Anti-tissue factor antibody conjugated with monomethyl auristatin E or deruxtecan in pancreatic cancer models.

Antibody-drug conjugates (ADCs) have been recognized as a promising class of cancer therapeutics. Tissue factor (TF), an initiator of the blood coagulation pathway, has been investigated regarding its relationship with cancer, and several preclinical and clinical studies have presented data on anti-TF ADCs, including tisotumab vedotin, which was approved in 2021. However, the feasibility of other payloads in the design of anti-TF ADCs is still unclear because no reports have compared payloads with different cytotoxic mechanisms. For ADCs targeting other antigens, such as Her2, optimizing the payload is also an important issue in order to improve in vivo efficacy. In this study, we prepared humanized anti-TF Ab (clone.1084) conjugated with monomethyl auristatin E (MMAE) or deruxtecan (DXd), and evaluated the efficacy in several cell line- and patient-derived xenograft models of pancreatic cancer. As a result, optimizing the drug / Ab ratio was necessary for each payload in order to prevent pharmacokinetic deterioration and maximize delivery efficiency. In addition, MMAE-conjugated anti-TF ADC showed higher antitumor effects in tumors with strong and homogeneous TF expression, while DXd-conjugated anti-TF ADC was more effective in tumors with weak and heterogeneous TF expression. Analysis of a pancreatic cancer tissue array showed weak and heterogeneous TF expression in most TF-positive specimens, indicating that the response rate to pancreatic cancer might be higher for DXd- than MMAE-conjugated anti-TF ADC. Nevertheless, our findings indicated that optimizing the ADC payloads individually in each patient could maximize the potential of ADC therapeutics.

Tumor Targeting of 211At-Labeled Antibody under Sodium Ascorbate Protection against Radiolysis.

Astatine-211 (211At) is an alpha emitter applicable to radioimmunotherapy (RIT), a cancer treatment that utilizes radioactive antibodies to target tumors. In the preparation of 211At-labeled monoclonal antibodies (211At-mAbs), the possibility of radionuclide-induced antibody denaturation (radiolysis) is of concern. Our previous study showed that this 211At-induced radiochemical reaction disrupts the cellular binding activity of an astatinated mAb, resulting in attenuation of in vivo antitumor effects, whereas sodium ascorbate (SA), a free radical scavenger, prevents antibody denaturation, contributing to the maintenance of binding and antitumor activity. However, the influence of antibody denaturation on the pharmacokinetics of 211At-mAbs relating to tumor accumulation, blood circulation time, and distribution to normal organs remains unclear. In this study, we use a radioactive anti-human epidermal growth factor receptor 2 (anti-HER2) mAb to demonstrate that an 211At-induced radiochemical reaction disrupts active targeting via an antigen-antibody interaction, whereas SA helps to maintain targeting. In contrast, there was no difference in blood circulation time as well as distribution to normal organs between the stabilized and denatured immunoconjugates, indicating that antibody denaturation may not affect tumor accumulation via passive targeting based on the enhanced permeability and retention effect. In a high-HER2-expressing xenograft model treated with 1 MBq of 211At-anti-HER2 mAbs, SA-dependent maintenance of active targeting contributed to a significantly better response. In treatment with 0.5 or 0.2 MBq, the stabilized radioactive mAb significantly reduced tumor growth compared to the denatured immunoconjugate. Additionally, through a comparison between a stabilized 211At-anti-HER2 mAb and radioactive nontargeted control mAb, we demonstrate that active targeting significantly enhances tumor accumulation of radioactivity and in vivo antitumor effect. In RIT with 211At, active targeting contributes to efficient tumor accumulation of radioactivity, resulting in a potent antitumor effect. SA-dependent protection that successfully maintains tumor targeting will facilitate the clinical application of alpha-RIT.

Antibody therapeutics and immunoregulation in cancer and autoimmune disease.

Cancer and autoimmune disease are closely related, and many therapeutic antibodies are widely used in clinics for the treatment of both diseases. Among them, the anti-CD20 antibody has proven to be effective against both lymphoid malignancy and autoimmune disease. Moreover, immune checkpoint blockade using the anti-PD1/PD-L1/CTLA4 antibody has improved the prognosis of patients with refractory solid tumors. At the same time, however, over-enhancement of immunoreaction can induce autoimmune reaction. Although anti-TNF antibody therapies represent a breakthrough in the treatment of autoimmune diseases, optimal management is required to control the serious associated issues, including development and progression of cancer, and it is becoming more and more important to control the immunoreaction. In addition, next-generation antibody therapeutics such as antibody-drug conjugates and bispecific antibodies, are anticipated to treat uncontrolled cancer and autoimmune disease. IL-7R signaling plays an important role in the development and progression of both lymphoid malignancy and autoimmune disease. In addition, abnormal homing activity and steroid resistance caused by IL-7R signaling may worsen prognosis. Therefore, anti-IL-7R targeting antibody therapies that enable suppression of such pathophysiological status have the potential to be beneficial for the treatment of both diseases. In this review, we discuss current antibody therapeutics in cancer and autoimmune disease, and describe a new therapeutic strategy for immunoregulation including IL-7R targeting antibodies.

Radioimmunotherapy with an 211 At-labeled anti-tissue factor antibody protected by sodium ascorbate.

Tissue factor (TF), the trigger protein of the extrinsic blood coagulation cascade, is abundantly expressed in various cancers including gastric cancer. Anti-TF monoclonal antibodies (mAbs) capable of targeting cancers have been successfully applied to armed antibodies such as antibody-drug conjugates (ADCs) and molecular imaging probes. We prepared an anti-TF mAb, clone 1084, labeled with astatine-211 (211 At), as a promising alpha emitter for cancer treatment. Alpha particles are characterized by high linear energy transfer and a range of 50-100 µm in tissue. Therefore, selective and efficient tumor accumulation of alpha emitters results in potent antitumor activities against cancer cells with minor effects on normal cells adjacent to the tumor. Although the 211 At-conjugated clone 1084 (211 At-anti-TF mAb) was disrupted by an 211 At-induced radiochemical reaction, we demonstrated that astatinated anti-TF mAbs eluted in 0.6% or 1.2% sodium ascorbate (SA) solution were protected from antibody denaturation, which contributed to the maintenance of cellular binding activities and cytocidal effects of this immunoconjugate. Although body weight loss was observed in mice administered a 1.2% SA solution, the loss was transient and the radioprotectant seemed to be tolerable in vivo. In a high TF-expressing gastric cancer xenograft model, 211 At-anti-TF mAb in 1.2% SA exerted a significantly greater antitumor effect than nonprotected 211 At-anti-TF mAb. Moreover, the antitumor activities of the protected immunoconjugate in gastric cancer xenograft models were dependent on the level of TF in cancer cells. These findings suggest the clinical availability of the radioprotectant and applicability of clone 1084 to 211 At-radioimmunotherapy.

Mechanism of action of a T cell-dependent bispecific antibody as a breakthrough immunotherapy against refractory colorectal cancer with an oncogenic mutation.

T cell-dependent bispecific antibody (TDB)-induced T cell activation, which can eliminate tumor cells independent of MHC engagement, is expected to be a novel breakthrough immunotherapy against refractory cancer. However, the mechanism of action of TDBs has not been fully elucidated thus far. We focused on TDB-induced T cell-tumor cell contact as an important initial step in direct T cell-mediated tumor cell killing via transport of cytotoxic cell proteases (e.g., granzymes) with or without immunological synapse formation. Using an anti-EGFR/CD3 TDB, hEx3, we visualized and quantified T cell-tumor cell contact and demonstrated a correlation between the degree of cell contact and TDB efficacy. We also found that cytokines, including interferon-gamma (IFNγ) and tumor necrosis factor-alpha (TNFα) secreted by activated T cells, damaged tumor cells in a cell contact-independent manner. Moreover, therapeutic experiences clearly indicated that hEx3, unlike conventional anti-EGFR antibodies, was effective against colorectal cancer (CRC) cells with mutant KRAS, BRAF, or PIK3CA. In a pharmacokinetic analysis, T cells spread gradually in accordance with the hEx3 distribution within tumor tissue. Accordingly, we propose that TDBs should have four action steps: 1st, passive targeting via size-dependent tumor accumulation; 2nd, active targeting via specific binding to tumor cells; 3rd, T cell redirection toward tumor cells; and 4th, TDB-induced cell contact-dependent (direct) or -independent (indirect) tumor cell killing. Finally, our TDB hEx3 may be a promising reagent against refractory CRC with an oncogenic mutation associated with a poor prognosis.

High expression of TMEM180, a novel tumour marker, is associated with poor survival in stage III colorectal cancer.

BACKGROUND: Transmembrane protein 180 (TMEM180) is a newly identified colorectal cancer (CRC)-specific molecule that is expressed very rarely in normal tissue and up-regulated under hypoxic conditions. We developed a monoclonal antibody (mAb) against TMEM180 and decided to examine the medical significance using the mAb. METHODS: A total of 157 patients (86 men and 71 women; median age 63.0 years) with stage III CRC who underwent curative surgery were analyzed for TMEM180 expression as a retrospective cohort design. Immunohistochemistry with anti-TMEM180 mAb was conducted on frozen sections, and the data were evaluated for any correlation with clinicopathological indices or prognosis. SW480 CRC cells were examined to investigate the relationship between the expression of TMEM180 and tumourigenesis of xenografts. RESULTS: In total, 92 cases had low TMEM expression and 65 had high TMEM180 expression. For disease-free survival, hazard ratio in high-TMEM180 cases was 1.449 (95% confidential interval = 0.802-2.619) higher than in low-TMEM180 cases, but the difference was not significant (p = 0.219). For cancer specific survival, hazard ratio in high-TMEM180 cases was 3.302 (95% confidential interval = 1.088-10.020), significantly higher than in low-TMEM180 cases (p = 0.035). In an assay examining in vitro colony-forming activity in soft agar, SW480-WT cells clearly formed colonies, but neither KD1 nor KD2 cells did. The in vivo tumour-initiating activity of SW480 cell lines was positively correlated with the level of TMEM180 expression. CONCLUSION: These results indicate that TMEM180 is a useful marker for clinical prognosis in patients with CRC. We believe that these fundamental data warrant further basic and translational studies of TMEM180, and its mAb, for development of therapeutics against CRC.

A Fluorescent Probe for Rapid, High-Contrast Visualization of Folate-Receptor-Expressing Tumors In†Vivo.

Folate receptors (FRs) are membrane proteins involved in folic acid uptake, and the alpha isoform (FR-α) is overexpressed in ovarian and endometrial cancer cells. For fluorescence imaging of FRs in vivo, the near-infrared (NIR) region (650-900 nm), in which tissue penetration is high and autofluorescence is low, is optimal, but existing NIR fluorescent probes targeting FR-α show high non-specific tissue adsorption, and require prolonged washout to visualize tumors. We have designed and synthesized a new NIR fluorescent probe, FolateSiR-1, utilizing a Si-rhodamine fluorophore having a carboxy group at the benzene moiety, coupled to a folate ligand moiety through a negatively charged tripeptide linker. This probe exhibits very low background fluorescence and afforded a tumor-to-background ratio (TBR) of up to 83 in FR-expressing tumor-bearing mice within 30 min. Thus, FolateSiR-1 has the potential to contribute to the research in the field of biology and the clinical medicine.

Significant antitumor effect of an antibody against TMEM180, a new colorectal cancer-specific molecule.

The present state of therapy for colorectal cancer (CRC) is far from satisfactory, highlighting the need for new targets for this disease. We identified a new CRC-specific molecule, TMEM180, a predicted 11-pass transmembrane protein that apparently functions as a cation symporter. We developed an anti-TMEM180 mAb and then succeeded in humanizing the mAb. Immunohistochemistry (IHC) in CRC with the mAb showed a similar positivity rate as compared with anti-epidermal growth factor receptor mAb, and IHC with anti-TMEM180 mAb did not show staining in major organs used in this study. Immune electron microscopy clearly indicated that TMEM180 was present on the tumor exosome. The TMEM180 promoter region contains 10 hypoxia-responsive element consensus sequences; accordingly, SW480 cells upregulated TMEM180 under low-oxygen conditions. Anti-TMEM180 mAb has in vitro antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity activity, and SW480 CRC xenografts were eradicated by the mAb. These data indicate that TMEM180 may be a new CRC marker and that a mAb against this protein could be used as antibody-based therapy against CRC.

Evaluation of the antitumor mechanism of antibody-drug conjugates against tissue factor in stroma-rich allograft models.

Tissue factor (TF) is known to be overexpressed in various cancers including pancreatic cancer. The upregulation of TF expression has been observed not only in tumor cells, but also in tumor stromal cells. Because of the potential of TF as a delivery target, several studies investigated the effectiveness of Ab-drug conjugates (ADCs) against TF for cancer therapy. However, it is still unclear whether anti-TF ADC can exert toxicity against both tumor cells and tumor stromal cells. Here, we prepared ADC using a rat anti-mouse TF mAb (clone.1157) and 2 types of in vivo murine pancreatic cancer models, one s.c. and other orthotopic with an abundant tumor stroma. We also compared the feasibility of bis-alkylating conjugation (bisAlk) with that of conventional maleimide-based conjugation (MC). In the s.c. models, anti-TF ADC showed greater antitumor effects than control ADC. The results also indicated that the bisAlk linker might be more suitable than the MC linker for cancer treatments. In the orthotopic model, anti-TF ADC showed greater in vivo efficacy and more extended survival time control ADC. Treatment with anti-TF ADC (20 mg/kg, three times a week) did not affect mouse body weight changes in any in vivo experiment. Furthermore, immunofluorescence staining indicated that anti-TF ADC delivered agents not only to TF-positive tumor cells, but also to TF-positive tumor vascular endothelial cells and other tumor stromal cells. We conclude that anti-TF ADC should be a selective and potent drug for pancreatic cancer therapy.

Characterization of Antibody Products Obtained through Enzymatic and Nonenzymatic Glycosylation Reactions with a Glycan Oxazoline and Preparation of a Homogeneous Antibody-Drug Conjugate via Fc N-Glycan.

Glycan engineering of antibodies has received considerable attention. Although various endo-ß- N-acetylglucosaminidase mutants have been developed for glycan remodeling, a side reaction has been reported between glycan oxazoline and amino groups. In this study, we performed a detailed characterization for antibody products obtained through enzymatic and nonenzymatic reactions with the aim of maximizing the efficiency of the glycosylation reaction with fewer side products. The reactions were monitored by an ultraperformance liquid chromatography system using an amide-based wide-pore column. The products were characterized by liquid chromatography coupled with tandem mass spectrometry. The side reactions were suppressed by adding glycan oxazoline in a stepwise manner under slightly acidic conditions. Through a combination of an azide-carrying glycan transfer reaction under optimized conditions and a bio-orthogonal reaction, a potent cytotoxic agent monomethyl auristatin E was site-specifically conjugated at N-glycosylated Asn297 with a drug-to-antibody ratio of 4. The prepared antibody-drug conjugate exhibited cytotoxicity against HER2-expressing cells.

Collagen type I induces EGFR-TKI resistance in EGFR-mutated cancer cells by mTOR activation through Akt-independent pathway.

Primary resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a serious problem in lung adenocarcinoma patients harboring EGFR mutations. The aim of this study was to examine whether and how collagen type I (Col I), the most abundantly deposited matrix in tumor stroma, affects EGFR-TKI sensitivity in EGFR-mutant cells. We evaluated the EGFR-TKI sensitivity of EGFR-mutated cancer cells cultured with Col I. Changes in the activation of downstream signaling molecules of EGFR were analyzed. We also examined the association between the Col I expression in tumor stroma in surgical specimens and EGFR-TKI response of postoperative recurrence patients with EGFR mutations. Compared to cancer cells without Col I, the survival rate of cancer cells cultured with Col I was significantly higher after EGFR-TKI treatment. In cancer cells cultured with and without Col I, EGFR-TKI suppressed the levels of phosphorylated (p-)EGFR, p-ERK1/2, and p-Akt. When compared to cancer cells without Col I, expression of p-P70S6K, a hallmark of mTOR activation, was dramatically upregulated in cancer cells with Col I. This activation was maintained even after EGFR-TKI treatment. Simultaneous treatment with EGFR-TKI and mTOR inhibitor abrogated Col I-induced resistance to EGFR-TKI. Patients with Col I-rich stroma had a significantly shorter progression-free survival time after EGFR-TKI therapy (238 days vs 404 days; P < .05). Collagen type I induces mTOR activation through an Akt-independent pathway, which results in EGFR-TKI resistance. Combination therapy using EGFR-TKI and mTOR inhibitor could be a possible strategy to combat this resistance.

Utility of epirubicin-incorporating micelles tagged with anti-tissue factor antibody clone with no anticoagulant effect.

Tissue factor (TF), an initiator of the extrinsic blood coagulation cascade, is overexpressed in different types of cancer. Tissue factor overexpression is also known as a poor prognostic factor in pancreatic cancer. We recently developed anti-TF antibody (clone1849)-conjugated epirubicin-incorporating micelles (NC-6300), and reported that this anti-TF1849-NC-6300 showed enhanced antitumor activity against TF-high expressed human pancreatic cancer cells, when compared with NC-6300 alone. However, clone 1849 antibody inhibited TF-associated blood coagulation activity. We studied another anti-TF antibody, clone 1859, which had no effect on blood coagulation and prepared anti-TF1859-NC-6300. In addition, to determine the optimum size of the antibody fragment to conjugate with NC-6300, three forms of the 1859 antibody (whole IgG, F[ab']2 , and Fab') were conjugated to NC-6300. The antitumor effect of each anti-TF1859-NC-6300 was studied in vitro and in vivo, using two human pancreatic cancer cell lines, BxPC3 with high-expressed TF, and SUIT2 with low levels of TF. In vitro, all forms of anti-TF1859-NC-6300 showed higher cytocidal effects than NC-6300 in BxPC3, whereas this enhanced effect was not observed in SUIT2. Likewise, all forms of anti-TF1859-NC-6300 significantly suppressed tumor growth when compared to NC-6300 in the BxPC3, but not in the SUIT2, xenograft model. Among the three forms of conjugates, anti-TF1859-IgG-NC-6300 had a higher antitumor tendency in TF-high expressed cells. Thus, we have confirmed an enhanced antitumor effect of anti-TF1859-NC-6300 in a TF-high expressing tumor; anti-TF1859-IgG-NC-6300 could be used to simplify the manufacturing process of the antibody-micelle conjugation for future clinical studies.

Antitumor effect of antitissue factor antibody-MMAE conjugate in human pancreatic tumor xenografts.

Tissue factor (TF) triggers the extrinsic blood coagulation cascade and is highly expressed in various types of cancer. In this study, we investigated the antitumor effect of an antibody-drug conjugate (ADC) consisting of an anti-TF monoclonal antibody and monomethyl auristatin E (MMAE). MMAE was conjugated to an anti-human TF or anti-mouse TF antibody using a valine-citrulline linker that could be potentially hydrolyzed by cathepsin B in the acidic environment of the lysosome. The cytotoxic and antitumor effects of the ADCs against four pancreatic cancer cell lines were analyzed. Both the ADC with the anti-human TF antibody and that with the anti-mouse TF antibody were stable under physiological conditions. The anti-human ADC was internalized in TF-expressing human tumor cell lines, followed by effective MMAE release. The half maximal inhibitory concentration (IC50 ) of MMAE was approximately 1 nM for all of the cell lines used. Meanwhile, the IC50 of anti-human ADC was 1.15 nM in the cell lines showing high TF expression, while exceeding 100 nM in the cells showing low TF expression levels. Anti-human ADC with passive and active targeting ability exerted significant suppression of tumor growth as compared to that observed in the saline group (p < 0.01). Also significant tumor growth suppressions were seen at the anti-mouse ADC and control ADC groups compared to the saline group (p < 0.01) due to EPR effect. Because various clinical human cancers express highly amount of TF, this new anti-TF ADC may deserve a clinical evaluation.

Enhanced antitumor effect of anti-tissue factor antibody-conjugated epirubicin-incorporating micelles in xenograft models.

For the creation of a successful antibody-drug conjugate (ADC), both scientific and clinical evidence has indicated that highly toxic anticancer agents (ACA) should be conjugated to a monoclonal antibody (mAb) to administer a reasonable amount of ADC to patients without compromising the affinity of the mAb. For ordinary ACA, the conjugation of a mAb to ACA-loaded micellar nanoparticles is clinically applicable. Tissue factor (TF) is often overexpressed in various cancer cells and tumor vascular endothelium. Accordingly, anti-TF-NC-6300, consisting of epirubicin-incorporating micelles (NC-6300) conjugated with the F(ab')2 of anti-TF mAb was developed. The in vitro and in vivo efficacy and pharmacokinetics of anti-TF-NC-6300 were compared to NC-6300 using two human pancreatic cancer cell lines, BxPC3 (high TF expression) and SUIT2 (low TF expression), and a gastric cancer cell line, 44As3 (high TF expression). The intracellular uptake of epirubicin was faster and greater in BxPC3 cells treated with anti-TF-NC-6300, compared with NC-6300. Anti-TF-NC-6300 showed a superior antitumor activity in BxPC3 and 44As3 xenografts, compared with NC-6300, while the activities of both micelles were similar in the SUIT2 xenograft. A higher tumor accumulation of anti-TF-NC-6300 compared to NC-6300 was seen, regardless of the TF expression levels. However, anti-TF-NC-6300 appeared to be localized to the tumor cells with high TF expression. These results indicated that the enhanced antitumor effect of anti-TF-NC6300 may be independent of the tumor accumulation but may depend on the selective intratumor localization and the preferential internalization of anti-TF-NC-6300 into high TF tumor cells.

Effect of combined treatment with the epirubicin-incorporating micelles (NC-6300) and 1,2-diaminocyclohexane platinum (II)-incorporating micelles (NC-4016) on a human gastric cancer model.

Anticancer agent-incorporating polymeric micelles accumulate effectively in tumors via the enhanced permeability and retention effect to exert potent antitumor effects. However, combined use of such micelles has not been elucidated. We compared the effect of combining the epirubicin-incorporating micelle NC-6300 and 1,2-diaminocyclohexane platinum (II) (oxaliplatin parent complex)-incorporating micelle NC-4016 (NCs) with that of epirubicin and oxaliplatin (E/O) in 44As3Luc cells using the combination index method. The in vivo antitumor activities of NCs and E/O were evaluated in mice bearing 44As3Luc xenografts. Pharmacokinetic analysis was performed by high-performance liquid chromatography and mass spectrometry. Cardiotoxicity of NC-6300 and epirubicin was assessed by echocardiography. Neurotoxicity of NC-4016 and oxaliplatin was evaluated by examining the paw withdrawal response to noxious mechanical stimuli. NCs showed a highly synergistic activity equivalent to E/O. In vivo, NCs exhibited higher antitumor activity in the subcutaneous tumor model and longer overall survival in the orthotopic tumor model than E/O (p < 0.001, p = 0.015, respectively). The intratumor concentrations of epirubicin and platinum were significantly higher following NCs than following E/O administration. Moreover, the micelles showed lower cardiotoxicity and neurotoxicity than the corresponding conventional drugs. The combined use of the micelles was associated with remarkable efficacy and favorable toxicities in the human gastric cancer model, and warrants the conduct of clinical trials.

Preparation and characterization of anti-tissue factor single-chain variable fragment antibody for cancer diagnosis.

Tissue factor (TF), which serves as the initiator of the extrinsic blood coagulation cascade, has been found to be overexpressed in various solid tumors, especially brain tumors, pancreatic cancer, and gastric cancer. Overexpression of TF is considered to contribute to the high incidence of thrombotic complications and poor prognosis in patients with such cancers. Therefore, detection or targeting of TF may be a promising approach for the diagnosis and treatment of solid tumors that are known to overexpress the protein. Here, we used the recombinant DNA technology to develop an anti-TF single-chain Fv (scFv) of small size and high affinity for its target. The biochemical characteristics of the anti-TF scFv were evaluated using surface plasmon resonance (SPR) sensing and flow cytometry. The data obtained showed that the affinity of the anti-TF scFv was 2.04 × 10(-8) (KD), and that the protein showed significant binding to the cancer cells. Then, Alexa 647-labeled anti-TF scFv and anti-TF IgG were administered to mice bearing chemically induced spontaneous tumors. The maximum tumor to background ratios of anti-TF scFv and anti-TF IgG were obtained 3 and 24 h after the injections, respectively. This study indicates anti-TF scFv may be suitable as an imaging probe for the diagnosis of solid tumors.

Quantitative Analysis of the Concentration of Trifluridine in Tumor Hypoxic Regions Using a Novel Platform Combining Functional Endoscopy and Mass Spectrometry.

Hypoxic regions in solid tumors are highly resistant to drugs and thus represents an obstacle in drug discovery. Currently, however, there are technical barriers in sampling human hypoxic tumors and examining drug delivery with high sensitivity and accuracy. Herein, we present a new platform combining functional endoscopy and highly sensitive liquid chromatography-mass spectrometry (LC-MS) to assess drug delivery to hypoxic regions. Because oxygen saturation endoscopic imaging (OXEI), a functional endoscopy, can evaluate lesions and hypoxia in real-time by simultaneously acquiring a pseudocolor map of oxygen saturation and conventional endoscopic images, this platform can be used to evaluate drug delivery with human samples from hypoxic regions. As the first clinical application of this platform, the relationship between hypoxic regions and the concentration of trifluridine (FTD) incorporated into DNA was evaluated in patients with advanced gastric cancer treated with FTD/tipiracil (FTD/TPI; n = 13) by obtaining and analysis of tissue samples by OXEI and LC-MS and vascular maturity index by CD31/α-SMA staining ex vivo. The results showed that the concentration of FTD was significantly higher in the normoxic region than in the hypoxic region (P < 0.05) and there were significantly more immature vessels in hypoxic regions than in normoxic regions (P < 0.05). These results indicate that the platform was sufficiently sensitive to evaluate differences in drug anabolism in different oxygenic regions of human tumor tissue. This new platform allows quantitative drug analysis in hypoxic regions and is expected to initiate a new era of drug discovery and development.

Potential mechanisms of interstitial lung disease induced by antibody-drug conjugates based on quantitative analysis of drug distribution.

Antibody-drug conjugates (ADCs) are a rapidly advancing category of therapeutic agents with notable anti-cancer efficacy. However, the emergence of interstitial lung disease (ILD) as a severe ADC-associated adverse event highlights the need to better understand the underlying mechanisms. In this study, xenograft model mice with tumors expressing different levels of the trophoblast antigen 2 (TROP2) were generated by subcutaneously transplanting the various TROP2-expression cancer lines. The mice received different doses of TROP2-eribulin, a novel TROP2-targeting ADC, composed of an anti-TROP2 antibody and the eribulin payload, joined by a cleavable linker. The concentration and distribution of TROP2-eribulin, as well as the pharmacokinetics of eribulin release, were assessed in tumor and lung tissues. Analysis of tumor tissue showed that the concentration of released eribulin was approximately 10-fold higher in NCI-H2110 (high TROP2 expression) than in A549 (low TROP2 expression), while analysis of lung tissue showed that TROP2-eribulin was distributed in lung tissue in a dose-dependent manner of TROP2-eribulin regardless of TROP2 expression, with significantly more eribulin released in the high-dose group than in the other dose groups (P < 0.05). Immunofluorescence assay analysis showed that TROP2-eribuilin localized to alveolar macrophages. In the analysis using human- leukemia monocytic cell, the concentration of eribulin released from TROP2-eribuilin was significantly reduced by the use of an Fc receptor inhibitor (P < 0.05). These results revealed that Fcγ-receptor-mediated uptake by alveolar macrophages releases cytotoxic payload into lung tissue, helping to clarify the pathogenesis of ADC-induced ILD.

Real-Time Fluorescence Monitoring System for Optimal Light Dosage in Cancer Photoimmunotherapy.

Background/Objectives: Near-infrared photoimmunotherapy (NIR-PIT) was recently approved for the treatment of unresectable locally advanced or recurrent head and neck cancers in Japan; however, only one clinical dose has been validated in clinical trials, potentially resulting in excessive or insufficient dosing. Moreover, IRDye700X (IR700) fluorescence intensity plateaus during treatment, indicating a particular threshold for the antitumor effects. Therefore, we investigated the NIR laser dose across varying tumor sizes and irradiation methods until the antitumor effects of the fluorescence decay rate plateaued. Methods: Mice were subcutaneously transplanted with A431 xenografts and categorized into control, clinical dose (cylindrical irradiation at 100 J/cm², frontal irradiation at 50 J/cm²), and evaluation groups. The rate of tumor IR700 fluorescence intensity decay to reach predefined rates (-0.05%/s or -0.2%/s) until the cessation of light irradiation was calculated using a real-time fluorescence imaging system. Results: The evaluation group exhibited antitumor effects comparable to those of the clinical dose group at a low irradiation dose. Similar results were observed across tumor sizes and irradiation methods. Conclusions: In conclusion, the optimal antitumor effect of NIR-PIT is achieved when the fluorescence decay rate reaches a plateau, indicating the potential to determine the appropriate dose for PIT using a real-time fluorescence monitoring system.

Tailored immunoconjugate therapy depending on a quantity of tumor stroma.

The purpose of this study was to clarify the appropriate combination of targeting antibody and conjugate-design of anti-tumor immunoconjugate depending on a quantity of tumor stroma. Most human solid tumors including pancreatic cancer (PC) forming hypovascular and stroma-rich tumor hinders the penetration of monoclonal antibodies (mAbs) into the cells, and that leads to failure of the conventional cell-targeting immunoconjugate strategy. To overcome this drawback, SN-38 as topoisomerase 1 inhibitor was conjugated to a mAb to collagen 4, a plentiful component of the tumor stroma via ester-bond. The immunoconjugate, which was able to release SN-38 in physiological condition outside the cells, was effective to stroma-rich PC-tumor. On the other hand, anti-CD 20 mAb-PEG-SN-38 via carbamate-bond as conventional immunoconjugate, enabled SN-38 to be released by a carboxylesterase inside of the tumor cell following the internalization, showed strong anti-tumor activity against malignant lymphoma as hypervascular and stroma-poor tumor. The conjugate-design, in parallel with the choice of targeting antibodies, should be selected to maximize the therapeutic effect in each individual tumor having a distinct stromal structure.