Monomethyl auristatin E, a potent cytotoxic payload for development of antibody-drug conjugates against breast cancer

Background: Breast cancer is a heterogeneous disease characterized by differential responses to targeted and chemotherapeutic agents. Antibody-drug conjugates are one of the promising strategies for the treatment of breast cancer. Monomethyl auristatin E [MMAE] is a highly potent microtubule inhibitor and a common payload used for development of antibody-drug conjugates. The purpose of this study was to investigate the cytotoxic effects of MMAE on breast cancer cell lines

Materials and Methods: MDA-MB-468 and MDA-MB-453 cells were treated with MMAE at various concentrations [1, 10, 100, and 1000 ng/ml], and cytotoxicity was measured after 48 and 72 hours using an MTT assay

Results: Our findings indicated that MMAE possesses dose- and time-dependent cytotoxic activities against human breast cancer cells. The morphological features of the treated cells were supportive of the cytotoxic activity of MMAE. The results of the MTT assay showed that MMAE has a significant cytotoxicity against MDA-MB-468 and, to a lesser degree, MDA-MB-453 cells

Conclusion: MMAE can be used as a highly cytotoxic payload for development of antibody-drug conjugates against breast cancer

Evaluation of factors influencing antibody reduction for development of antibody drug conjugates

Background: Reduction/alkylation is one of the leading strategies for the development of antibody drug conjugates [ADCs]. Precise control of the reduction process would not only yield a defined number of free thiols per antibody but also result in development of more homogenous conjugates

Methods: In the present study, we investigated the effect of various dithiothreitol [DTT] concentrations, temperature conditions, and DTT exposure times on antibody reduction. After antibody reduction, the Ellman's test and SDS-PAGE analysis were used to evaluate free thiols produced and confirm the reduction process, respectively

Results: DTT concentration seems to be a potential factor in the reduction process. Concentrations of 0.1, 1, 5, 10, 20, 50, and 100 mM DTT at 37[degree]C for 30 minutes resulted in approximately 0.4, 1.2, 5.4, 7, 8, 8, and 8 thiols per antibody, respectively

Conclusion: Optimized site specific conjugation can provide better process control and reproducibility for the development of disulfide-based ADCs

Monomethyl auristatin E exhibits potent cytotoxic activity against human cancer cell lines skbr3 and hek293

Background: Monomethyl auristatin E [MMAE] is a synthetic analog of dolastatin 10, a compound originally isolated from the marine mollusk. MMAE, as a highly potent microtubule inhibitor, exerts its potent cytotoxic effect by inhibiting microtubule assembly, tubulin-dependent GTP hydrolysis and microtubes polymerization. This molecule, by itself, lacks the tumor specificity required to elicit therapeutic benefit. Nevertheless, the extremely cytotoxic potential of MMAE could be harnessed in the form of MMAE-antibody conjugates. The aim of the present study was to evaluate the cytotoxic activity of MMAE against breast [SKBR3] and kidney [HEK293] cancer cell lines in an in vitro cell-based assay

Materials and Methods: SKBR3 and HEK293 cells were treated with different concentrations ranging from 0.002048, 0.01024, 0.0512, 0.256, 1.28, 6.4, 32, 160, 800 and 4000 nM of MMAE, and cell viability was determined after 72 hours using an MTT colorimetric assay. The effect of MMAE was regularly monitored by direct observation using an invert microscope

Results: Microscopic observation showed that there was a concentration-dependent increase in cell death

Results from the MTT assay revealed a statistically significant loss of viability [P<0.0001] at concentrations ranging from 0.01024 to 4000 nM in SKBR3 cells, and 0.0512 to 4000 nM in HEK293 cells. Our findings showed that MMAE inhibited the growth of SKBR3 and HEK293 cells in a concentration-dependent manner, with IC50 values of 3.27 +/- 0.42 and 4.24 +/- 0.37 nM, respectively

Conclusion: MMAE was able to significantly inhibit cell growth at nanomolar concentrations, emphasizing its great potential for the development of antibody-drug conjugates

Heat shock proteins enriched-promastigotes of leishmania major inducing Th2 immune response in BALB/c mice

Heat shock proteins [HSP] are highly conserved molecules with many immunological functions. They are highly immunogenic with important role in cancer immunotherapy and in vaccine development against infectious diseases. As adjuvant, HSP can augment the immunogenicity of weak antigens and can stimulate antigen presenting cells. Although vaccines have been successful for many infectious diseases, progress in leishmaniasis has not been achieved. In this report, the protective effect of HSP-enriched soluble leishmania antigen [SLA] was determined. BALB/c mice were immunized 3× with HSP-enriched SLA and SLA alone and 10 days after final boost. They were infected with 10[6] stationary phase promastigote of Leishmania major and immunological responses were followed until nine weeks. No significant differences were observed in lymphocyte proliferation, footpad swelling, parasite burden, nitric oxide or IL-12 cytokine between HSP-enriched or SLA groups. Although the levels of IFN-gamma, IL-4, TGF-beta, IgG1 and IgG2b were increased in both groups, IFN-gamma was significantly higher in SLA group and IgG2a in HSP-enriched SLA. These results indicate that HSP direct the immune system towards Th2 pattern and does not have protective role in L. major infection

Functional Recombinant Extra Membrane Loop of Human CD20, an Alternative of the Full Length CD20 Antigen

Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length transmembrane protein CD20. In this study, we expressed the extra membrane loop of hCD20 [exCD20] consisting of a non-glycosylated 47-amino acids region. The exCD20 coding sequence was amplified by PCR and cloned in pET32a[+] expression vector. The desired protein was expressed in fusion with thioredoxin and 6 His tag in E. coll Origami strain. ELISA and Western-blotting data were performed to indicate the functionality of this protein. We have obtained the exCD20 recombinant protein which can be detected in ELISA and Western-blot experiments. This recombinant fusion protein was soluble and stable without aggregation and misfolding problems. Conclusion: The recombinant extra membrane loop of human CD20 protein in fusion with thioredoxin [exCD20] can be used in function assays and some applications such as ELISA, immuneblotting, affinity purification, immunization, screening, and development of anti-CD20 antibodies

Candida albicans structural and secreted proteins modulate CD4/CD8 ratio in tumor infiltrating lymphocytes of spontaneous adenocarcinoma bearing mice

Candida albicans is one of the most important opportunistic pathogens that suppress immunologic mechanisms of the host. It is speculated that structural and secretory proteins of C. albicans have immunomodulatory effects in cancer. To evaluate the effects of C. albicans structural and secreted proteins on intratumoral CD4/CD8 ratio as well as the survival rate in BALB/c tumor model. Structural and secretory proteins from C. albicans were isolated and examined for their effects on tumor growth and survival of adenocarcinoma bearing mice. The results indicated that in mice treated with C. albicans structural protein, the survival rate significantly decreased compared with the control groups. Also, mice treated with secretory proteins showed a decrease in survival rate but it was not statistically significant [p>0.05]. Investigating the frequency of tumor infiltrated CD4+ and CD8+ T lymphocytes indicated that the percentages of tumor infiltrated CD4+ T lymphocytes in response to structural and secreted proteins were higher compared to the control groups. Our study suggests that C. albicans structural and secreted proteins modulate intratumor T lymphocyte infiltration

Production and characterization of monoclonal antibodies recognizing a common 57-kDA antigen of leishmania species

The therapy of leishmania infection is difficult and each year 1.5 million new cases of cutaneous leishmaniasis and 500,000 new cases of visceral leishmaniasis are estimated, therefore, there is a need for an effective vaccine. Monoclonal antibody [mAb] is one of the suitable methods for isolation and purification of leishmania antigens. In this report, we produced several mAb against leishmania infantum antigens for antigen purification to be used as candidate vaccine. BALB/c mice were injected with freeze-thawed promastigote twice together with Freund adjuvant. Three days before fusion, antigen in saline was injected into the tail vain and then mice were killed and the spleen lymphocytes were fused with myeloma SP2/0. Five mAb against promastigote form of Leishmania infantum parasite were obtained. Western-blot analysis showed that these mAb recognize a band of 57- kDa protein either in parasite lysate or on whole L. infantum, L. tropica, L. major and L. donovani. It seems that the 57 kDa-protein is the major surface leishmania antigen [gp63] that is neither stage-specific nor differentially regulated. These mAb do not recognize the recombinant gp63 antigen and seems recognizing only the native form of a gp63 isoform. The IgG1 mAb was purified by affinity column and was used to purify 57 kDa antigens from Leishmania lysate. Since these antibodies recognizing one specific protein band in 4 different strains of leishmania, they could be used for leishmania diagnostic kits and also for purification of antigen to be tested for its protective effect against leishmania infection

Partial purification of a potent immunosuppressive factor excreted from leishmania major promastigote and amastigote

Recent scientific evidence indicates that distinct patterns of susceptibility in BALB/c mice to Leishmania major infection are attributable to the differential expansion of distinct CD4+ T-cell subsets and their cytokines production. Production of the Th1 cytokine IFN- gamma is associated with resistance, whereas production of the Th2 cytokines IL-4 and IL-10 are associated with extreme susceptibility. The major host immune defense mechanism against Leishmania is activation of macrophages by INF-gamma derived from T cells. The inability of susceptible hosts to mount the immune response necessary to activate macrophage and destroy the parasites may be due to the parasite-specific proteins that are able to suppress the immune system. In the present study, we have semi-purified the excreted antigens of Leishmania major promastigote and amastigote by column chromatography. The isolated fraction showed a potent immunosuppressive activity on normal BALB/c mice lymphocytes stimulated with mitogens. Fifteen microgram of the isolated fraction caused 81% suppression of lymphocyte proliferation. These data may suggest that the parasite by secreting immunosuppressive factor down regulate the immune system and as a result survive in the body

Identification of two epitopes on the outer surface protein A of the Lyme disease spirochete borrelia burgdorferi

A murine IgM monoclonal antibody [MA-2C6] with kappa-light chains directed against an antigenic determinant of outer surface protein A [OspA] of the Lyme disease spirochete, Borrelia burgdorferi, is produced. This antibody could bind specifically to OspA antigen of several isolates of B. burgdorferi, but not to the non-Lyme disease bacteria such as T. pallidum and B. hermsii. Antibody MA-2C6 was purified by ion-exchange chromatography and used for purification of OspA antigen from Borrelia burgdorferi cell lysate. This antibody together with an IgG1 monoclonal antibody specific for OspA, that was previously characterized, were used to test whether these antibodies recognize different epitopes on OspA antigen of Borrelia burgdorferi. For this test, ELISA double antibody binding was used. Two antibodies were added to the antigen either separately or simultaneously, and the amount of bound antibody was quantitatively measured by the use of rabbit anti-mouse IgG conjugated with alkaline phosphatase. Additivity of the bound enzymatic activity was observed when the monoclonal antibodies bind to distinct epitopes. With this test, two distinct epitopes were recognized on the OspA molecule. This antibody can be used not only for the purification and subtyping of OspA, but also for neutralization and immunotherapy

Effects of antiproliferative protein [APP] on modulation of cytosolic protein phosphorylation of prostatic carcinoma cell line LNCaP

Antiproliferative protein [APP] isolated from conditioned media of two androgen-independent prostatic carcinoma cell lines, PC3 and Du-145 was shown to inhibit selectively cell proliferation of androgen-dependent prostate cancer cell line LNCaP in a dose dependent manner. This protein was further purified with HPLC using hydrophobic interaction column [phenyl 5PW] and was used to study the modulation of protein phosphorylation of LNCaP cells. The data indicated that antiproliferative protein could partially change the cytosolic protein phosphorylation. When compared with control LNCaP cells, in APP-treated cells 4 new proteins [88, 46, 30 and 18 kDa] were phosphorylated, while a 72 kDa phosphoprotein was de-phosphorylated. The same results were obtained when two types of protein kinase activators were used. Protein kinase activators showed that the above changes of protein phosphorylation are not due to the protein kinase C or DNA protein kinase activity. These data may indicate the inhibition of LNCaP cell's proliferation by APP may be is due to the modulation of protein kinases and as a result due to interference on second messenger pathway