AJICAP-M: Traceless Affinity Peptide Mediated Conjugation Technology for Site-Selective Antibody-Drug Conjugate Synthesis.

A traceless site-selective conjugation method, "AJICAP-M", was developed for native antibodies at sites using Fc-affinity peptides, focusing on Lys248 or Lys288. It produces antibody-drug conjugates (ADCs) with consistent drug-to-antibody ratios, enhanced stability, and simplified manufacturing. Comparative in vivo assessment demonstrated AJICAP-M's superior stability over traditional ADCs. This technology has been successfully applied to continuous-flow manufacturing, marking the first achievement in site-selective ADC production. This manuscript outlines AJICAP-M's methodology and its effectiveness in ADC production.

Biological Evaluation of Maytansinoid-Based Site-Specific Antibody-Drug Conjugate Produced by Fully Chemical Conjugation Approach: AJICAP®.

BACKGROUND: Trastuzumab-emtansine (T-DM1, commercial name: Kadcyla) is well-known antibody-drug conjugate (ADC) and was first approved for human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. This molecular format consisting of trastuzumab and maytansinoid payload (emtansine) is very simple, however, T-DM1 has wide heterogeneity due to non-specific conjugation, lowering its therapeutic index (TI). METHODS: To overcome this issue during the chemical modification of the random conjugation approach to generate T-DM1, we developed a novel chemical conjugation technology termed "AJICAP®" for modification of antibodies in site-specific manner by IgG Fc-affinity peptide based reagents. RESULTS: In this study, we compared site-specific maytansinoid-based ADCs synthesized by AJICAP and T-DM1 in rat safety studies. The results indicated an increase in the maximum tolerated dose, demonstrating an expansion of the AJICAP-ADC therapeutic index compared with that of commercially available T-DM1. Gram scale preparation of this AJICAP-ADC and the initial stability study are also described. CONCLUSIONS: Trastuzumab-AJICAP-maytansinoid produced by this unique chemical conjugation methodology showed higher stability and tolerability than commercially available T-DM1.

Chemical Site-Specific Conjugation Platform to Improve the Pharmacokinetics and Therapeutic Index of Antibody-Drug Conjugates.

To overcome a lack of selectivity during the chemical modification of native non-engineered antibodies, we have developed a technology platform termed "AJICAP" for the site-specific chemical conjugation of antibodies through the use of a class of IgG Fc-affinity reagents. To date, a limited number of antibody-drug conjugates (ADCs) have been synthesized via this approach, and no toxicological study was reported. Herein, we describe the compatibility and robustness of AJICAP technology, which enabled the synthesis of a wide variety of ADCs. A stability assessment of a thiol-modified antibody synthesized by AJICAP technology indicated no appreciable increase in aggregation or decomposition upon prolonged storage, indicating that the unexpectedly stable thiol intermediate has a great potential intermediate for payload or linker screening or large-scale manufacturing. Payload conjugation with this stable thiol intermediate generated several AJICAP-ADCs. In vivo xenograft studies indicated that the AJICAP-ADCs displayed significant tumor inhibition comparable to benchmark ADC Kadcyla. Furthermore, a rat pharmacokinetic analysis and toxicology study indicated an increase in the maximum tolerated dose, demonstrating an expansion of the AJICAP-ADC therapeutic index, compared with stochastic conjugation technology. This is the first report of the therapeutic index estimation of site-specific ADCs produced by utilizing Fc affinity reagent conjugation. The described site-specific conjugation technology is a powerful platform to enable next-generation ADCs through reduced heterogeneity and enhanced therapeutic index.

[Three Cases of Resectable Advanced Gastric Cancer Treated with Neoadjuvant Chemotherapy].

Three patients diagnosed with HER2-negative resectable advanced gastric cancer with extensive regional lymph node metastases were treated with neoadjuvant chemotherapy(NAC), followed by gastrectomy with D2lymph node dissection. One patient received four 21-day courses of S-1 plus oxaliplatin(G-SOX), and pathological effect(PE)was Grade 3. Two patients received four 21-day courses of capecitabine plus oxaliplatin(CapeOX), and each PE was Grade 2and Grade 1a, respectively. One patient in poor PE was with recurrent liver and peritoneal metastases. This suggested that for resectable advanced gastric cancer with extensive regional lymph node metastases, NAC by SOX or CapeOX was effective for some patients.

Catalytic Cyclopropanation by Myoglobin Reconstituted with Iron Porphycene: Acceleration of Catalysis due to Rapid Formation of the Carbene Species.

Myoglobin reconstituted with iron porphycene catalyzes the cyclopropanation of styrene with ethyl diazoacetate. Compared to native myoglobin, the reconstituted protein significantly accelerates the catalytic reaction and the kcat/Km value is 26-fold enhanced. Mechanistic studies indicate that the reaction of the reconstituted protein with ethyl diazoacetate is 615-fold faster than that of native myoglobin. The metallocarbene species reacts with styrene with the apparent second-order kinetic constant of 28 mM-1 s-1 at 25 °C. Complementary theoretical studies support efficient carbene formation by the reconstituted protein that results from the strong ligand field of the porphycene and fewer intersystem crossing steps relative to the native protein. From these findings, the substitution of the cofactor with an appropriate metal complex serves as an effective way to generate a new biocatalyst.

[A Case of HER2-Positive Unresectable Gastric Cancer with Multiple Lymph Node and Liver Metastases Controlled Effectively by Combination Chemotherapy with Capecitabine, Oxaliplatin, and Trastuzumab].

A 77-year-old woman was diagnosed with HER2-positive unresectable gastric cancer with multiple lymph node and liver metastases(cT3-4, cN3, cM1[HEP, LYM], cStage IV ). Four courses of combination chemotherapy with capecitabine, oxaliplatin, and trastuzumab(XELOX plus Tras)were administered. Though all lesions showed a complete or partial response, anorexia and body weight loss appeared because of the stenosis in the primary gastric lesion. After another course, these symptoms became worse and she underwent laparoscopic gastrojejunostomy. She progressed favorably after the surgery, her anorexia improved and her weight increased. Thirty-four days after the surgery, the same chemotherapy was continued. At present, the metastases are well controlled 12months after the initial treatment. It is suggested that XELOX plus Tras is an effective chemotherapy regimen for HER2-positive unresectable gastric cancer.

Metformin inhibits the radiation-induced invasive phenotype of esophageal squamous cell carcinoma.

Esophageal cancer is one of the most aggressive tumor types because of its invasiveness and metastatic potential. Several reports have described an association between increased invasiveness after ionizing radiation (IR) treatment and epithelial-to-mesenchymal transition (EMT). The biguanide metformin is reported to prevent transforming growth factor-ß (TGF-ß)-induced EMT and proliferation of cancer. This study examined whether IR induces EMT and promotes the invasive potential of TE-9 esophageal squamous cell carcinoma cells and the effect of metformin on IR-induced EMT. After IR exposure, TE-9 cells showed a spindle-shaped morphology and lost cell-cell adhesion. Immunoblotting showed that IR induced expression of mesenchymal markers (vimentin and N-cadherin), transcription factors (Slug, Snail, and Twist), and matrix metalloproteinases. A scratch wound assay and Matrigel invasion assay showed that IR enhanced the invasive potential and migratory capacity of TE-9 cells. Expression of hypoxia-related factor-1α and TGF-ß was increased after IR. IR also induced phosphorylation of Smad2 and Smad3. Metformin inhibited radiation-induced EMT-like morphological changes, and enhanced invasion and migration of TE-9 cells. Metformin inhibited IR-induced phosphorylation of Smad2 and Smad3. Although phosphorylation of AMP-activated protein kinase was enhanced by IR and metformin, phosphorylation of mammalian target of rapamycin was enhanced by IR and suppressed by metformin. These results indicated that metformin suppressed IR-induced EMT via suppression of the TGF-ß-Smad phosphorylation pathway, and a part of the non-Smad pathway. Metformin might be useful to prevent IR-induced invasion and metastasis of esophageal squamous cell carcinoma.

[Experience with Gemcitabine plus Nab-Paclitaxel Therapy for Advanced Metastatic Pancreatic Cancer in Our Hospital].

A 71-year-old-man was referred to our hospital because of jaundice of the skin. On biochemical examination of blood, we identified an elevation in the levels of AST and ALT, the serum level of biliary enzymes, and the serum levels of tumor markers. We found pancreatic head cancer with invasion to the main blood vessels and duodenum, with liver metastases, on abdominal CT. We made a diagnosis of unresectable advanced pancreatic head cancer with distant metastasis, and we initiated gemcitabine plus nab-paclitaxel therapy as first-line chemotherapy. The serum CA19-9 level decreased gradually after initiating therapy; both the primary tumor and liver metastases slightly reduced in size after 3 courses of first-line therapy, as assessed on abdominal CT. Hair loss, peripheral neuropathy, and neutropenia were observed as adverse events, but treatment continued as it was tolerable. Finally, because his disease condition worsened after 7 courses of the therapy, we switched to TS-1 as second-line therapy. Eleven months have elapsed since treatment was initiated, and the patient is continuing secondline therapy while maintaining PS.

Multiple liver metastases of pancreatic solid pseudopapillary tumor treated with resection following chemotherapy and transcatheter arterial embolization: A case report.

A 33-year-old female was diagnosed with a solid pseudopapillary tumor (SPT) of the pancreas and multiple liver metastases at the Department of Gastroenterological Surgery, Ishikawa Prefectural Central Hospital (Kanazawa, Japan). Distal pancreatectomy and postoperative systemic chemotherapy with gemcitabine (GEM) and S-1, an oral fluoropyrimidine derivative, was administered, however, liver metastases became enlarged and local recurrence occurred. Therefore, the patient was referred to the Department of Gastroenterologic Surgery at the Graduate School of Medicine (Kanazawa, Japan) for hepatic arterial infusion (HAI) chemotherapy. Oral S-1 (80 mg/m2) was administered as well as HAI chemotherapy with GEM (1,000 mg/standard liver volume). Following 18 cycles, tumor sizes were reduced and 18-fluorodeoxyglucose positron emission tomography (18FDG-PET) examination revealed obvious reduction of tumor FDG uptake. Transarterial tumor embolization (TAE) was performed for the previously unresectable right subphrenic liver tumor, and the other tumors were surgically resected. The resected tumors were diagnosed as liver metastases and a local recurrence of SPT in the postoperative pathological examination, which revealed that the resected tumors were composed of sheets of bland cells, which were positive for CD10, CD56, vimentin, neuron-specific enolase and α-antitrypsin. The postoperative course was uneventful, and the patient is currently under observation at an outpatient clinic; postoperative adjuvant chemotherapy with oral S-1 has continued, and additional TAE is planned. In the future, if the middle segment of the liver becomes enlarged, surgery for the residual right lobe tumor may be possible. This case demonstrates one method of SPT treatment: Preoperative HAI chemotherapy with GEM, plus oral S-1 and TAE. If complete resection can be achieved, the majority of patients with SPT have a favorable prognosis. In patients with unresectable metastases from SPT, it is crucial to conduct systematic multimodal treatment to maximize treatment success.

[A case report of hepatic arterial infusion chemotherapy and RFA for liver metastasis from pancreatic cancer].

A 71-year-old female patient was administered 2 courses of neoadjuvant chemotherapy (GS therapy) for pancreatic body cancer, and underwent pancreatic body and tail resection. She was diagnosed as having T3N0M0, Stage III disease, and adjuvant chemotherapy with gemcitabine was started. However, a solitary 9 mm liver metastasis was found using CT imaging 3 months after the operation. We started hepatic arterial infusion chemotherapy (GEM+5-FU), with additional treatment using RFA after 5 courses, and a CR was achieved. The HAI regimens were changed to GEM+S-1 and a further 18 courses were administered. After HAI, adjuvant chemotherapy (S-1) was continued, but 2 further liver metastases were found. The patient was still alive 4 years after surgery and continued to undergo radiation chemotherapy.

Oral treatment with a novel small molecule alpha 4 integrin antagonist, AJM300, prevents the development of experimental colitis in mice.

BACKGROUND AND AIMS: Inhibition of lymphocyte trafficking by treatment with an anti-α4 integrin antibody has been clinically validated as a therapeutic approach for inflammatory bowel disease (IBD), and the orally effective 'anti-α4 integrin therapy' may be more convenient in clinical practice. The aim of this study was to investigate the pharmacological profile and anti-inflammatory effect of a novel, orally active small molecule α4 integrin antagonist, AJM300. METHODS: The binding specificity/potency of HCA2969 (the active metabolite of AJM300) were investigated in vitro. The pharmacodynamics for α4 integrin antagonism of AJM300 was investigated in mice. The anti-inflammatory effect of AJM300 fed in a diet and the anti-α4 integrin monoclonal antibody was evaluated in a mouse colitis model induced by transfer of IL-10 deficient T cells. RESULTS: HCA2969 selectively inhibited the in vitro binding of α4 integrin (α4ß7/α4ß1) to the cell adhesion molecules. Oral treatment with AJM300 dose-dependently inhibited lymphocyte homing to Peyer's patches and increased the peripheral lymphocyte count in the same dose range. AJM300 dose-dependently prevented the development of experimental colitis in mice. A significant inhibition of colon weight increase was accompanied by inhibition of T-cell infiltration into the lamina propria of colon. The maximum efficacy of AJM300 (1% diet) was comparable to that achieved by the saturated α4 integrin blockade with antibody. CONCLUSIONS: Oral treatment with the selective small molecule α4 integrin antagonist (AJM300) prevented the development of colitis and its efficacy was comparable to that of the anti-α4 integrin antibody.

Competing effects of rare gas atoms in matrix isolation spectroscopy: a case study of vibrational shift of BeO in Xe and Ar matrices.

We investigate the vibrational shift of beryllium oxide (BeO) in Xe matrix as well as in Ar matrix environments by mixed quantum-classical simulation and examine the origin of spectral shift in details. BeO is known to form strong chemical complex with single rare gas atom, and it is predicted from the gas phase calculations that vibrational frequencies are blueshifted by 78 cm(-1) and 80 cm(-1) upon formation of XeBeO and ArBeO, respectively. When the effects of other surrounding rare gas atoms are included by Monte Carlo simulations, it is found that the vibrational frequencies are redshifted by 21 cm(-1) and 8 cm(-1) from the isolated XeBeO and ArBeO complexes, respectively. The vibrational shift of XeBeO in Ar matrix is also calculated and compared with experimental data. In all simulations examined in this paper, the calculated vibrational frequency shifts from the isolated BeO molecule are in reasonable agreement with experimental values. The spectral shift due to the rare-gas-complex formation of RgBeO (Rg = Xe or Ar) is not negligible as seen in the previous studies, but it is shown in this paper that the effects of other surrounding rare gas atoms should be carefully taken into account for quantitative description of the spectral shifts and that these two effects are competing in vibrational spectroscopy of BeO in matrix environments.

Redox state of albumin is not associated with colloid osmotic pressure.

Serum albumin exists in oxidized and reduced forms. Although the oxidation of albumin affects some of its functions, the relationship between oxidized albumin and colloid osmotic pressure (COP) remains unclear. The aim of this study was to determine whether there is an association between oxidized albumin and COP. Blood samples from 20 healthy volunteers were divided into two aliquots in order to prepare reduced (n=20) and oxidized albumin samples (n=20). This was achieved by treatment with L-cysteine and a redox-stabilizing agent before and after incubation at 37°C for 24 h. The percentage of oxidized albumin was determined by high-performance liquid chromatography. COP was measured using a colloid osmometer. Reduced and oxidized albumin samples showed 100% of reduced and 100% of oxidized albumin, respectively. There were no significant differences in albumin level and total protein level between the reduced and the oxidized albumin samples. No significant change was seen in COP between the reduced and the oxidized albumin samples (reduced albumin, 17.4±0.2 mmHg; oxidized albumin, 17.3±0.2 mmHg; P=0.465). Therefore, there is no significant difference in COP between reduced and oxidized albumin samples.

[Structure and function changes of oxidized human serum albumin: physiological significance of the biomarker and importance of sampling conditions for accurate measurement].

Human serum albumin (HSA) exists in both reduced and oxidized forms, and the percentage of oxidized albumin increases in several diseases; however, little is known regarding the pathological and physiological significance of oxidation due to poor characterization of the precise structural and functional properties of oxidized HSA. Here, we characterize both structural and functional differences between reduced and oxidized HSA. Using LC-ESITOFMS and FTMS analysis, we determined that the major structural change in oxidized HSA in healthy human plasma is a disulfide-bonded cysteine at the thiol of Cys34 of reduced HSA. Based on this structural information, we prepared standard samples of purified HSA, e.g. nonoxidized (intact purified HSA which mainly exists in reduced form), mildly oxidized and highly oxidized HSA. Using these standards, we demonstrated several differences in functional properties of HSA, including protease susceptibility, ligand-binding affinity and antioxidant activity. From these observations, we conclude that an increased level of oxidized HSA may impair HSA function in a number of pathological conditions. In addition, we determined blood and plasma sampling conditions for accurate measurement of the oxidized albumin ratio in plasma using EST-TOFMS screening.

The influence of medial reattachment of the torn cuff tendon for retracted rotator cuff tears.

We reattached the torn rotator cuff medial to the anatomic cuff insertion site if it was retracted. The purpose was to correlate the amount of medial reattachment of the cuff with shoulder function. We evaluated 63 shoulders in which repaired cuffs were followed with magnetic resonance imaging at a mean follow-up of 8 years. The amount of medial reattachment of the cuff tendon was determined by use of a T2-weighted oblique coronal view, which passed through the center of the humeral head, and was defined as the NCA angle (where N indicates the new cuff insertion point, C indicates the center of the humeral head, and A indicates the anatomic cuff insertion point). Theoretically, the more medially the cuff tendon was reattached, the greater the NCA angle. Neither the Japanese Orthopaedic Association shoulder score nor isometric strength of forward elevation was correlated with the NCA angle. The NCA angle was significantly correlated (P = .001) with the active forward elevation angle, which dramatically decreased at 30 degrees of the NCA angle, approximately 13 mm from the original cuff insertion point, assuming a humeral head radius of 25 mm.

High-affinity near-infrared fluorescent small-molecule contrast agents for in vivo imaging of prostate-specific membrane antigen.

Surgical resection remains a definitive treatment for prostate cancer. Yet, prostate cancer surgery is performed without image guidance for tumor margin, extension beyond the capsule and lymph node positivity, and without verification of other occult metastases in the surgical field. Recently, several imaging systems have been described that exploit near-infrared (NIR) fluorescent light for sensitive, real-time detection of disease pathology intraoperatively. In this study, we describe a high-affinity (9 nM), single nucleophile-containing, small molecule specific for the active site of the enzyme PSMA. We demonstrate production of a tetra-sulfonated heptamethine indocyanine NIR fluorescent derivative of this molecule using a high-yield LC/MS purification strategy. Interestingly, NIR fluorophore conjugation improves affinity over 20-fold, and we provide mechanistic insight into this observation. We describe the preparative production of enzymatically active PSMA using a baculovirus expression system and an adenovirus that co-expresses PSMA and GFP. We demonstrate sensitive and specific in vitro imaging of endogenous and ectopically expressed PSMA in human cells and in vivo imaging of xenograft tumors. We also discuss chemical strategies for improving performance even further. Taken together, this study describes nearly complete preclinical development of an optically based small-molecule contrast agent for image-guided surgery.

Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping.

The use of near-infrared or infrared photons is a promising approach for biomedical imaging in living tissue. This technology often requires exogenous contrast agents with combinations of hydrodynamic diameter, absorption, quantum yield and stability that are not possible with conventional organic fluorophores. Here we show that the fluorescence emission of type II quantum dots can be tuned into the near infrared while preserving absorption cross-section, and that a polydentate phosphine coating renders them soluble, disperse and stable in serum. We then demonstrate that these quantum dots allow a major cancer surgery, sentinel lymph node mapping, to be performed in large animals under complete image guidance. Injection of only 400 pmol of near-infrared quantum dots permits sentinel lymph nodes 1 cm deep to be imaged easily in real time using excitation fluence rates of only 5 mW/cm(2). Taken together, the chemical, optical and in vivo data presented in this study demonstrate the potential of near-infrared quantum dots for biomedical imaging.

Functional near-infrared fluorescence imaging for cardiac surgery and targeted gene therapy.

Cardiac revascularization is presently performed without real-time visual assessment of myocardial blood flow or perfusion. Moreover, gene therapy of the heart cannot, at present, be directed to specific territories at risk for myocardial infarction. We have developed a surgical imaging system that exploits the low autofluorescence, deep tissue penetration, low tissue scatter, and invisibility of near-infrared (NIR) fluorescent light. By completely isolating visible and NIR light paths, one is able to visualize, simultaneously, the anatomy and/or function of the heart, or any desired tissue. In rat model systems, we demonstrate that the heptamethine indocyanine-type NIR fluorophores IR-786 and the carboxylic acid form of IRDye78 can be injected intravenously in the living animal to provide real-time visual assessment of myocardial blood flow or perfusion intraoperatively. This imaging system may prove useful for the refinement of revascularization techniques, and for the administration of cardiac gene therapy.