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.

Bispecific Antibodies Produced via Chemical Site-Specific Conjugation Technology: AJICAP Second-Generation.

Bispecific antibodies (BisAbs) are biotherapeutics that amalgamate the specificities of two distinct antibodies into one molecule, however, their engineering requires genetic modification and remains time-consuming. Therefore, we used AJICAP second-generation technology, which drives the production of site-specific conjugation without genetic modification requirements, to generate BisAbs. Using haloketone chemistry as an alternative to maleimide chemistry, we successfully produced site-specific antibody conjugates. Pharmacokinetic studies revealed that the haloketone-based antibody conjugate was stable in the rat plasma. The resultant BisAbs were rigorously evaluated, and surface plasmon resonance measurements and flow cytometry analyses confirmed that the antigen binding remained intact. Additionally, the affinity for the neonatal Fc receptor (FcRn) was retained after conjugation. Further cytotoxicity evaluation emphasized the pronounced activity of the generated BisAbs. This novel approach introduces a fully chemical, site-specific strategy capable of producing BisAbs, heralding a new era in the field of biotherapeutics.

p70S6K/Akt dual inhibitor DIACC3010 is efficacious in preclinical models of gastric cancer alone and in combination with trastuzumab.

The PI3K-Akt-mTOR (PAM) pathway is implicated in tumor progression in many tumor types, including metastatic gastric cancer (GC). The initial promise of PAM inhibitors has been unrealized in the clinic, presumably due, in part, to the up-regulation of Akt signaling that occurs when the pathway is inhibited. Here we present that DIACC3010 (formerly M2698), an inhibitor of two nodes in the PAM pathway, p70S6K and Akt 1/3, blocks the pathway in in vitro and in vivo preclinical models of GC while providing a mechanism that inhibits signaling from subsequent Akt up-regulation. Utilizing GC cell lines and xenograft models, we identified potential markers of DIACC3010-sensitivity in Her2-negative tumors, i.e., PIK3CA mutations, low basal pERK, and a group of differentially expressed genes (DEGs). The combination of DIACC3010 and trastuzumab was evaluated in Her2-positive cell lines and models. Potential biomarkers for the synergistic efficacy of the combination of DIACC3010 + trastuzumab also included DEGs as well as a lack of up-regulation of pERK. Of 27 GC patient-derived xenograft (PDX) models tested in BALB/c nu/nu mice, 59% were sensitive to DIACC3010 + trastuzumab. Of the 21 HER2-negative PDX models, DIACC3010 significantly inhibited the growth of 38%. Altogether, these results provide a path forward to validate the potential biomarkers of DIACC3010 sensitivity in GC and support clinical evaluation of DIACC3010 monotherapy and combination with trastuzumab in patients with HER2- negative and positive advanced GCs, respectively.

Chromatographic Analysis of the N-Glycan Profile on Therapeutic Antibodies Using FcγRIIIa Affinity Column Chromatography.

N-Linked glycosylation on IgG has a profound impact on antibody functions. The relationship between the N-glycan structure and the binding affinity of FcγRIIIa, relating to antibody-dependent cell-mediated cytotoxicity (ADCC) activity, is important for the efficient development of a therapeutic antibody. Here, we report an influence of the N-glycan structure of IgGs, Fc fragments, and antibody-drug conjugates (ADCs) on FcγRIIIa affinity column chromatography. We compared the retention time of several IgGs with heterogeneous and homogeneous N-glycans. IgGs with a heterogeneous N-glycan structure provided several peaks in column chromatography. On the other hand, homogeneous IgGs and ADCs gave a single peak in column chromatography. The length of glycan on IgG also affected the retention time of the FcγRIIIa column, suggesting that the length of glycan is also impacted by binding affinity to FcγRIIIa, resulting in ADCC activity. This analytic methodology provides evaluation of the binding affinity of FcγRIIIa and ADCC activity, not only full-length IgG but also Fc fragments, which are difficult to measure in a cell-based assay. Furthermore, we showed that the glycan-remodeling strategy controls the ADCC activity of IgGs, Fc fragment, and ADCs.

Expansion of mixed immune cells using CD3/CD161 co-stimulation for the treatment of cancer.

Adoptive cell transfer (ACT) is a type of personalized immunotherapy in which expanded immune cells are administered to patients with cancer. However, single-cell populations, such as killer T cells, dendritic cells, natural killer (NK) cells, and NKT (NKT) cells, have been generally used, and their effectiveness remains limited. Here, we established a novel culture method via CD3/CD161 co-stimulation and successfully expanded CD3+/CD4+ helper T cells, CD3+/CD8+ cytotoxic T cells (CTLs), CD3-/CD56+ NK cells, CD3+/CD1d+ NKT cells, CD3+/CD56+ NKT cells, CD3+/TCRγδ+ T cells, and CD3-/CD11c+/HLA-DR+ dendritic cells in peripheral blood mononuclear cells from healthy donors; their respective numbers were 155.5, 1132.5, 5.7, 117.0, 659.2, 325.6, and 6.8 times higher than those before expansion. These mixed immune cells showed strong cytotoxicity against cancer cell lines Capan-1 and SW480. Moreover, both CD3+/CD8+ CTLs and CD3+/CD56+ NKT cells killed tumor cells in cell contact-dependent and -independent manners via granzyme B and interferon-γ/TNF-α, respectively. Furthermore, the cytotoxicity of the mixed cells was significantly superior to that of CTLs or NKTs alone. A bet-hedging CTL-NKT circuitry is one potential mechanism underlying this cooperative cytotoxicity. Collectively, CD3/CD161 co-stimulation may be a promising culture method to expand multiple, distinct immune cell populations for the treatment of cancer.

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.

Quantitative analysis of drug distribution in heterogeneous tissues using dual-stacking capillary electrophoresis-mass spectrometry.

BACKGROUND AND PURPOSE: Intratumour heterogeneity frequently leads to drug resistance, which is a major issue in drug discovery. Drug distribution is one of the key factors for elucidating the resistance mechanism; however, quantitative and regional drug measurement is challenging. Here, we developed a novel ultra-sensitive analytical method and applied it to HER3-targeting antibody-drug conjugate patritumab deruxtecan (HER3-DXd), aiming to explore its payload (DXd) distribution within heterogeneous tissues. EXPERIMENTAL APPROACH: The developed analytical method is named LDMS-CE-MS, a capillary electrophoresis-mass spectrometry (CE-MS) coupled with a novel sample preconcentration/separation method called "large-volume dual-sample stacking by micelle collapse and sweeping (LDMS)". First, the analytical performance of LDMS-CE-MS for DXd detection was evaluated. Subsequently, we evaluated the bystander effect of HER3-DXd, where tumour tissues were excised from xenograft models and clinical specimens after administration of HER3-DXd. HER3-high expression, adjacent, and HER3-low expression regions were then sampled by laser microdissection to quantify the released DXd. KEY RESULTS: LDMS concentrated DXd by 1000-fold and separated it from the hydrophilic bio-matrix through continuous capture and release by the charged micelles, allowing quantification at sub-attomole-level. DXd concentrations decreased in the order of antigen-high expression > adjacent > antigen-low expression regions in the tumour xenograft model, whereas in clinical specimens, adjacent and antigen-high expression regions had approximately the same concentration. These distributions represent a bystander effect. CONCLUSIONS AND IMPLICATIONS: Our LDMS-CE-MS successfully visualized the attomole-level drug distributions in heterogeneous clinical specimens. This new platform opens a new era of quantitative pharmacokinetic analysis, facilitating drug discovery and development.

Molecular design of near-infrared (NIR) fluorescent probes targeting exopeptidase and application for detection of dipeptidyl peptidase 4 (DPP-4) activity.

Monitoring the activities of proteases in vivo is an important requirement in biological and medical research. Near-infrared (NIR) fluorescent probes are particularly useful for in vivo fluorescence imaging, due to the high penetration of NIR and the low autofluorescence in tissue for this wavelength region, but most current NIR fluorescent probes for proteases are targeted to endopeptidase. Here, we describe a new molecular design for NIR fluorescent probes that target exopeptidase by utilizing the >110 nm blueshift of unsymmetrical Si-rhodamines upon amidation of the N atom of their xanthene moiety. Based on this molecular design, we developed Leu-SiR640 as a probe for leucine amino peptidase (LAP). Leu-SiR640 shows a one order of magnitude larger fluorescence increment (669-fold) upon reaction with LAP than existing NIR fluorescent probes. We similarly designed and synthesized EP-SiR640, a NIR fluorescent probe that targets dipeptidyl peptidase 4 (DPP-4). We show that this probe can monitor DPP-4 activity not only in living cells but also in mouse organs and tumors. This probe could also detect esophageal cancer in human clinical specimens, based on the overexpression of DPP-4 activity.

Identification of CD73 as the Antigen of an Antigen-Unknown Monoclonal Antibody Established by Exosome Immunization, and Its Antibody-Drug Conjugate Exerts an Antitumor Effect on Glioblastoma Cell Lines.

Development of antibodies against the native structure of membrane proteins with multiple transmembrane domains is challenging because it is difficult to prepare antigens with native structures. Previously, we successfully developed a monoclonal antibody against multi-pass membrane protein TMEM180 by exosome immunization in rats. This approach yielded antibodies that recognized cancer-specific antigens on the exosome. In this study, we performed immunoprecipitation using magnetic beads to identify the antigen of one of the rat antibody clones, 0614, as CD73. We then converted antibody 0614 to human chimeric antibody 0614-5. Glioblastoma (GB) was the cancer type with the highest expression of CD73 in the tumor relative to healthy tissue. An antibody-drug conjugate (ADC) of 0614-5 exerted an antitumor effect on GB cell lines according to expression of CD73. The 0614-5-ADC has potential to be used to treat cancers with high CD73 expression. In addition, our strategy could be used to determine the antigen of any antibody produced by exosome immunization, which may allow the antibody to advance to new antibody therapies.

Protection from contamination by 211At, an enigmatic but promising alpha-particle-emitting radionuclide.

PURPOSE: 211At, a promising alpha-particle-emitting radionuclide, can easily volatilize and contaminate the environment. To safely manage this unique alpha-particle-emitting radionuclide, we investigated the permeability of four types of plastic films and two types of rubber gloves against 211At and identified suitable materials that prevent contamination by 211At. METHODS: Four types of plastic films, polyethylene, polyvinylidene chloride, polyvinyl chloride, and a laminated film, and two types of rubber gloves, latex and nitrile, were examined. Small pieces of filter paper were covered with these materials, and a drop containing 100 kBq of 211At was placed on them. The radioactivity of the pieces of filter paper under the materials was evaluated by measuring counts using a gamma counter and obtaining autoradiograms 3.5 h later. These experiments were also performed using 225Ac, 125I, 111In, 201Tl, and 99mTc. RESULTS: 211At solution easily penetrated polyethylene, polyvinyl chloride, and latex rubber. Similar results were obtained for 125I, while other radionuclides did not penetrate films or gloves. These results suggest that halogenic radionuclides under anionic conditions are likely to penetrate plastic films and rubber gloves. CONCLUSION: Our evaluation revealed that, when 211At solution is used, the protection by polyvinylidene chloride, a laminated film, or nitrile rubber would be more effective than that by polyethylene, polyvinyl chloride, or latex rubber.

Evaluation of Fluorescence Intensity and Antitumor Effect Using Real-Time Imaging in Photoimmunotherapy.

Photoimmunotherapy (PIT) is a promising tumor-selective treatment method that uses light-absorbing dye-conjugated antibodies and light irradiation. It has been reported that IR700 fluorescence changes with light irradiation. The purpose of this study was to investigate the fluorescence intensity and antitumor effect of PIT using real-time fluorescence observation of tumors and predict the required irradiation dose. The near-infrared camera system LIGHTVISION was used to image IR700 during PIT treatment. IR700 showed a sharp decrease in fluorescence intensity in the early stage of treatment and almost reached a plateau at an irradiation dose of 40 J/cm. Cetuximab-PIT for A431 xenografts was performed at multiple doses from 0-100 J/cm. A significant antitumor effect was observed at 40 J/cm compared to no irradiation, and there was no significant difference between 40 J/cm and 100 J/cm. These results suggest that the rate of decay of the tumor fluorescence intensity correlates with the antitumor effect by real-time fluorescence imaging during PIT. In addition, when the fluorescence intensity of the tumor plateaued in real-time fluorescence imaging, it was assumed that the laser dose was necessary for treatment.

The natural sulfoglycolipid derivative SQAP improves the therapeutic efficacy of tissue factor-targeted radioimmunotherapy in the stroma-rich pancreatic cancer model BxPC-3.

α-Sulfoquinovosylacyl-1,3-propanediol (SQAP) is a semi-synthetic derivative of natural sulfoglycolipid that sensitizes tumors to external-beam radiotherapy. How SQAP affects internal radiotherapy, however, is not known. Here, we investigated the effects of SQAP for radioimmunotherapy (RIT) targeting tissue factor (TF) in a stroma-rich refractory pancreatic cancer mouse model, BxPC-3. A low dose of SQAP (2 mg/kg) increased tumor uptake of the 111In-labeled anti-TF antibody 1849, indicating increased tumor perfusion. The addition of SQAP enhanced the growth-inhibitory effect of 90Y-labeled 1849 without leading to severe body weight changes, allowing for the dose of 90Y-labeled 1849 to be reduced to half that when used alone. Histologic analysis revealed few necrotic and apoptotic cells, but Ki-67-positive proliferating cells and increased vascular formation were detected. These results suggest that the addition of a low dose of SQAP may improve the therapeutic efficacy of TF-targeted RIT by increasing tumor perfusion, even for stroma-rich refractory pancreatic cancer.

T Cell Bispecific Antibodies: An Antibody-Based Delivery System for Inducing Antitumor Immunity.

As a breakthrough immunotherapy, T cell bispecific antibodies (T-BsAbs) are a promising antibody therapy for various kinds of cancer. In general, T-BsAbs have dual-binding specificity to a tumor-associated antigen and a CD3 subunit forming a complex with the TCR. This enables T-BsAbs to crosslink tumor cells and T cells, inducing T cell activation and subsequent tumor cell death. Unlike immune checkpoint inhibitors, which release the brake of the immune system, T-BsAbs serve as an accelerator of T cells by stimulating their immune response via CD3 engagement. Therefore, they can actively redirect host immunity toward tumors, including T cell recruitment from the periphery to the tumor site and immunological synapse formation between tumor cells and T cells. Although the low immunogenicity of solid tumors increases the challenge of cancer immunotherapy, T-BsAbs capable of immune redirection can greatly benefit patients with such tumors. To investigate the detailed relationship between T-BsAbs delivery and their T cell redirection activity, it is necessary to determine how T-BsAbs deliver antitumor immunity to the tumor site and bring about tumor cell death. This review article discusses T-BsAb properties, specifically their pharmacokinetics, redirection of anticancer immunity, and local mechanism of action within tumor tissues, and discuss further challenges to expediting T-BsAb development.

TMEM180 contributes to SW480 human colorectal cancer cell proliferation through intra-cellular metabolic pathways.

TMEM180, a novel colon cancer-specific protein with a 12-transmembrane topology, is upregulated at low oxygen. Previously, we established a humanized monoclonal antibody against TMEM180 aimed at clinical trials. Prior to such trials, it is necessary to clarify the function of TMEM180 in cancer. To compare SW480 human colon cancer cells and their TMEM180-knockdown derivatives, we analyzed proliferation and oxygen consumption, and also performed phosphorylation proteomics, metabolomics, and next-generation sequencing (NGS). The preliminary results revealed that TMEM180 appeared to promote the growth of colon cancer but had almost no effect on oxygen consumption or expression of phosphorylated proteins. By contrast, glycolysis differed dramatically between SW480 and TMEM180-knockdown cells. The NGS analysis revealed that TMEM180 promotes enzyme expression in nitric oxide (NO) synthesis system, suggesting that it promotes glucose and glutamine metabolism, thereby contributing to cancer growth. Overall, the results of this study warrant further basic studies of TMEM180 molecule.

Stabilization of an 211At-Labeled Antibody with Sodium Ascorbate.

211At, an α-particle emitter, has recently attracted attention for radioimmunotherapy of intractable cancers. However, our sodium dodecyl sulfate polyacrylamide gel electrophoresis and flow cytometry analyses revealed that 211At-labeled immunoconjugates are easily disrupted. Luminol assay revealed that reactive oxygen species generated from radiolysis of water caused the disruption of 211At-labeled immunoconjugates. To retain their functions, we explored methods to protect 211At-immunoconjugates from oxidation and enhance their stability. Among several other reducing agents, sodium ascorbate most safely and successfully protected 211At-labeled trastuzumab from oxidative stress and retained the stability of the 211At-labeled antibody and its cytotoxicity against antigen-expressing cells for several days.

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.

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.

Antitumor effect of humanized anti­tissue factor antibody­drug conjugate in a model of peritoneal disseminated pancreatic cancer.

Tissue factor (TF) is an attractive target for cancer therapy due to its overexpression in multiple types of malignancies. In addition, TF has been reported to play functional roles in both cancer development and metastasis. Several groups have already developed antibody­drug conjugates (ADCs) against TF for use as cancer treatments, and have demonstrated their efficacies in conventional subcutaneous xenograft models and patient­derived xenograft models. However, no previous studies have investigated the effectiveness of anti­TF ADC in an advanced­stage cancer model. The present study developed an original humanized anti­TF monoclonal antibody conjugated with monomethyl auristatin E, and evaluated its in vivo efficacy in a pancreatic cancer xenograft model with peritoneal dissemination. In vitro assays demonstrated that the anti­TF ADC had potent binding affinity and cytotoxic activity against human pancreatic cancer cells that strongly expressed TF antigens. The anti­TF ADC also exhibited greater antitumor effect than that of a control ADC in conventional subcutaneous xenograft models, with efficacy depending on the TF expression in the tumor tissues. Furthermore, the anti­TF ADC significantly inhibited tumor growth in an orthotopic xenograft model, and extended the survival period in a murine peritoneal dissemination model. These results indicated that anti­TF ADC has the potential to be an effective treatment not only for primary tumors, but also for those that are widely disseminated. Therefore, it can be concluded that ADC targeting TF may be a promising agent for advanced pancreatic cancer therapy.

Reinforcement of antitumor effect of micelles containing anticancer drugs by binding of an anti-tissue factor antibody without direct cytocidal effects.

It has been preclinically and clinically proven that anticancer agent-incorporating (ACA-incorporating) polymeric micelles selectively accumulate in tumor via the enhanced permeability and retention (EPR) effect, yielding a wider therapeutic window and greater safety than conventional low-molecular weight ACAs. To increase the antitumor effect of these safer micelle formulations, epirubicin-incorporating polymer micelles (NC-6300) conjugated with monoclonal antibodies (mAbs) have been prepared. In this study, we used two types of mAb: an anti-tissue factor (TF) mAb that does not exert a direct cytocidal effect, and an anti-HER2 mAb that has a direct cytocidal effect. We compared the antitumor effects and pharmacological properties of the two types of antibody conjugated to NC-6300. Immunomicelles conjugated to anti-TF mAb exerted greater antitumor activity toward TF-positive stomach cancer than the combination of anti-TF mAb and NC-6300, and were distributed more uniformly throughout TF-positive tumor tissue than NC-6300. On the other hand, immunomicelles conjugated to anti-HER2 mAb did not exert significant antitumor activity toward HER2-positive stomach cancer relative to the combined use of anti-HER2 mAb and NC-6300. Thus, this immunomicelle-based strategy may be useful for antibodies that target cancer as pilot molecules even when the antibodies themselves do not have an antitumor effect.