the present invention, a pharmaceutically acceptable carrier refers to an inert carrier typically used in drug formulations that does not have a negative effect on the antibody-light absorber conjugate of the present invention, and examples include, but are not limited to, water, buffered saline, dextrose, glycerol, ethanol, etc. The pharmaceutical composition of the present invention may further contain additives such as diluents, excipients, emulsifiers, etc., as long as they do not impair the objectives of the present invention.

[0025] The form of the pharmaceutical composition of the present invention is not particularly limited, and can be appropriately selected depending on the purpose from among tablets, powders, granules, capsules, enteric-coated capsules, suppositories, liquids, suspensions, gels, and the like.

[0026] The method of administration of the pharmaceutical composition of the present invention is not particularly limited and can be appropriately determined taking into consideration the dosage form, the age and sex of the patient, the severity of the disease, etc. For example, administration methods such as intravenous administration, oral administration, and tube administration can be suitably used.

[0027] As used herein, "treating a tumor" refers to alleviating, improving, inhibiting the worsening or progression of, or eliminating one or more symptoms caused by the presence of a tumor. The in vivo effect of the antibody-light absorber conjugate of the present invention or a pharmaceutical composition containing the same on an EpCAM-expressing tumor can be evaluated, for example, by administering the antibody-light absorber conjugate of the present invention or a pharmaceutical composition containing the same to a tumor-bearing animal and measuring changes in tumor size, etc.

[0028] Tumors expressing EpCAM can be identified by immunostaining a pathological specimen of the tumor using a known EpCAM antibody, etc. In the present invention, the tumor expressing EpCAM is preferably an epithelial cancer, more preferably a cancer selected from the group consisting of colorectal cancer, gastric cancer, esophageal cancer, prostate cancer, breast cancer, lung cancer, gallbladder cancer, pancreatic cancer, and endometrial cancer.

[0029] <Method for producing antibody-light absorber conjugate> One aspect of the present invention relates to a method for producing the antibody-light absorber conjugate.

[0030] The production method of the present invention includes a step of reacting an antibody with a light absorber under neutral conditions at 4°C for 24 hours or at 20°C for 24 hours, thereby making it possible to stably adjust the DAR of the antibody-light absorber conjugate to a preferred range, for example, 2 to 3. The production method of the present invention preferably includes a step of reacting an antibody against EpCAM or an EpCAM mutant with a light absorber that is a phthalocyanine derivative under neutral conditions at 4°C for 24 hours or at 20°C for 24 hours.

[0031] <Method for treating EpCAM-expressing tumors> One aspect of the present invention relates to a method for treating EpCAM-expressing tumors in a subject in need thereof, the method comprising administering the antibody-light absorber conjugate or the pharmaceutical composition to the subject.

[0032] In the present invention, the subject is preferably a mammal, more preferably a human.

[0033] The method of the present invention for treating a tumor expressing EpCAM may be a method comprising repeatedly administering the antibody-photoabsorber conjugate or pharmaceutical composition of the present invention to a human in photoimmunotherapy (PIT). The administration regimen can be appropriately determined taking into consideration the age, sex, weight, severity of the disease, etc. of the subject. For example, one administration of the antibody-photoabsorber conjugate or pharmaceutical composition followed by two separate administrations of light irradiation constitutes one treatment, and the treatment can be repeated once or multiple times every three or four weeks.

[0034] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the embodiments shown in the examples.

[0035] Example 1

[0036] Methods: On June 23, 2018, PubMed online (https: / / www.ncbi.nlm.nih.gov / pubmed / ) was searched for articles using the terms "EpCAM" or "immunohistochemical staining." All articles reviewed in this study were published after January 2010. We searched for cancer antigens with high and uniform expression levels regardless of cancer type, and selected and compared reliable data. We listed the expression levels of EpCAM in each cancer type and verified that EpCAM is the optimal antigen for photoimmunotherapy. Furthermore, we determined the optimal EpCAM antibody. Furthermore, we performed immunohistochemical staining on pathological specimens from nine surgically resected Barrett's esophageal cancer cases at our institution.

[0037] Results: A literature search was performed on PubMed to find target antigens expressed in many types of cancer by immunohistochemical staining, and the EpCAM antigen was found to be suitable. Specifically, high expression rates of EpCAM antigen were observed in colorectal cancer, gastric cancer, esophageal cancer, prostate cancer, breast cancer, gallbladder cancer, pancreatic cancer, and endometrial cancer (P.T. Went. et al. 2004, C.G. Rao. et al. Int J Oncol. 2005, P.T. Went. et al. (Br J Cancer. 2006, G. Spizzo. et al. J Clin Pathol 2011, I J Goosens-Beumer. et al. BJ Cancer 2014). Furthermore, the antigen was uniformly expressed throughout the tumor (P.T. Went. et al. Hum Pathol. 2004, P.T. Went. et al. Br J Cancer 2014). Cancer. 2006). The results are shown in Table 1.

[0038] Based on these results, we selected adecatumumab, a human antibody against the EpCAM antigen whose safety has been confirmed in clinical trials, as the optimal antibody for photoimmunotherapy. Adecatumumab (AMG401) manufactured by AMGEN was used in the experiment.

[0039] Figures 11A and 11B show pathological specimens of Barrett's esophageal cancer. Immunohistochemical staining for EpCAM, HER2, and EGFR was performed on nine cases of Barrett's esophageal cancer. The results are shown in Figure 1. HER2 and EGFR were expressed only in a portion of the cancer, and expression was also observed in normal mucosa. However, EpCAM was expressed uniformly throughout the tumor at moderate to strong levels in all cases (100%), with no expression in normal mucosa. These results suggest that EpCAM-IR-700 is an optimal antibody for photoimmunotherapy.

[0040] Example 2

[0041] Method: 1 mg of adecatumumab (AMG401, AMGEN), a human monoclonal IgG1 antibody, was mixed with 53 μg of IR-700 (LI-COR Biosciences) under neutral conditions at 4°C or 20°C using a Rotator RT5 (TAITEC). After 2 or 24 hours of reaction, the mixture was purified using Zeba™ Spin Desalting Columns, 7K MWCO, 5 mL (Thermo Scientific) to obtain the adecatumumab-IR-700 conjugate. Absorbance was measured using a NANODROP ONE (Thermo Scientific) to calculate the number of IR-700 molecules bound per adecatumumab antibody.

[0042] Results: The results are shown in Table 2.

[0043] Previous reports have reported that the binding conditions for antibodies and IR-700 are a weak alkaline (pH 8.5) reaction at room temperature for 2 hours. However, the inventors chose a neutral environment, which causes less denaturation of the antibody protein, and succeeded in producing an adecatumumab-IR-700 conjugate. Furthermore, they compared a reaction condition of 4°C for 24 hours and a reaction condition of 20°C for 2 hours in a neutral environment, and established conditions for producing an adecatumumab-IR-700 conjugate with little variation in DAR.

[0044] Example 3

[0045] Method: 6-8 week-old female BALB / c-nu / nu mice (Charles River Laboratories Japan) were inoculated with 2 × 10 HT29 cells, which highly express EpCAM antigen, on the left dorsal side. ７ The tumor diameter was calculated by multiplying the major axis by the minor axis by the minor axis by 0.5, and the tumor diameter was 70-130 mm. ３ Mice that reached the target age were used. Seven mice were used in each group. The mice were administered 100 μg / mouse of the adecatumumab-IR-700 conjugate and the control antibody conjugate, CD20-IR-700 conjugate, via the tail vein. 24 and 48 hours after administration of the conjugates, they were exposed to 690 nm light at an output of 150 mW / cm. ２The treatment effects were compared between the PBS-administered group (A), the CD20-IR-700 conjugate group (B), and the adecatumumab-IR-700 conjugate group (C).

[0046] Results: The results are shown in Figure 12. The PBS group, CD20-IR-700 conjugate group, and adecatumumab-IR-700 conjugate group each had n=7, and the antitumor effect was examined over time for 23 days after administration of mAb-IR-700. There was a significant tumor growth inhibition ( ＊＊ P = 0.002 and ＊ P=0.009; one-way ANOVA (Turkey's multiple comparisons) was observed.

[0047] A previous report (Isoda Y. et al. Oncotarget. 2018) reported that photoimmunotherapy using EpCAM-IR-700 had a therapeutic effect even with a control antibody conjugate, and that the therapeutic effect was nonspecific. However, our results in this study demonstrated that the control antibody CD20-IR-700 conjugate did not have a significant antitumor effect, while the adecatumumab-IR-700 conjugate had a significant therapeutic effect compared to the PBS group and the CD20-IR-700 conjugate group, demonstrating a specific antitumor effect.

[0048] Example 4

[0049] Method: 6-8 week-old female BALB / c-nu / nu mice (Charles River Laboratories Japan) were inoculated with 2 × 10 HT29 cells, which highly express EpCAM antigen, on the left dorsal side. ７ The tumor diameter was calculated by multiplying the major axis by the minor axis by 0.5, and the tumor diameter was 70-130 mm. ３ Mice that reached this age were used. Six mice were used in each group. The adecatumumab-IR-700 conjugate was administered to the tail vein of each mouse at 100 μg / mouse, and 24 and 48 hours after antibody administration, 690 nm light was applied at an output of 150 mW / cm. ２So, a total of 100 J / cm ２ Group (C) was irradiated with 100 μg of adecatumumab-IR-700 conjugate per mouse, and 24 hours after administration of the conjugate, the mice were irradiated with 690 nm light at an output of 150 mW / cm. ２ The therapeutic effects were compared between the group (B) irradiated with a total of 100 J / cm2 and the PBS group (A).

[0050] Results: The results are shown in Figure 13. The PBS group (A), the group irradiated once after 24 hours (B), and the group irradiated once each after 24 and 48 hours (C) showed significant tumor growth inhibition ( ＊＊ P = 0.001 and ＊ A one-way ANOVA (Turkey's multiple comparison) was observed (P<0.001). A significant difference was observed between Groups B and C from Day 34 onward, and the significance was P=0.003 on Day 44. This demonstrated that not only light irradiation 24 hours after administration of the adecatumumab-IR-700 conjugate but also additional light irradiation 48 hours later resulted in a more significant antitumor effect.

[0051] Example 5

[0052] Method: 6-8 week-old female BALB / c-nu / nu mice (Charles River Laboratories Japan) were inoculated with 2 × 10 HT29 cells, which highly express EpCAM antigen, on the left dorsal side. ７ The tumor diameter was calculated by multiplying the major axis by the minor axis by 0.5, and the tumor diameter was 70-130 mm. ３ Group A was the PBS group, and Group B was the group exposed to 690 nm light at an output of 150 mW / cm. ２ So, a total of 100 J / cm ２ Group C was irradiated twice, 24 and 48 hours later, with 100 μg of adecatumumab-IR-700 conjugate administered per animal. Group D was irradiated with 100 μg of adecatumumab-IR-700 conjugate per animal, and 24 and 48 hours after administration of the conjugate, with 690 nm light at an output of 150 mW / cm. ２ So, a total of 100 J / cm ２ Each group was treated every seven days, and the therapeutic effects were compared.

[0053] Results: The results are shown in Figure 14. No significant difference in antitumor effect was observed among the PBS group (A), the light irradiation only group (B), and the adecatumumab-IR-700 conjugate administration only group (C). However, the adecatumumab-IR-700 conjugate administration and light irradiation group (D) showed significant tumor growth inhibition ( ＊ P = 0.007, ＊＊ P<0.001 and ＊＊＊ P<0.001; one-way ANOVA (Turkey's multiple comparisons) was observed.

[0054] In a previous report (Mitsunaga M. et al. Nat Med. 2011), the antitumor effect of photoimmunotherapy was significantly different from that of the control group, but tumor growth was observed in the treatment group. However, the inventors have now revealed a method in which repeating the treatment every week results in almost no tumor growth and a groundbreaking therapeutic effect.

[0055] Example 6

[0056] Materials and Methods: Patients: Patients with EGJ EAC who underwent surgery at the National Cancer Center Hospital East between April 2015 and March 2020 were enrolled in this study. Patients with a history of systemic chemotherapy and / or radiation therapy for EGJ or other cancers were excluded. A total of 46 patients met all study criteria and were included in this study. The majority of enrolled cases were classified as Siewert type II, with 67% of cases having a Barrett's epithelial background. The pathological T stage was T1 in 46% and T2 or higher in 54% (Table 3).

[0057] EGFR, HER2, and EpCAM immunohistochemical staining: For each patient, the section containing the deepest infiltration from the surgical resection specimen was selected as the tumor area, and normal areas of the esophagus and stomach were evaluated using sections adjacent to the tumor.

[0058] The expression of epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and epithelial cell adhesion molecule (EpCAM) was assessed using immunohistochemical (IHC) staining of surgically excised spinous processes. Formalin-fixed, paraffin-embedded tissue samples were immunohistochemically stained using a VENTANA BenchMark ULTRA automated slide stainer (VENTANA, Roche, Basal, Switzerland) according to the manufacturer's instructions. The antibodies used in this study for EGFR, HER2, and EpCAM were EGFR (clone 3C6), VENTANA PATHWAY HER2 (clone 4B5), and EpCAM (clone BER-EP4), respectively.

[0059] Evaluation of EGFR, HER2, and EpCAM Expression and Uniformity: EACs at EGJs and adjacent non-neoplastic esophageal epithelium (Barrett's epithelium and non-Barrett's epithelium) and gastric epithelium were evaluated by IHC staining by two physicians, including an expert gastrointestinal pathologist. The staining intensity of EGFR, HER2, and EpCAM was scored using the following classification system: [0] negative, [1+] weak staining, [2+] moderate staining, and [3+] strong staining (Figure 1). Samples in which tumors with a score of 2+ or 3+ accounted for 10% or more of the evaluated tumors were considered positive in this study.

[0060] Of the tumors judged to be positive, those with a score of 0 or 1+ accounting for 30% or more of the total were classified as heterologous, and the rest were classified as homologous.

[0061] Cells and cell culture: The OE-19 and EGJ adenocarcinoma cell lines used in this study were purchased from the European Collection of Authenticated Cell Cultures (ECACC). These cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Thermo Fisher Scientific, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% penicillin-streptomycin-amphotericin B suspension (Fujifilm Wako Pure Chemical Industries, Osaka, Japan) under 5% CO. ２ The cells were cultured in a 37% CO atmosphere.

[0062] Conjugation of adecatumumab to OE-19: EpCAM expression in OE-19 was analyzed by flow cytometry. ５ Cells were incubated with 1.0 μg of adecatumumab for 1 hour at 4°C. After washing with phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and 2 mM EDTA (BE-PBS), cells were incubated with Alexa Fluor 488-conjugated anti-human IgG (Thermo Fisher Scientific) for 30 minutes at 4°C. As a control, 2 × 10 ５ Cells were incubated with Alexa Fluor 488-conjugated anti-human IgG alone for 30 minutes at 4°C. Stained cells were analyzed using Guava easyCyte 10HT (Merck Millipore, Billerica, MA). Data were analyzed using FlowJo software (Tree Star Inc. Ashland, OR).

[0063] Synthesis of IR-700-conjugated antibody: In this study, adecatumumab (Amgen, CA), a recombinant human IgG1 monoclonal antibody targeting human EpCAM, was used. The photoabsorber used was IRDye 700DX NHS Ester (IR-700; C74H96N12Na4O27S6Si3, molecular weight: 1954.22), purchased from LI-COR Bioscience (Lincoln, NE). All chemicals used in this study were reagent grade. IR-700 was conjugated to the antibody using the IRDye® 700DX Protein Labeling Kit (LI-COR Bioscience) according to previously established methods. Briefly, 1 mg of adecatumumab was incubated with 53.0 μg of IR-700. This mixture was purified using a Zeba™ Desalting Spin Column, 7K MWCO (Thermo Fisher Scientific) to obtain adecatumumab-IR-700. The number of fluorescent molecules bound to each antibody molecule was determined as follows: Protein and IR-700 concentrations were measured by measuring absorbance at 280 nm and 689 nm using a spectrophotometer (NanoDrop One; Thermo Fisher Scientific). On average, two molecules of IR-700 bound to adecatumumab (dye-antibody ratio; DAR2). Furthermore, the DAR was measured when the amount of IR-700 was increased to 2 and 5 times the recommended amount.

[0064] Binding Ability of Adecatumumab-IR-700 (DAR 2,4,7): Adecatumumab and adecatumumab-IR-700 (DAR 2,4,7) were analyzed by SDS-PAGE, and binding ability was assessed using flow cytometry as described in "The conjugation of adecatumumab to OE-19."

[0065] In vitro fluorescence microscopy imaging and PIT procedure: Fluorescence microscopy observations were performed using a BZ-X700 fluorescence microscope (Keyence, Osaka, Japan). OE-19 cells were seeded in a polymer-based (Thermo Fisher Scientific) 96-well optical-bottom plate and cultured at 37°C for 24 hours. Adecatumumab-IR-700 (2.5 μg) or control IgG-IR-700 (2.5 μg) was added to the culture medium and incubated at 37°C for 10 minutes. The cells were then washed with PBS and observed under a fluorescence microscope. Next, the sample was irradiated with a 690 nm continuous wave laser (MLL-III-690; CNI Optoelectronics Technology, Changchun, China) at 100 mW / cm using an optical power meter (PM100, Thorlabs, Newton, NJ, USA). ２ The power density of the laser was measured at 100 J / cm. ２ The cells were observed under a microscope before and after irradiation (immediately after irradiation and 3 hours later).

[0066] Animal model: Five-week-old female BALB / c nu / nu mice (Charles River Japan, Yokohama, Japan) were purchased. Mice were anesthetized with isoflurane and 4 × 10 mice suspended in 100 μL of PBS were injected. ６ OE-19 cells were inoculated into the left dorsum. Tumor volume was calculated using the following formula: TV = (L × W ２ ) / 2 (L and W are the length and width of the subcutaneous tumor, respectively). Tumor volume and body weight were measured every 2 days, and when the tumor size reached 1000 mm ３ Mice that reached 100 mg / kg or lost more than 20% of their body weight were humanely euthanized. Animal experiments were conducted with the approval of the National Cancer Center Animal Experiment Committee and in accordance with the "Guidelines for the Care and Use of Laboratory Animals" established by the committee. These guidelines meet the ethical standards set forth by law and conform to the Japanese Guidelines for the Use of Laboratory Animals.

[0067] In vivo fluorescence imaging: To determine the biodistribution of adecatumumab-IR-700 (DAR 2, 4, 7), fluorescence images were obtained using an IVIS® Imaging System (Caliper Life Science) with an excitation filter of 660 nm and an emission filter of 710 nm. In this study, OE-19 xenograft tumors were grown to approximately 300 mm ３ After injection of 150 μg of adecatumumab-IR-700 (DAR 2, 4, 7), fluorescent images were obtained daily up to day 6. All fluorescent images were analyzed using Living Image® Software.

[0068] In vivo real-time fluorescence imaging during PIT: To perform fluorescence imaging during PIT, a LIGHT VISION (Shimadzu Corporation, focused wavelength 810-840 nm, excitation light was a 690 nm laser (150 mW / cm) from a laser irradiation device (MLL-III-690) used in PIT therapy. A front diffuser for surface irradiation of the tumor was placed approximately 30 cm above the tumor. The LIGHT VISION camera head was placed approximately 50 cm from the observation target. ３ Mice bearing OE-19 tumors were injected with 100 μg of adecatumumab-IR-700 (DAR 2, 4, 7) and then treated with PIT 24 hours later. Fluorescence images and tumor fluorescence intensity were obtained during treatment. Fluorescence imaging was performed continuously under the same conditions, including exposure time, camera sensitivity, and binning. The obtained fluorescence images were analyzed using Fiji software. All pixel values ​​were normalized to 100% maximum intensity.

[0069] In vivo PIT with adecatumumab-IR-700 (DAR 2, 4, 7): In vivo experiments were performed using mice implanted with OE-19 cells. Mice with OE-19 xenograft tumors and tumor volumes of approximately 100 mm ３Mice were selected and randomly divided into four groups (n = 8 per group). Each of the four groups received an intravenous injection of 100 μL of PBS and 100 μg of adecatumumab-IR-700 (DAR 2, 4, 7). At 24 and 48 hours after injection, the mice were subjected to laser irradiation at 100 J / cm. Laser irradiation was performed under isoflurane anesthesia using a 690 nm continuous wave laser at a power density of 150 mW / cm. After PIT treatment, tumor volumes reached 1000 mm. ３ Tumor volume and body weight were measured every two days until the tumor volume reached 100 mg / kg.

[0070] Histological analysis: Histological changes were evaluated 24 hours after PIT. Mice bearing OE-19 tumors were euthanized 24 hours after irradiation. Tumors were excised, fixed in 10% formalin, and embedded in paraffin. Serial sections (4 μm) were mounted on glass slides and stained with hematoxylin and eosin. Pathological images were analyzed using a NanoZoomer 2.0HT (Hamamatsu Photonics) and image display software NDP.view2 (Hamamatsu Photonics).

[0071] result:

[0072] Expression of EGFR, HER2, and EpCAM: The EGFR, HER2, and EpCAM expression status was evaluated using immunohistochemical staining for a portion of the resected specimens from each case. The EGFR, HER2, and EpCAM positivity rates in the tumor area were 22%, 13%, and 98%, respectively (Table 4).

[0073] HER2 expression was negative in the diffuse type, but EGFR and EpCAM expression was observed at similar rates in each tissue type.

[0074] Normal esophageal mucosa adjacent to the tumor was divided into Barrett's epithelium and non-Barrett's epithelium, and the expression of EGFR, HER2, and EpCAM was evaluated in each. Barrett's epithelium was observed in 31 of 46 cases (67%), and the EGFR positivity rate in Barrett's epithelium was 16%, the HER2 positivity rate was 32%, and the EpCAM positivity rate was 97%. Meanwhile, no expression of any of these items was observed in non-Barrett's esophageal squamous epithelium. When normal gastric mucosa adjacent to the tumor was similarly evaluated, EGFR and HER2 expression was 0%, and EpCAM expression was 17% (Table 5).

[0075] The homogeneity was 30% for EGFR, 0% for HER, and 93% for EpCAM, respectively (Figure 2).

[0076] Endoscopic and histological images of two representative cases are shown in Figure 3. The tumor area ratios in Figure 3C (1) evaluated using scores 0, 1, 2, and 3 were "100%, 0%, 0%, 0%, 0%, " "100%, 0%, 0%, " "0%, 0%, 0%, 0%, 100%" for EGFR, HER2, and EpCAM, respectively. Therefore, their expression was negative, negative, and positive, respectively. The uniformity of EpCAM with positive expression was determined to be positive. The Barrett's epithelium ratios evaluated using scores 1, 2, and 3 were "100%, 0%, 0%, 0%, 0%, " "100%, 0%, 0%, 0%, 0%, " "0%, 50%, 50%, 0%, 0%" for EGFR, HER2, and EpCAM, respectively. Therefore, their expression was negative, negative, and positive, respectively. Similarly, the tumors in Figure 3C(2) were positive, negative, and positive, respectively. Homogeneity was determined as EGFR negative and EpCAM positive. The Barrett's epithelium was EGFR negative, HER2 negative, and EpCAM positive, respectively.

[0077] The experiment was carried out in vivo / vitro, and EpCAM, which has a high expression rate and uniformity, was used.

[0078] Conjugation of adecatumumab to OE-19 cells: The binding of adecatumumab to OE-19 cells was examined by flow cytometry and found to exhibit high binding affinity, whereas the secondary antibody alone did not bind to OE-19 cells (Figure 4).

[0079] Synthesis of IR-700-conjugated adecatumumab: The schematic structure of adecatumumab-IR-700 (DAR2), in which adecatumumab was conjugated to IR-700, is shown in Figure 5A. When IR-700 was used at twice the recommended amount, 4-5 molecules of IR-700 bound to adecatumumab (DAR4), and when IR-700 was used at five times the recommended amount, 6-7 molecules of IR-700 bound (DAR7) (Figure 5B).

[0080] Binding Ability of Adecatumumab-IR-700 (DAR 2, 4, 7): Adecatumumab and adecatumumab-IR-700 (DAR 2, 4, 7) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of adecatumumab-IR-700 (DAR 2, 4, 7) and unconjugated adecatumumab were similar, approximately 150 kDa (Figure 5C).

[0081] The binding ability of adecatumumab IR-700 (DAR 4 or 7) to the EpCAM antigen on OE-19 cells was reduced compared to adecatumumab or adecatumumab IR-700 (DAR 2) (Figure 5D).

[0082] In vitro fluorescence microscopy and PIT: To examine the effects of PIT, time-lapse fluorescence microscopy imaging was performed. OE-19 cells formed blister cells 3 hours after laser irradiation with adecatumumab-IR-700. However, no morphological changes were observed when IgG-IR-700 was used as a control (Figure 6).

[0083] Biodistribution of adecatumumab-IR-700 (DAR 2, 4, 7): We found that adecatumumab-IR-700 (DAR 2, 4, 7) localized to OE-19 tumors after intravenous injection ( Figure 7 ). Serial imaging analysis showed that the IR-700 signal reached a maximum one day after injection of adecatumumab-IR-700 (DAR 2, 4, 7), followed by a gradual decrease in signal. The tumor-to-background ratios during the measurement periods from day 1 to day 6 were approximately 1.8 for DAR 2, approximately 1.6 for DAR 4, and approximately 1.2 for DAR 7, respectively ( Figure 8 ).

[0084] In vivo real-time fluorescence imaging during PIT: Observation of the fluorescence image during the laser irradiation period showed that the fluorescence intensity of the tumor was significantly higher than the normal fluorescence intensity at the start of laser irradiation (0 J), and decreased to the same level as the normal fluorescence intensity at the end of laser irradiation (100 J) (Figure 9).

[0085] In vivo PIT with adecatumumab-IR-700 (DAR 2, 4, 7): One animal in the DAR 4 group died during tumor measurement, and one animal in the DAR 7 group was unable to walk or eat due to paw edema, resulting in death the day after PIT. Therefore, n = 7 was used for evaluation in the DAR 4 and DAR 7 groups. The PIT-treated group had smaller tumor volumes and a longer prognosis than the control group (PBS). However, no significant differences were observed between DAR 2, 4, and 7 (Figure 10A). There was also no significant difference in body weight change between the groups (Figure 10B). As a side effect, edema was observed at DAR 4 (25%, 2 / 8) and DAR 7 (100%, 8 / 8) (Figures 10C and 10D).

[0086] Histological analysis of PIT: Histological changes were evaluated 24 hours after PIT with PBS and adecatumumab-IR-700 (DAR 2, 4, 7) (Fig. 10E, F).

[0087] Discussion: When the expression and uniformity of EGFR, HER2, and EpCAM in esophagogastric junction (EGJ) adenocarcinoma were evaluated, EpCAM showed the highest values ​​for both expression and uniformity. Therefore, the usefulness of PIT for EGJ adenocarcinoma was investigated in vitro / vivo using the EpCAM antigen, and the results suggested that EpCAM antigen is useful for PIT for EGJ adenocarcinoma.

Claims

1. An antibody-photoabsorber conjugate for repeated administration in photoimmunotherapy (PIT) to humans, wherein the antibody is an antibody against EpCAM or an EpCAM variant, and the photoabsorber is a phthalocyanine derivative.

2. The antibody-photoabsorber conjugate of claim 1, wherein the antibody is adecatumumab and the photoabsorber is IR-700.

3. The antibody-photoabsorber conjugate according to claim 1 or 2, wherein the number of IR-700 bound per molecule of adecatumumab (DAR) is 2 to 3.

4. The antibody-light absorber conjugate according to any one of claims 1 to 3, wherein the PIT comprises one administration of the antibody-light absorber conjugate followed by two separate administrations of light irradiation as one treatment, and the treatment is repeated once or multiple times every three or four weeks.

5. A pharmaceutical composition for treating a tumor expressing EpCAM, comprising an antibody-photoabsorber conjugate described in any one of claims 1 to 4 and a pharma- ceutically acceptable carrier.

6. The pharmaceutical composition of claim 5, wherein the tumor is an epithelial cancer.

7. The pharmaceutical composition of claim 6, wherein the epithelial cancer is a cancer selected from the group consisting of colorectal cancer, gastric cancer, esophageal cancer, prostate cancer, breast cancer, lung cancer, gallbladder cancer, pancreatic cancer and endometrial cancer.

8. A method for producing an antibody-photoabsorber conjugate for repeated administration in photoimmunotherapy (PIT) to humans, comprising a step of reacting an antibody against EpCAM or an EpCAM mutant with a photoabsorber which is a phthalocyanine derivative under neutral conditions at 4°C for 24 hours or at 20°C for 24 hours.

9. A method for treating a tumor expressing EpCAM in a subject in need thereof, comprising administering to the subject an antibody-photoabsorber conjugate described in any one of claims 1 to 4 or a pharmaceutical composition described in any one of claims 5 to 7.

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