Theranostic application of 64Cu/177Lu-labeled anti-Trop2 monoclonal antibody in pancreatic cancer tumor models.

PURPOSE: Pancreatic cancer is a malignant tumor with a high degree of malignancy, strong heterogeneity, and high lethality. Trop2 is a transmembrane glycoprotein associated with the occurrence, development, and poor prognosis of pancreatic cancer. This study aims to develop 64Cu/177Lu-labeled anti-Trop2 monoclonal antibody (hIMB1636) for positron emission tomography (PET) imaging and radioimmunotherapy (RIT) application in pancreatic cancer tumor models. METHODS: The binding kinetics of hIMB1636 to Trop2 antigen was measured by Biolayer interferometry (BLI). Western blotting was used to screen the Trop2 expression of pancreatic cancer cell lines. Flow cytometry and cell immunofluorescence were used to evaluate the binding ability of hIMB1636 and Trop2 on the cell surface. hIMB1636 were conjugated with p-SCN-Bn-NOTA (NOTA) and DOTA-NHS-ester (DOTA) for 64Cu and 177Lu radiolabeling respectively. ImmunoPET imaging and RIT studies were performed using 64Cu-NOTA-hIMB1636 and 177Lu-DOTA-hIMB1636 in subcutaneous pancreatic cancer tumor models. RESULTS: hIMB1636 had a strong binding affinity to Trop2 according to the results of BLI. The T3M-4 cell line showed the strongest expression of Trop2 and specific binding ability of hIMB1636 according to the results of Western blotting, flow cytometry, and cell immunofluorescence. The radiochemical purity of 64Cu-NOTA-hIMB1636 and 177Lu-DOTA-hIMB1636 exceeded 95%. PET imaging showed gradually an accumulation of 64Cu-NOTA-hIMB1636 in T3M-4 tumor models. The maximum tumor uptake was 8.95 ± 1.07%ID/g (n = 4) at 48 h post injection (p.i.), which had significant differences with T3M-4-blocked and PaTu8988-negative groups (P < 0.001). The high-177Lu-hIMB1636 group demonstrated the strongest tumor suppression with standardized tumor volume about 94.24 ± 14.62% (n = 5) at 14 days p.i., significantly smaller than other groups (P < 0.05). Ex vivo biodistribution and histological staining verified the in vivo PET imaging and RIT results. CONCLUSIONS: This study demonstrated that 64Cu/177Lu-labeled hIMB1636 could noninvasively evaluate the expression level of Trop2 and inhibit the Trop2-overexpressed tumor growth in pancreatic cancer tumor models. Further clinical evaluation and translation of Trop2-targeted drug may be of great help in the stratification and management of pancreatic cancer patients.

Novel 5-Fluorouracil Carbonate-Loaded Liposome: Preparation, In Vitro, and In Vivo Evaluation as an Antitumor Agent.

5-Fluorouracil (5-FU) is a chemotherapeutic drug against many types of cancers, especially colorectal cancer. However, its short plasma half-life and serious adverse reactions limit its wide clinical applications. To overcome these shortcomings, a novel lipophilic 5-FU carbonate [XL-01, (5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl) methyl tetradecyl carbonate] was designed, synthesized, and encapsulated into liposome (LipoXL-01) by a thin-film dispersion method through formulation screening and optimization. LipoXL-01 was characterized by a particle size of around 100 nm, polydispersity index of 0.200, ζ-potential value of -41 mV, encapsulation efficiency of 93.9%, and drug-loading efficiency of 11.6%. The cellular uptake of LipoXL-01 was increased in a concentration-dependent manner on HCT15 cells. LipoXL-01 could enhance the induction of cell apoptosis and the inhibition of cell migration and arrest the ability of the cell cycle at the S-phase on HCT15 cells better than 5-FU. Additionally, LipoXL-01 exhibited a slow drug release profile with a cumulative release rate of 12% in 8 h. The results of pharmacokinetic and biodistribution studies revealed that LipoXL-01 had a long plasma half-life (7.21 h) and a high tumor accumulation (733 nmol/g at 8 h). The in vivo antitumor effect study also showed that LipoXL-01 had more potent efficacy than 5-FU (65 vs 48% of the tumor-inhibition rate). Simultaneously, negligible systemic toxicity was observed via analyzing the body weight as well as hematological and pathological parameters in the tested mice. The current study suggested that LipoXL-01 might be a promising nanocandidate for chemotherapy of colorectal cancer.

EGFR-targeting, ß-defensin-tailored fusion protein exhibits high therapeutic efficacy against EGFR-expressed human carcinoma via mitochondria-mediated apoptosis.

Defensins play an essential role in innate immunity. In this study, a novel recombinant ß-defensin that targets the epidermal growth factor receptor (EGFR) was designed and prepared. The EGFR-targeting ß-defensin consists of an EGF-derived oligopeptide (Ec), a ß-defensin-1 peptide (hBD1) and a lidamycin-derived apoprotein (LDP), which serves as the "scaffold" for the fusion protein (Ec-LDP-hBD1). Ec-LDP-hBD1 effectively bound to EGFR highly expressed human epidermoid carcinoma A431 cells. The cytotoxicity of Ec-LDP-hBD1 to EGFR highly expressed A431 cells was more potent than that to EGFR low-expressed human lung carcinoma A549 and H460 cells (the IC50 values in A431, A549, and H460 cells were 1.8 ± 0.55, 11.9 ± 0.51, and 5.19 ± 1.21 µmol/L, respectively); in addition, the cytotoxicity of Ec-LDP-hBD1 was much stronger than that of Ec-LDP and hBD1. Moreover, Ec-LDP-hBD1 suppressed cancer cell proliferation and induced mitochondria-mediated apoptosis. Its in vivo anticancer action was evaluated in athymic mice with A431 and H460 xenografts. The mice were administered Ec-LDP-hBD1 (5, 10 mg/kg, i.v.) two times with a weekly interval. Administration of Ec-LDP-hBD1 markedly inhibited the tumor growth without significant body weight changes. The in vivo imaging further revealed that Ec-LDP-hBD1 had a tumor-specific distribution with a clear image of localization. The results demonstrate that the novel recombinant EGFR-targeting ß-defensin Ec-LDP-hBD1 displays both selectivity and enhanced cytotoxicity against relevant cancer cells by inducing mitochondria-mediated apoptosis and exhibits high therapeutic efficacy against the EGFR-expressed carcinoma xenograft. This novel format of ß-defensin, which induces mitochondrial-mediated apoptosis, may play an active role in EGFR-targeting cancer therapy.

A new TROP2-targeting antibody-drug conjugate shows potent antitumor efficacy in breast and lung cancers.

Trophoblast cell surface antigen 2 (Trop2) is considered to be an attractive therapeutic target in cancer treatments. We previously generated a new humanized anti-Trop2 antibody named hIMB1636, and designated it as an ideal targeting carrier for cancer therapy. Lidamycin (LDM) is a new antitumor antibiotic, containing an active enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). AE and LDP can be separated and reassembled, and the reassembled LDM possesses cytotoxicity similar to that of native LDM; this has made LDM attractive in the preparation of gene-engineering drugs. We herein firstly prepared a new fusion protein hIMB1636-LDP composed of hIMB1636 and LDP by genetic engineering. This construct showed potent binding activities to recombinant antigen with a KD value of 4.57 nM, exhibited binding to Trop2-positive cancer cells and internalization and transport to lysosomes, and demonstrated powerful tumor-targeting ability in vivo. We then obtained the antibody-drug conjugate (ADC) hIMB1636-LDP-AE by molecular reconstitution. In vitro, hIMB1636-LDP-AE inhibited the proliferation, migration, and tumorsphere formation of tumor cells with half-maximal inhibitory concentration (IC50) values at the sub-nanomolar level. Mechanistically, hIMB1636-LDP-AE induced apoptosis and cell-cycle arrest. In vivo, hIMB1636-LDP-AE also inhibited the growth of breast and lung cancers in xenograft models. Moreover, compared to sacituzumab govitecan, hIMB1636-LDP-AE showed more potent antitumor activity and significantly lower myelotoxicity in tumors with moderate Trop2 expression. This study fully revealed the potent antitumor efficacy of hIMB1636-LDP-AE, and also provided a new preparation method for LDM-based ADC, as well as a promising candidate for breast cancer and lung cancer therapeutics.

Elucidating the development, characterization, and antitumor potential of a novel humanized antibody against Trop2.

Trophoblast cell surface antigen 2 (Trop2) has emerged as a potential target for effective cancer therapy. In this study, we report a novel anti-Trop2 antibody IMB1636, developed using hybridoma technology. It exhibited high affinity and specificity (KD = 0.483 nM) in binding both antigens and cancer cells, as well as human tumor tissues. hIMB1636 could induce endocytosis, and enabled targeted delivery to the tumor site with an in vivo retention time of 264 h. The humanized antibody hIMB1636, acquired using CDR grafting, exhibited the potential to directly inhibit cancer cell proliferation and migration, and to induce ADCC effects. Moreover, hIMB1636 significantly inhibited the growth of MDA-MB-468 xenograft tumors in vivo. Mechanistically, hIMB1636 induced cell cycle arrest and apoptosis by regulating cyclin-related proteins and the caspase cascade. In comparison to commercialized sacituzumab, hIMB1636 recognized a conformational epitope instead of a linear one, bound to antigen and cancer cells with similar binding affinity, induced significantly more potent ADCC effects against cancer cells, and displayed superior antitumor activities both in vitro and in vivo. The data presented in this study highlights the potential of hIMB1636 as a carrier for the formulation of antibody-based conjugates, or as a promising candidate for anticancer therapy.

hIMB1636-MMAE, a Novel TROP2-Targeting Antibody-Drug Conjugate Exerting Potent Antitumor Efficacy in Pancreatic Cancer.

Herein, we first prepared a novel anti-TROP2 antibody-drug conjugate (ADC) hIMB1636-MMAE using hIMB1636 antibody chemically coupled to monomethyl auristatin E (MMAE) via a Valine-Citrulline linker and then reported its characteristics and antitumor activity. With a DAR of 3.92, it binds specifically to both recombinant antigen (KD ∼ 0.687 nM) and cancer cells and could be internalized by target cells and selectively kill them with IC50 values at nanomolar/subnanomolar levels by inducing apoptosis and G2/M phase arrest. hIMB1636-MMAE also inhibited cell migration, induced ADCC effects, and had bystander effects. It displayed significant tumor-targeting ability and excellent tumor-suppressive effects in vivo, resulting in 5/8 tumor elimination at 12 mg/kg in the T3M4 xenograft model or complete tumor disappearance at 10 mg/kg in BxPc-3 xenografts in nude mice. Its half-life in mice was about 87 h. These data suggested that hIMB1636-MMAE was a promising candidate for the treatment of pancreatic cancer with TROP2 overexpression.

Antitumor efficacy of the scFv-based fusion protein and its enediyne-energized analogue directed against epidermal growth factor receptor.

Epidermal growth factor receptor (EGFR), overexpressed in many epithelial tumors, plays important roles in the formation and the development of tumors, and thus it is regarded as a promising target for cancer therapy. Single-chain variable fragment (scFv), an engineered antibody fragment, is generally used for constructing antibody-targeted drugs, owing to its low immunogenicity and high penetration capability into solid tumors. A fusion protein ER(Fv-LDP), consisting of an anti-EGFR scFv and the apoprotein (LDP) of lidamycin (LDM), was prepared and then assembled with the active chomophore [active enediyne (AE)] of LDM to generate enediyne-energized analogue ER(Fv-LDP-AE). The fusion protein ER(Fv-LDP) bound specifically to EGFR-overexpressing cancer cells and internalized into the cytoplasm through receptor-mediated endocytosis. ER(Fv-LDP) possessed cytotoxicity against carcinoma cell lines, which was hundreds of times more potent than the separate moiety of ER(Fv) and LDP. The enediyne-energized fusion protein ER(Fv-LDP-AE) also showed stronger cytotoxicity to target-relevant cancer cells than LDM in vitro. In human epidermoid carcinoma A431 xenografts, ER(Fv-LDP) presented higher antitumor efficacy than that of ER(Fv), LDP, and their mixture, with tumor growth inhibition rates of 63.6, 46.7, 48.5, and 49.9%, respectively. The enediyne-energized fusion protein ER(Fv-LDP-AE) at a dose of 0.4 mg/kg inhibited tumor growth by 89.2%, while no significant body weight loss was seen in treated animals. The results show that an anti-EGFR scFv-based fusion protein and its enediyne-energized analogue can be prepared by DNA recombination and molecular reconstitution. Both ER(Fv-LDP) and ER(Fv-LDP-AE) are effective against EGFR-overexpressing cancer xenograft in athymic mice. An integrated technical platform for scFv-based enediyne-energized fusion proteins has been established.

[An overview of antibody-based cancer therapy].

The use of monoclonal antibodies (mAbs) for cancer therapy has achieved considerable success in recent years. Approximate 17 monoclonal antibodies have been approved as cancer therapeutics since 1997. Antibody-drug conjugates (ADC) are powerful new treatment options for cancer, and naked antibodies have recently achieved remarkable success. The safety and effectiveness of therapeutic mAbs in oncology vary depending on the nature of the target antigen and the mechanisms of tumor cell killing. This review provides a summary of the current state of antibody-based cancer therapy, including the mechanisms of tumor cell killing by antibodies, tumor antigens as antibody targets, clinical effectiveness of antibodies in cancer patients and nanoparticles-based ADCs.

Rational Design and Systemic Appraisal of an EGFR-Targeting Antibody-Drug Conjugate LR-DM1 for Pancreatic Cancer.

By harnessing the payload DM1 and a monoclonal antibody LR004 through a noncleavable linker succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate, we designed and evaluated an antibody-drug conjugate LR-DM1 with an appropriate drug-antibody ratio of 3.6. LR-DM1, which was targeted toward the epidermal growth factor receptor for pancreatic cancer, exhibited potent antiproliferation activity in vitro with a half-maximal inhibitory concentration value of 7.03 nM for Capan-2 cells. Particularly, it displayed prominent tumor growth inhibition in vivo under 20 mg/kg LR-DM1 dosage in a single administration or multiple administrations without apparent abnormality of pathological observation. Moreover, LR-DM1 possessed a relatively broad therapeutic index with a half-lethal dose above 300 mg/kg, which was over 15-fold higher than the highest administration dosage of 20 mg/kg. This initial study on LR-DM1 holds promise for further development of a new antibody drug conjugate that is transformative for treatment of patients concerned.

The recombinant defensin/HSA fusion protein that inhibits NF-κb associated with intensive macropinocytosis shows potent efficacy against pancreatic cancer.

KRAS mutation and NF-κB both play crucial role in pancreatic cancer; in addition, defensin, the peptide mediator in innate immunity, can inhibit NF-κB. Assuming a strategy that targets both NF-κB and concomitantly the mutated KRAS indirectly via intensive macropinocytosis, we designed and generated a recombinant protein DF2-HSA which consists of two molecules of human beta-defensin 2 (HBD2) and a moiety of human serum albumin (HSA). As shown, the recombinant protein DF2-HSA markedly down-regulated NF-κB in both KRAS mutant MIA PaCa-2 cells and wild type BxPC-3 cells. Determined by confocal microscopy, the uptake of DF2-HSA in MIA PaCa-2 cells was more intense than that in BxPC-3 cells. The uptake was blocked by the specific inhibitor EIPA, indicating that DF2-HSA internalized via macropinocytosis. DF2-HSA displayed more potent cytotoxicity to cancer cells than HBD2. DF2-HSA induced apoptosis in cancer cells. Notably, DF2-HSA inhibited tumor cell spheroid formation, an effect comparable to that of salinomycin. DF2-HSA inhibited tumor cell migration and invasion. As detected with scanning electron microscopy, DF2-HSA strongly depleted filopodia on cell surface; and salinomycin induced similar changes. By in vivo imaging, DF2-HSA displayed intense tumor-site accumulation and lasting retention for over 14 days; however, HBD2 showed much less tumor-site accumulation and a shorter retention time for only 24 h. DF2-HSA suppressed the growth of pancreatic cancer MIA PaCa-2 xenograft in athymic mice; and its combination with gemcitabine achieved higher antitumor efficacy. In summary, the recombinant defensin/HSA fusion protein that inhibits NF-κb associated with intensive macropinocytosis is highly effective against pancreatic cancer.

Development of a novel multi-functional integrated bioconjugate effectively targeting K-Ras mutant pancreatic cancer.

Folate receptor (FR) overexpression occurs in a variety of cancers, including pancreatic cancer. In addition, enhanced macropinocytosis exists in K-Ras mutant pancreatic cancer. Furthermore, the occurrence of intensive desmoplasia causes a hypoxic microenvironment in pancreatic cancer. In this study, a novel FR-directed, macropinocytosis-enhanced, and highly cytotoxic bioconjugate folate (F)-human serum albumin (HSA)-apoprotein of lidamycin (LDP)-active enediyne (AE) derived from lidamycin was designed and prepared. F-HSA-LDP-AE consisted of four moieties: F, HSA, LDP, and AE. F-HSA-LDP presented high binding efficiency with the FR and pancreatic cancer cells. Its uptake in wild-type cells was more extensive than in K-Ras mutant-type cells. By in vivo optical imaging, F-HSA-LDP displayed prominent tumor-specific biodistribution in pancreatic cancer xenograft-bearing mice, showing clear and lasting tumor localization for 360 h. In the MTT assay, F-HSA-LDP-AE demonstrated potent cytotoxicity in three types of pancreatic cancer cell lines. It also induced apoptosis and caused G2/M cell cycle arrest. F-HSA-LDP-AE markedly suppressed the tumor growth of AsPc-1 pancreatic cancer xenografts in athymic mice. At well-tolerated doses of 0.5 and 1 mg/kg, (i.v., twice), the inhibition rates were 91.2% and 94.8%, respectively (P<0.01). The results of this study indicate that the F-HSA-LDP multi-functional bioconjugate might be effective for treating K-Ras mutant pancreatic cancer.

Promoted antitumor therapy on pancreatic cancer by a novel recombinant human albumin-bound miriplatin nanoparticle.

Pancreatic cancer is an aggressive and highly lethal disease with a very poor prognosis. Our previous study found miriplatin can inhibit proliferation of various tumor cells, including pancreatic cancer cells. For the chemotherapy of pancreatic cancer, a novel recombinant human serum albumin (rHSA)-bound miriplatin nanoparticles (rHSA-miPt) were constructed by emulsion-diffusion evaporation method. The optimal formulation was composed of 150 mg of rHSA and 30 mg of miriplatin. The key parameters in rHSA-miPt production were 10 min of high-pressure homogenization in a solution with volume ratio of 10:2 of 5% glucose and chloroform. The rHSA-miPt was characterized with a particle size of 61 ± 10 nm, a zeta potential value of -18 ± 5 mV, encapsulation efficiency of 98.4%, drug loading of 16.4%, T1/2 of 13.3 h and Vd of 0.5 L in Sprague Dawley rats. The concentrations of platinum (Pt) in the tumors were 15 and 22-fold higher than those in the blood at 24 and 72 h in tumor-bearing mice, respectively. The internalization of rHSA-miPt through caveolae-dependent pathway. In vitro, the half-maximal inhibitory concentration (IC50) of rHSA-miPt was 12.7 µM vs more than 100 µM of gemcitabine (Gem). The inhibition rate of tumor growth was 76% of rHSA-miPt and 51% of Gem, respectively. Compared with Gem, rHSA-miPt was identified to be safer and less toxic based on body weight loss in mice (0% vs 20%), the survival rate of mice (100% vs 80%) and hematological and biochemical parameters of the mice including leukocytes, lymphocytes, neutrophils, monocytes, serum alanine aminotransferase and aspartate aminotransferase. The present study revealed that rHSA-miPt might be a promising candidate for pancreatic cancer therapy.

Excellent effects and possible mechanisms of action of a new antibody-drug conjugate against EGFR-positive triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive subtype and occurs in approximately 15-20% of diagnosed breast cancers. TNBC is characterized by its highly metastatic and recurrent features, as well as a lack of specific targets and targeted therapeutics. Epidermal growth factor receptor (EGFR) is highly expressed in a variety of tumors, especially in TNBC. LR004-VC-MMAE is a new EGFR-targeting antibody-drug conjugate produced by our laboratory. This study aimed to evaluate its antitumor activities against EGFR-positive TNBC and further studied its possible mechanism of antitumor action. METHODS: LR004-VC-MMAE was prepared by coupling a cytotoxic payload (MMAE) to an anti-EGFR antibody (LR004) via a linker, and the drug-to-antibody ratio (DAR) was analyzed by HIC-HPLC. The gene expression of EGFR in a series of breast cancer cell lines was assessed using a publicly available microarray dataset (GSE41313) and Western blotting. MDA-MB-468 and MDA-MB-231 cells were treated with LR004-VC-MMAE (0, 0.0066, 0.066, 0.66, 6.6 nmol/L), and the inhibitory effects of LR004-VC-MMAE on cell proliferation were examined by CCK-8 and colony formation. The migration and invasion capacity of MDA-MB-468 and MDA-MB-231 cells were tested at different LR004-VC-MMAE concentrations (2.5 and 5 nmol/L) with wound healing and Transwell invasion assays. Flow cytometric analysis and tumorsphere-forming assays were used to detect the killing effects of LR004-VC-MMAE on cancer stem cells in MDA-MB-468 and MDA-MB-231 cells. The mouse xenograft models were also used to evaluate the antitumor efficacy of LR004-VC-MMAE in vivo. Briefly, BALB/c nude mice were subcutaneously inoculated with MDA-MB-468 or MDA-MB-231 cells. Then they were randomly divided into 4 groups (n = 6 per group) and treated with PBS, naked LR004 (10 mg/kg), LR004-VC-MMAE (10 mg/kg), or doxorubicin, respectively. Tumor sizes and the body weights of mice were measured every 4 days. The effects of LR004-VC-MMAE on apoptosis and cell cycle distribution were analyzed by flow cytometry. Western blotting was used to detect the effects of LR004-VC-MMAE on EGFR, ERK, MEK phosphorylation and tumor stemness marker gene expression. RESULTS: LR004-VC-MMAE with a DAR of 4.02 were obtained. The expression of EGFR was found to be significantly higher in TNBC cells compared with non-TNBC cells (P < 0.01). LR004-VC-MMAE inhibited the proliferation of EGFR-positive TNBC cells, and the IC50 values of MDA-MB-468 and MDA-MB-231 cells treated with LR004-VC-MMAE for 72 h were (0.13 ± 0.02) nmol/L and (0.66 ± 0.06) nmol/L, respectively, which were significantly lower than that of cells treated with MMAE [(3.20 ± 0.60) nmol/L, P < 0.01, and (6.60 ± 0.50) nmol/L, P < 0.001]. LR004-VC-MMAE effectively inhibited migration and invasion of MDA-MB-468 and MDA-MB-231 cells. Moreover, LR004-VC-MMAE also killed tumor stem cells in EGFR-positive TNBC cells and impaired their tumorsphere-forming ability. In TNBC xenograft models, LR004-VC-MMAE at 10 mg/kg significantly suppressed tumor growth and achieved complete tumor regression on day 36. Surprisingly, tumor recurrence was not observed until the end of the experiment on day 52. In a mechanistic study, we found that LR004-VC-MMAE significantly induced cell apoptosis and cell cycle arrest at G2/M phase in MDA-MB-468 [(34 ± 5)% vs. (12 ± 2)%, P < 0.001] and MDA-MB-231 [(27 ± 4)% vs. (18 ± 3)%, P < 0.01] cells. LR004-VC-MMAE also inhibited the activation of EGFR signaling and the expression of cancer stemness marker genes such as Oct4, Sox2, KLF4 and EpCAM. CONCLUSIONS: LR004-VC-MMAE showed effective antitumor activity by inhibiting the activation of EGFR signaling and the expression of cancer stemness marker genes. It might be a promising therapeutic candidate and provides a potential therapeutic avenue for the treatment of EGFR-positive TNBC.

Synthesis, characterization, and targeted chemotherapy of SCT200-linker-monomethyl auristatin E conjugates.

Antibody-drug conjugates (ADCs) are currently among the most successful and important strategies for treating patients with solid tumors. ADCs are composed of a monoclonal antibody and warhead, which are conjugated via a linker. Currently, monomethyl auristatin E (MMAE) is the most widely applied warhead in the development of ADCs. However, MMAE-based ADCs are generally constructed using the MC-VC-PABC linker, and this design has limited structural diversity and some disadvantages. Accordingly, in this study, we generated three types of novel linker-MMAE (with alterations in the spacer, catabolizing area, and self-immolative compared with MC-VC-PABC-MMAE) in ADCs, termed SCT200-linker-MMAE conjugates, and then evaluated the linker-drug plasma stability and the rate of drug release by cathepsin B. The binding ability, internalization rates, and efficacy of all SCT200-linker-MMAE ADCs were systematically studied, and the expression of apoptosis-associated proteins and the therapeutic efficacies of SCT200-M-2, -C-2, and -C-4 were evaluated. The results showed that the activities of some of these ADCs were increased for epidermal growth factor receptor-positive tumors. Moreover, the novel linkers designed in this study can be linked with other antibodies to treat other types of cancer. Overall, these findings provide important insights into the application of SCT200-based linkers in ADCs.

Preliminary biological evaluation of 123I-labelled anti-CD30-LDM in CD30-positive lymphomas murine models.

Overexpression of CD30 has been reported on the surface of some T-cell lymphomas, especially on Hodgkin's lymphoma (HL) and anaplastic large cell lymphoma (ALCL). CD30 targeted immunotherapy has good clinical therapy response. We have produced a novel antibody drug conjugates (ADCs)-anti-CD30-LDM, which shows attractive tumour-targeting capability and extremely potent antitumor efficacy. To further investigate biological characteristics and promote clinical translation of anti-CD30-LDM, we constructed a radiolabeled 123I-anti-CD30-LDM to evaluate the biodistribution characteristics. The anti-CD30-LDM was radioiodinated by the Iodogen method. The radiochemical purity of 123I-anti-CD30-LDM was more over 98%, and the specific activity of 240.5 MBq/mg. The stability and the specificity of 123I-anti-CD30-LDM were evaluated in vitro. Cellular binding assays were used to evaluate the binding capabilities in CD30-positive Karpas299 cells and CD30-negative Raji cells. B-NDG mice bearing Karpas 299 and Raji xenografts were used for in vivo biodistribution studies. Our results demonstrated that anti-CD30-LDM as an ideal ADC targeted to CD30, which was labelled easily with 123I and obtained the sufficient yields. The 123I-anti-CD30-LDM preserved specific binding to CD30 in vitro and uptake in tumour xenografts in B-NDG mice. These results are encouraging for anti-CD30-LDM as a promising clinical translational candidate for various CD30 positive lymphomas and other diseases.

Dexamethasone enhances the antitumor efficacy of Gemcitabine by glucocorticoid receptor signaling.

Gemcitabine (Gem) is currently used as the first-line therapy for liver and pancreatic cancer but has limited efficacy in most cases. Dexamethasone (Dex) have been applied as a chemoprotectant and chemosensitizer in cancer chemotherapy. This study further explored the potential of combination of Gem and Dex and tested the hypothesis that glucocorticoid receptor signaling is essential for the synergistic antitumor activity. In the HepG2 and AsPC-1 xenograft models, the combination treatment showed a significantly synergistic antitumor activity. Immunohistochemistry of post-treatment tumors showed a significant decrease in proliferation and angiogenesis as compared to either of the treatments alone. Dex alone and the combination with Gem inhibited the expression of glucocorticoid receptor. The combination of Dex and Gem showed synergistic cytotoxicity in cell lines in vitro. The antiproliferative synergism is prevented by used glucocorticoid receptor (GR) small interfering RNA, demonstrating that the glucocorticoid receptor is required for the antiproliferative synergism of Gem and Dex. The inhibition of glucocorticoid receptor signaling pathway and induction of apoptosis via activation of caspases 3, 8 and 9, PARP, contributed to the synergistic effect of this combination therapy. These results demonstrate that Dex could potentiate the antitumor efficacy of Gem. The synergistic antitumor activity of the combination of Dex and Gem was through glucocorticoid receptor signaling. Taken together, a combination of Dex and Gem shows a significant synergistic antitumor activity and lesser toxicity both in vitro and in vivo and could be a combination chemotherapy for the treatment of highly expression of glucocorticoid receptor patients.

[Construction and screening of phage antibody libraries against epidermal growth factor receptor and soluble expression of single chain Fv].

Recent studies have shown that epidermal growth factor receptor (EGFR) is an important target for cancer therapy. The present study prepared single chain Fv (scFv) directed against EGFR. Balb/c mice were immunized by human carcinoma A431 cells, and total RNA of the splenic cells was extracted. VH and VL gene fragments were amplified by RT-PCR and further joined into scFv gene with a linker, then scFv gene fragments were ligated into the phagemid vector pCANTAB 5E. The phagemid containing scFv were transformed into electro-competent E. coli TG1 cells. The recombinant phage antibody library was constructed through rescuing the transformed cells with help phage M13K07. The specified recombinant phages were enriched through 5 rounds of affinity panning and the anti-EGFR phage scFv clones were screened and identified with ELISA. A total of 48 clones from the library were selected randomly and 45 clones were identified positive. After infecting E. coli HB2151 cells with one positive clone, soluble recombinant antibodies about 27 kD were produced and located in the periplasm and the supernatant. The result of sequencing showed that the scFv gene was 768 bp, which encoded 256 amino acid residues. VH and VL including 3 CDRs and 4 FRs, respectively, were all homologous to mouse Ig. The soluble scFv showed the specific binding activity to purified EGFR and EGFR located in carcinoma cell membrane. The successful preparation of anti-EGFR scFv will provide an EGFR targeted molecule for the development of antibody-based drugs and biological therapy of cancer.

[Anti-type IV collagenase single-chain antibody ameliorates bleomycin-induced experimental pulmonary fibrosis in mice].

OBJECTIVE: To study the effect of anti-type IV collagenase single-chain antibody scFv (3G11) on bleomycin (BLM)-induced pulmonary fibrosis. METHODS: C57BL/6 mice were divided into six groups (blank, saline control, BLM, low-dose treatment, intermediate-dose treatment, and high-dose treatment), the animal model of pulmonary fibrosis was induced with 400 ng/g intratracheal BLM. The day that treatment groups were injected with BLM was marked Day 0. Then at Days 1 - 7, scFv (3G11) was injected once intraperitoneally each day with three dosage [low-dose treatment (15 microg/g), intermediate-dose treatment (30 microg/g) and high-dose treatment (45 microg/g)]. At Day 21, 6 mice of each group (saline control, BLM, intermediate-dose treatment) were sacrificed and pathomorphological changes of left lungs evaluated with HE stained sections. Meanwhile, 6 mice of each group (blank, saline control, BLM, low-dose treatment, intermediate-dose treatment, and high-dose treatment) were sacrificed, the content of hydroxyproline was detected to measure the degree of collagen deposition. Macrophages were extracted from murine abdominal cavity and primarily cultured. And the inhibition of matrix metalloproteinase (MMP)-2 and MMP-9 excretion by different concentrations of scFv (3G11) (0, 15, 30, 45, and 60 micromol/L) was determined by gelatin zymography. RESULTS: By image analysis, the degree of fibrosis in the lungs of treated group was lighter than that of the BLM group. The percentages of the area of interalveolar septum (45.3% +/- 3.2%) and the number of nucleated cells (451 +/- 47) of treated group were remarkably reduced as compared with the BLM group (59.0% +/- 3.0%, 599 +/- 42, both P < 0.01). The content of hydroxyproline of the intermediate-dose treatment group [(0.82 +/- 0.05) microg/mg] and high-dose treatment group [(0.80 +/- 0.03) microg/mg] was lower than that of the BLM group [(0.92 +/- 0.07) microg/mg, P < 0.05, P < 0.01]. The result of gelatin zymography demonstrated that the excretion of MMP-2 and MMP-9 by macrophage was inhibited by scFv (3G11) at the concentration of 15 micromol/L. CONCLUSION: Single-chain antibody directed against type IV collagenase might inhibit the development of bleomycin-induced pulmonary fibrosis.

Crizotinib enhances anti-CD30-LDM induced antitumor efficacy in NPM-ALK positive anaplastic large cell lymphoma.

Combining antibody-drug conjugates (ADCs) with targeted small-molecule inhibitors can enhance antitumor effects beyond those attainable with monotherapy. In this study, we investigated the therapeutic combination of a CD30-targeting ADC (anti-CD30-lidamycin [LDM]) with a small-molecule inhibitor (crizotinib) of nucleophosmin-anaplastic lymphoma kinase NPM-ALK in CD30+/ALK+ anaplastic large cell lymphoma (ALCL). In vitro, anti-CD30-LDM showed strong synergistic antiproliferative activity when combined with crizotinib. Furthermore, treatment with anti-CD30-LDM plus crizotinib resulted in a stronger induction of cell apoptosis than monotherapy with either treatment. Western blot analysis revealed that ERK1/2 phosphorylation was increased in response to anti-CD30-LDM-induced DNA damage. Interestingly, the addition of crizotinib inhibited the expression of phosphorylated ERK1/2 and further augmented anti-CD30-LDM-mediated apoptosis, providing a potential synergistic mechanism for DNA-damaging agents combined with NPM-ALK inhibitors. In Karpas299 and SU-DHL-1 xenograft models, anti-CD30-LDM plus crizotinib was more effective in inhibiting tumor growth than either treatment alone. This research demonstrated for the first time that the combination of anti-CD30-LDM and crizotinib exhibits a synergistic inhibitory effect in tumor cells. These results provide scientific support for future clinical evaluations of anti-CD30-LDM, or other DNA-damaging agents, combined with NPM-ALK inhibitors.

An EGFR-targeting antibody-drug conjugate LR004-VC-MMAE: potential in esophageal squamous cell carcinoma and other malignancies.

Epidermal growth factor receptor (EGFR) is a rational target for cancer therapy, because its overexpression plays an important oncogenic role in a variety of solid tumors; however, EGFR-targeted antibody-drug conjugate (ADC) therapy for esophageal squamous cell carcinoma (ESCC) is exceedingly rare. LR004 is a novel anti-EGFR antibody with the advantages of improved safety and fewer hypersensitivity reactions. It may be of great value as a carrier in ADCs with high binding affinity and internalization ability. Here, we prepared an EGFR-targeting ADC, LR004-VC-MMAE, and evaluated its antitumor activities against ESCC and EGFR-positive cells. LR004 was covalently conjugated with monomethyl auristatin E (MMAE) via a VC linker by antibody interchain disulfide bond reduction. VC-MMAE was conjugated with LR004 with approximately 4.0 MMAE molecules per ADC. LR004-VC-MMAE showed a potent antitumor effect against ESCC and other EGFR-positive cells with IC50 values of nM concentrations in vitro. The in vivo antitumor effects of LR004-VC-MMAE were investigated in ESCC KYSE520 and A431 xenograft nude mice models. Significant activity was seen at 5 mg·kg-1 , and complete tumor regression was observed at 15 mg·kg-1 in the KYSE520 xenograft nude mice after four injections, while the naked antibody LR004 had little effect on inhibiting tumor growth. Similar promising results were obtained in the A431 models. In addition, the tumors also remained responsive to LR004-VC-MMAE for large tumor experiments (tumor volume 400-500 mm3 ). The study results demonstrated that LR004-VC-MMAE could be a potential therapeutic agent for ESCC and other EGFR-expressing malignancies. We also evaluated PK profile of LR004-VC-MMAE ADC in the mice model, which would provide qualitative guiding significance for the further research.