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.

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.

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.

Report of the use of patient-derived xenograft models in the development of anticancer drugs in Japan.

Cell line-derived xenograft (CDX) models created by implanting cancer cell lines into immunodeficient mice have contributed largely to the development of cancer drug therapies. However, cell lines often lose their original biological characteristics through many passages and cancer tissues in CDX models have many cancer cells and few cancer stromal cells, therefore CDX models are currently considered not suitable for predicting the results of clinical studies. Conversely, patient-derived xenograft (PDX) models are gaining importance, as human cancer biological characteristics and microenvironments are recreated by implanting tumor tissue into immunodeficient mice. These highly expected, evidently beneficial PDX models have been used in some basic research and are becoming more generalized. However, quality control and quality assurance criteria have not been established for them, and challenges and problems in the utilization of valuable PDX models in drug development have yet to be clarified. In this report, we conducted a questionnaire survey among researchers in Japanese academic institutions and pharmaceutical companies to understand the current status of PDX models in Japan. Based on the questionnaire results, we summarized the situations surrounding respondent's utilization and quality control in the development of anticancer drugs and proposed several measures to facilitate the utilization of PDX models in the development of anticancer drugs.

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.

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.

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.

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.

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.

Assessing the Correlation Between Visual Acuity and Critical Fusion Frequency in Acute Optic Neuritis Before and After Steroid Therapy.

Background Optic nerve diseases include inflammatory optic nerve diseases such as vasculitis, metabolic optic neuropathy, ischemic optic neuropathy, and optic neuritis. In this study, patients with acute optic neuritis are classified with better and poor visual acuity based on visual acuity after one month of steroid pulse therapy. To determine prognosis, initial visual acuity and critical fusion frequency (CFF) values will be compared with those recorded one month after treatment and at the last visit. Methods Visual acuity and CFF were evaluated one month after the start of treatment in patients diagnosed with acute optic neuritis, and follow-up was available for at least three months at Hiroshima University Hospital. Results All patients received steroid pulse therapy as initial treatment. After one month of treatment, visual acuity and CFF at the last visit were significantly improved in the group with improved visual acuity compared to the group with impaired visual acuity. Conclusions Visual acuity at the initial visit did not affect treatment outcome, and final visual acuity and CFF after one month of treatment for acute optic neuritis were better in patients with better visual acuity. Therefore, visual acuity values one month after treatment initiation may affect treatment outcomes.

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.

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.

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.

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.

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.

Influence of the dissociation rate constant on the intra-tumor distribution of antibody-drug conjugate against tissue factor.

Antibody-drug conjugates (ADCs) are currently considered to be promising agents for cancer therapy. However, especially in solid tumors, the uneven distribution of ADCs would decrease their efficacy in clinical studies. We suggest that in addition to optimizing ADC components, such as the linker structure and anticancer agent, it is necessary to consider the distribution of the ADC within tumor tissue. In this study, we established three kinds of anti-tissue factor (TF) ADCs: 1849ADC with a low kd, 444ADC with an intermediate kd, and 1084ADC with a high kd. All three of the anti-TF ADCs exhibited almost the same in vitro cytotoxicity and pharmacological and biochemical characteristics, although the binding kinetics parameters differed. In vivo, all ADCs exerted equivalent antitumor effects against small BxPC3 tumors. However, on larger BxPC3 tumors, 1084ADC (higher kd) exerted higher antitumor activity than 1849ADC (lower kd). Furthermore, immunofluorescence staining indicated that 1084ADC was distributed throughout the whole tumor, whereas 1849ADC was mainly localized close to tumor vessels. We conclude that the ADC with a higher kd increased the antitumor effect of because it penetrated and distributed evenly throughout the entire solid tumor. These findings highlight the importance of the kd of a mAb in ADC design.

Feasibility study of the Fab fragment of a monoclonal antibody against tissue factor as a diagnostic tool.

Tissue factor (TF) is expressed strongly in various types of cancer, especially cancers that are often refractory to treatment, such as pancreatic cancer. In this study, we compared the differences in the biophysical and pharmacological properties of whole IgG and the Fab fragment of anti-human TF monoclonal antibody (1849 antibodies), in order to determine their suitability for application in the diagnosis and treatment of cancers. In the biophysical examination, we investigated the characteristics of 1849-whole IgG and 1849-Fab by SPR sensing and confocal fluorescence microscopy analysis using recombinant human TF antigen and TF-overexpressing human pancreatic cancer cell line, BxPC3, respectively. After conjugation with Alexa-Flour-647, in vivo imaging was conducted in mice bearing BxPC3 xenograft tumors. Furthermore, the distribution of the conjugates in tumors and major organs was evaluated by ex vivo study. The in vitro experiments showed that 1849 antibodies had high affinity against TF antigen. In addition, 1849-Fab showed a faster dissociation rate from the antigen than 1849-whole IgG. In mice, 1849-Fab-Alexa-Flour-647 showed rapid renal clearance and faster tumor accumulation, achieving a high contrast signal over nearby normal tissues in the early phase and enhanced tumor penetration after administration. On the other hand, 1849-whole IgG-Alexa-Flour-647 showed slow clearance from the blood and sustained high tumor accumulation. These results suggest that 1849-Fab may be a useful tool for pancreatic cancer diagnosis.

Antibody fragment-conjugated polymeric micelles incorporating platinum drugs for targeted therapy of pancreatic cancer.

Antibody-mediated therapies including antibody-drug conjugates (ADCs) have shown much potential in cancer treatment by tumor-targeted delivery of cytotoxic drugs. However, there is a limitation of payloads that can be delivered by ADCs. Integration of antibodies to drug-loaded nanocarriers broadens the applicability of antibodies to a wide range of therapeutics. Herein, we developed antibody fragment-installed polymeric micelles via maleimide-thiol conjugation for selectively delivering platinum drugs to pancreatic tumors. By tailoring the surface density of maleimide on the micelles, one tissue factor (TF)-targeting Fab' was conjugated to each carrier. Fab'-installed platinum-loaded micelles exhibited more than 15-fold increased cellular binding within 1 h and rapid cellular internalization compared to non-targeted micelles, leading to superior in vitro cytotoxicity. In vivo, Fab'-installed micelles significantly suppressed the growth of pancreatic tumor xenografts for more than 40 days, outperforming non-targeted micelles and free drugs. These results indicate the potential of Fab'-installed polymeric micelles for efficient drug delivery to solid tumors.