Comparative study of pituitary and bacteria-derived human growth hormone by monoclonal antibodies

We have established hybridoma lines which secrete mouse monoclonal antibodies (Mabs) to human pituitary growth hormone, hGH. Using indirect competitive ELISA and indirect passive hemagglutination inhibition twelve different Mabs were characterized with regard to cross-reactivity with the hGH-related hormones, human chorionic somatomammotropin, hCS, and human prolactin, hPRL. The reactivity of these Mabs with pituitary hGH was compared to that with either bacterially-produced methionyl-hGH or to that of reduced and S-carboxymethylated hGH, which has an altered conformation. None of the Mabs reacted with hPRL. Four did not react with hCS whereas the others showed varying degree of cross-reactivity with hCS. All Mabs reacted more weakly with reduced and S-carboxymethylated hGH than with the native form of the hormone, which was not seen with conventional rabbit antisera to hGH. Thus in the case of hGH the Mabs are superior to conventional antisera in revealing small conformational differences. However the pituitary and bacterially-derived methionyl-hGH were indistinguishable as determined by the 12 Mabs.     

Demonstration of T4, T8, M1 and B7 determinants on human T cells with a rosette test: implications for the assay specificity of monoclonal antibodies

Under optimal test conditions significantly more freshly isolated human T cells reacted with OKT4, OKT8, OKM1 and OKB7 monoclonal antibodies (Mabs) in the indirect antiglobulin rosetting reaction (IARR) than by indirect immunofluorescence. Rabbit erythrocytes (E) coated with anti-mouse immunoglobulin were more sensitive indicator cells in the IARR than similarly coated sheep E. Treatment of T cells with neuraminidase further enhanced T cell reactivity in the IARR with each Mab so that an average of 60% or more of T cells were T4+, T8+ and M1+ and at least 40% had the T4+ T8+ phenotype. The various findings suggest that the rosette assay detects determinants on T cells that are expressed below the detection threshold of immunofluorescence. Moreover, these findings indicate that the cellular specificities of a particular Mab may change when one assay system is substituted for another or when the protocol of a particular assay is altered.     

Detection of non-HLA-DR determinants by alloreactive lymphocyte clones

Using the soft-agar colony assay, we have generated three MT3-associated clones: HJ1, HJ13, and HJ39, from an MLR combination of two unrelated individuals. Another clone, HJ37, appeared to recognize a novel HLA-D determinant. PLT inhibition studies with monoclonal anti-Ia-like antibodies (Mab) were conducted on clones HJ1, HJ39, and HJ37. Five different anti-DR Mab had no significant inhibitory effect on these clones. On the other hand, two Mab SG171 and Q5/13 which appear to react with DR and MT3 (I-A like) molecules strongly inhibited the two MT3-specific PLT clones. While SG171 and Q5/13 had little effect on HJ37, it was observed that a polymorphic Mab 17.15 had a strong inhibitory effect. These results, in concordance with biochemical data on Ia molecules precipitated by these Mab, suggest that these alloreactive clones may recognize non-DR PLT determinants. They also provide further indirect support that MT3 molecules represent the human homologue of murine I-A molecules.     

Fungal infections in solid organ transplantation: An update on diagnosis and treatment

Invasive fungal infections constitute an important cause of morbidity and mortality in solid organ transplantation recipients. Since solid organ transplantation is an effective therapy for many patients with end-stage organ failure, prevention and treatment of fungal infections are of vital importance. Diagnosis and management of these infections, however, remain difficult due to the variety of clinical symptoms in addition to the lack of accurate diagnostic methods. The use of fungal biomarkers can lead to an increased diagnostic accuracy, resulting in improved clinical outcomes. The evidence for optimal prophylactic approaches remains inconclusive, which results in considerable variation in the administration of prophylaxis. The implementation of a standard protocol for prophylaxis remains difficult as previous treatment regimens, which can alter the distribution of different pathogens, affect the outcome of antifungal susceptibility testing. Furthermore, the increasing use of antifungals also contributes to incremental costs and the risk of development of drug resistance. This review will highlight risk factors, clinical manifestations and timing of fungal infections and will focus predominately on the current evidence for diagnosis and management of fungal infections.     

Production, Binding and Cytotoxicity of Human/Mouse Chimeric Monoclonal Antibody-Neocarzinostatin Conjugate

A human/mouse chimeric Fab monoclonal antibody A7 (chFabA7) was covalently coupled to neocarzinostatin (NCS) by the SPDP method at various chFabA7:NCS substitution ratios. The antigen-binding activity of the conjugate, examined by ELISA using fixed antigen-positive colon cancer cells, was identical to that of the parent chFabA7 when one mole of NCS was conjugated, but was reduced with 2 or 3 moles of conjugated NCS. By means of a colony-forming assay, the cytocidal effect of the conjugate on antigen-positive cancer cells was found to be stronger than that of free NCS, whereas in antigen-negative cancer cells it was similar to that of free NCS. This effect was attenuated by adding an excess amount of monoclonal antibody A7. These findings indicate that the conjugate has an antigen-specific cytocidal action, and thus chFabA7-NCS is a promising tool for targeting cancer chemotherapy.     

Evaluation of the binding of the tricyclic isoxazole photoaffinity label LY475776 to multidrug resistance associated protein 1 (MRP1) orthologs and several ATP- binding cassette (ABC) drug transporters

Several of the ATP-binding cassette (ABC) transporters confer resistance to anticancer agents and/or antiviral agents when overexpressed in drug-sensitive cells. Recently a MRP1 (ABCC1) tricyclic isoxazole inhibitor, LY475776 was shown to be a glutathione-dependent photoaffinity label of human MRP1 and showed poor labeling of murine mrp1, an ortholog that does not confer anthracycline resistance. In the present study, the specificity of LY475776 was examined for its ability to modulate or photolabel orthologs of MRP1 and several other drug efflux transporters of the ABC transporter family. LY475776 modulated MRP1 and Pgp-mediated resistance (MDR, ABCB1) in, respectively, HeLa-T5 and CEM/VLB(100) cells to both vincristine and doxorubicin. LY475776 photolabeled 170kDa Pgp and was inhibited by the potent Pgp inhibitor LY335979 (Zosuquidar.3HCl). The labeling of the 190kDa MRP1 protein in membranes of HeLa-T5 cells was inhibited by substrates of MRP1 such as leukotriene C(4), vincrisine, and doxorubicin and by the inhibitor, MK571. LY475776 did not photolabel human MRP2 (ABCC2), MRP3 (ABCC3), MRP5 (ABCC5) or breast cancer resistance protein (ABCG2). Because LY475776 photolabels murine mrp1 less well than human MRP1 and binds to a region believed important for anthracycline binding, studies were conducted with monkey and canine MRP1 which also show a reduced ability to confer resistance to anthracyclines. Unlike murine mrp1, both orthologs were photolabeled well by LY475776. These studies indicate that the specificity of LY475776 is fairly limited to Pgp and MRP1 and further studies will help to define the binding regions.     

Accumulation of Antibody-Target Complexes and the Pharmacodynamics of Clotting after Single Intravenous Administration of Humanized Anti-Factor IX Monoclonal Antibody to Rats

SB-249417, a humanized monoclonal antibody (Mab) specific for the Gla domain of Factor IX, inhibits activation of this zymogen and blocks the activity of Factor IXa on Factor X, the subsequent enzyme in the clotting cascade. In the present study, the pharmacokinetics and pharmacodynamics of SB-249417 were investigated in male Sprague-Dawley rats after IV administration of single doses of 10, 50, or 250 mg/kg. Blood samples were collected for up to six weeks to assess total plasma Mab concentration and activated partial thromboplastin time (aPTT). A PK/PD model was developed using an empirical relationship between aPTT and the concentration of free Factor IX (inhibitory Emax model). The model assumed natural synthesis and degradation of the endogenous zymogen that was interrupted by the complexation of Factor IX with the antibody. Following antibody administration, aPTT values increased 5-fold above baseline at the earliest sampling time in all dose groups. Higher doses led to a longer duration of prolonged clotting time. Estimates of model parameters yielded a Kd for antibody-antigen interaction (38 nM) that was similar to the in vitro value. The estimated degradation half-life of Factor IX (8 hr) was consistent with historical estimates. The PK/PD model predicted that the maximum concentration of antibody-Factor IX complex (Cmax) and the time to Cmax (Tmax) would increase with increasing dose. The extent of accumulation, up to 10-fold greater than the concentration of endogenous Factor IX at baseline, was confirmed by Western Blot analysis of Protein A extracts. Complex Tmax was similar to the duration of pharmacological effect and suggests effects persisted until Factor IX synthesis produced sufficient antigen to saturate the antibody.     

Induction chemotherapy with sequential nimotuzumab plus concurrent chemoradiotherapy in advanced nasopharyngeal carcinoma: A retrospective real-world study

Many locally advanced nasopharyngeal carcinoma patients develop local recurrence or distant metastasis. Our retrospective real-world study aims to evaluate the efficacy and safety of curative sequential approach with induction chemotherapy followed by concurrent chemoradiation + nimotuzumab as first-line therapy in advanced nasopharyngeal carcinoma. From 2015 to 2021, the clinic data of 117 patients with advanced nasopharyngeal carcinoma (stage III–IV a) who were treated in the Affiliated Hospital of Guangdong Medical University were retrospectively reviewed. Fifty-four patients in observation group received taxanes, cisplatin, and 5-fluorouracil/taxanes and cisplatin induction chemotherapy and nimotuzumab (200 mg, weekly) combined with concurrent chemo-radiotherapy (cisplatin: 40 mg/m² weekly; intensity-modulated radiation therapy); 63 patients in control group received same therapy without nimotuzumab. There was no significant difference in patients’ characteristic baseline between 2 groups (P > .05). The complete response rate and objective response rate of the observational group was significantly higher than control group (46.30% vs 17.64%, P = .01; 96.30% vs 82.54%, P = .02). The median follow-up time was 24.77 (3.53–65.97) months. Both of the median progress free survival time and overall survival time were not reached. The 5-year progression-free survival rate of observation group was greater than control group (84.40% vs 63.70%, hazard ratios 0.365, 95% confidence intervals 0.147–0.909, P = .03). The 5-year overall survival rate of observation group and control group were 91.70% and 84.60%, respectively (P = .20). None of the patients withdrew from the study due to adverse events. Nimotuzumab combined with concurrent chemoradiotherapy as first-line therapy in advanced nasopharyngeal carcinoma can improve objective response rate and 5-year progress free survival rate with good safety profile.     

In Vitro and In Vivo Effect of HPMA Copolymer-bound Doxorubicin Targeted to Transferrin Receptor of B-cell Lymphoma 38C13

N -(2-Hydroxypropyl)methacrylamide (HPMA) copolymers containing the anticancer agent doxorubicin and targeted to the transferrin receptor either with anti-mouse CD71 monoclonal antibody (mAb) or with transferrin were synthesized to evaluate their binding and anti-proliferative activity in vitro and anti-tumor potential against 38C13 B-cell lymphoma in vivo. Both the doxorubicin and the targeting moieties were bound to HPMA copolymer chain by aminolysis via a Gly-Phe (d, l) -Leu-Gly spacer to ensure controlled intracellular release of the conjugated drug. We demonstrated that HPMA copolymer-bound doxorubicin targeted to the transferrin receptor with anti-mouse CD71 mAb strongly retards tumor growth, prolongs the survival and completely cures three out of nine experimental mice with established 38C13 tumors. The conjugate targeted with transferrin was less effective in vitro as well as in vivo. It completely cured only one out of seven experimental mice. Free or non-targeted HPMA copolymer-bound doxorubicin showed only a mild anti-tumor effect within the therapeutic schedule used. In vitro, HPMA copolymer-bound doxorubicin targeted with anti-mouse CD71 mAb shows approximately 4-fold higher cytotoxic effect than HPMA copolymer-bound doxorubicin targeted with transferin and 9-fold higher cytotoxic effect than non-targeted HPMA copolymer-bound doxorubicin.     

特异、快速、简便、经济的淋病检测试剂盒的研制

本研究成功地制备了多株淋球菌主要外膜蛋白的单抗，并以胶体金标记。制备兔抗外膜蛋白多抗。在硝酸纤维素膜条上打点包被多抗，加经处理的被检样品和胶体金标记的单抗，直接显色。与细菌培养法一同检测标本312例，两法检测结果符合率力99．36％。结果表明两者特异性、灵敏度基本一致，本试剂稳定性、重复性好，且操作简便，不用仪器，15分钟内即可得到结果。本研究具有很高的特异性和灵敏度，结果适用于快速临床检测。