Biomedical subjects

Manuel L Penichet

Publications and source records attributed to Manuel L Penichet.

14 recordsLinked to original sources

PubMed2007

Targeting aurora kinases as therapy in multiple myeloma.

The aurora kinases facilitate transit from G2 through cytokinesis and, thus, are targets in cancer therapy. Multiple myeloma (MM) is a malignancy characterized by genetic instability, suggesting a disruption of checkpoints that arrest cells at G2M when injury to the mitotic machinery occurs. Since deficient checkpoints would prevent cell cycle arrest and may render cells susceptible to apoptosis in mitosis and since aurora kinases are intermediaries in checkpoint pathways, we tested antimyeloma effects of 2 agents that inhibit aurora kinases. Both inhibited growth of MM lines and primary myeloma samples at nanomolar concentrations while having less of an effect on proliferating lymphocytes and hematopoietic cells. MM cells were not protected by IL-6 or activating mutations of Ras. Antimyeloma effects included induction of tetraploidy followed by apoptosis. Apoptosis correlated with inhibition of aurora activity as shown by reduction of histone 3B phosphorylation. Ectopic expression of aurora A protected MM cells against aurora inhibitors but had no effect on apoptosis induced by bortezomib. As expression of RHAMM in MM contributes to genetic instability, we tested effects of RHAMM. RHAMM overexpression enhanced sensitivity to apoptosis and RHAMM silencing decreased sensitivity. These results suggest potential for aurora kinase inhibitors in MM especially in patients in whom RHAMM is overexpressed.

Antineoplastic Agents↗

PubMed2006

The transferrin receptor part II: targeted delivery of therapeutic agents into cancer cells.

Traditional anti-cancer treatments consist of chemotherapeutic drugs that effectively eliminate rapidly dividing tumor cells. However, in many cases chemotherapy fails to eliminate the tumor and even when chemotherapy is successful, its systemic cytotoxicity often results in detrimental side effects. To overcome these problems, many laboratories have focused on the design of novel therapies that exhibit tumor specific toxicity. The transferrin receptor (TfR), a cell membrane-associated glycoprotein involved in iron homeostasis and cell growth, has been explored as a target to deliver therapeutics into cancer cells due to its increased expression on malignant cells, accessibility on the cell surface, and constitutive endocytosis. The TfR can be targeted by direct interaction with conjugates of its ligand transferrin (Tf) or by monoclonal antibodies specific for the TfR. In this review we summarize the strategies of targeting the TfR in order to deliver therapeutic agents into tumor cells by receptor-mediated endocytosis.

Antibiotics, Antineoplastic↗

PubMed2006

The transferrin receptor part I: Biology and targeting with cytotoxic antibodies for the treatment of cancer.

The transferrin receptor (TfR) is a cell membrane-associated glycoprotein involved in the cellular uptake of iron and in the regulation of cell growth. Iron uptake occurs via the internalization of iron-loaded transferrin (Tf) mediated by the interaction with the TfR. In addition, the TfR may also contain other growth regulatory properties in certain normal and malignant cells. The elevated levels of TfR in malignancies, its relevance in cancer, and the extracellular accessibility of this molecule make it an excellent antigen for the treatment of cancer using antibodies. The TfR can be targeted by monoclonal antibodies specific for the extracellular domain of the receptor. In this review, we summarize advancements in the basic physiology of the TfR including structure, function, and expression. We also discuss the efficacy of targeting the TfR using cytotoxic antibodies that inhibit cell growth and/or induce apoptosis in targeted malignant cells.

Animals↗

PubMed2006

Molecular events contributing to cell death in malignant human hematopoietic cells elicited by an IgG3-avidin fusion protein targeting the transferrin receptor.

We have previously reported that an anti-human transferrin receptor IgG3-avidin fusion protein (anti-hTfR IgG3-Av) inhibits the proliferation of an erythroleukemia-cell line. We have now found that anti-hTfR IgG3-Av also inhibits the proliferation of additional human malignant B and plasma cells. Anti-hTfR IgG3-Av induces internalization and rapid degradation of the TfR. These events can be reproduced in cells treated with anti-hTfR IgG3 cross-linked with a secondary Ab, suggesting that they result from increased TfR cross-linking. Confocal microscopy of cells treated with anti-hTfR IgG3-Av shows that the TfR is directed to an intracellular compartment expressing the lysosomal marker LAMP-1. The degradation of TfR is partially blocked by cysteine protease inhibitors. Furthermore, cells treated with anti-hTfR IgG3-Av exhibit mitochondrial depolarization and activation of caspases 9, 8, and 3. The mitochondrial damage and cell death can be prevented by iron supplementation, but cannot be fully blocked by a pan-caspase inhibitor. These results suggest that anti-hTfR IgG3-Av induces lethal iron deprivation, but the resulting cell death does not solely depend on caspase activation. This report provides insights into the mechanism of cell death induced by anti-TfR Abs such as anti-hTfR IgG3-Av, a molecule that may be useful in the treatment of B-cell malignancies such as multiple myeloma.

Amino Acid Chloromethyl Ketones↗

PubMed2006

Anti-HER2/neu IgG3-(IL-2) and anti-HER2/neu IgG3-(GM-CSF) promote HER2/neu processing and presentation by dendritic cells: implications in immunotherapy and vaccination strategies.

HER2/neu, a transmembrane glycoprotein overexpressed in several types of human cancers, is a potential target for active immunotherapy. However, this protein and especially its extracellular domain (ECD(HER2)), is weakly immunogenic and is poorly processed by dendritic cells (DCs). Previously, we showed that anti-HER2/neu IgG3-(IL-2) and anti-HER2/neu IgG3-(GM-CSF) fusion proteins can enhance the immunogenicity of ECD(HER2) in mice, and that the non-covalent physical association between each antibody fusion proteins and ECD(HER2) was critical to elicit optimal protective immunity against HER2/neu expressing tumors. We now use the professional antigen-presenting DCs to investigate the effect of the antibody fusion protein binding to ECD(HER2) on its trafficking and presentation. We found that when the extracellular domain of HER2/neu fused to ovalbumin (OVA-ECD(HER2)) is bound by HER2/neu-specific antibody-(IL-2) or antibody-(GM-CSF) fusion proteins, the bound antigen is more efficiently processed by murine bone-marrow-derived dendritic cells (BMDCs) and presented to OVA-specific T-cells than the unbound OVA-ECD(HER2). We also found that ECD(HER2) bound by anti-HER2/neu IgG3-(IL-2) is very efficiently internalized and that the internalized ECD(HER2) is not retained in the early endosomal compartments but traffics to the antigen-processing compartments. These results are consistent with our earlier in vivo studies and suggest that both antibody-(IL-2) and antibody-(GM-CSF) fusion proteins can be used to enhance the immune response to poorly immunogenic antigens including tumor-associated antigens (TAAs).

Animals↗

PubMed2006

Cytokines fused to antibodies and their combinations as therapeutic agents against different peritoneal HER2/neu expressing tumors.

We have previously generated antihuman HER2/neu-humanized IgG3 fused to interleukin-2 (IL-2), IL-12, or granulocyte macrophage colony-stimulating factor (GM-CSF) [monofunctional fusion proteins (mono-AbFP)] or fused to IL-2 and IL-12 or IL-12 and GM-CSF [bifunctional fusion proteins (bi-AbFP)]. These AbFPs retained cytokine and antigen-binding activities. We have now further characterized the AbFPs and determined the heparin-binding activity of the fused cytokines, their ability to trigger IFN-gamma secretion and natural killer (NK) activation, and their direct antitumor efficacy. Flow cytometry revealed heparin-binding activity in the AbFPs containing IL-12 and IL-2, although this activity seems to be decreased in the bi-AbFPs. However, both bi-AbFPs retained the capacity to stimulate IL-12-dependent IFN-gamma secretion in the NK cell line KY-1, and IL-12/IL-2 bi-AbFP induced NK activity in splenocytes. The antitumor effectiveness of bi-AbFPs and mono-AbFP combinations was studied in mice challenged i.p. with three different human HER2/neu murine syngeneic models (D2F2/E2, CT26-HER2/neu, and MC38-HER2/neu). Although a significant variability in the profile of antitumor response was observed in the different tumor models, the combination of IL-12 and GM-CSF mono-AbFPs protected 100% of D2F2/E2-challenged and 75% of CT26-HER2/neu-challenged mice. In contrast, bi-AbFPs protected less than the combination of mono-AbFPs and, in some models, even less than mono-AbFPs alone. However, in all cases, most of long-term survivors showed protection after s.c. rechallenge with the tumors and later with the parental tumors not expressing HER2/neu. These results show that, although the pattern of protection is tumor model dependent, treatments with AbFPs can effectively generate high levels of protection against peritoneal tumors expressing HER2/neu, which may be relevant in patients with primary or metastatic peritoneal carcinomatosis that may be observed in ovarian, colon, stomach, bladder, lung, and breast cancers.

Animals↗

PubMed2005

Insights into the mechanism of anti-tumor immunity in mice vaccinated with the human HER2/neu extracellular domain plus anti-HER2/neu IgG3-(IL-2) or anti-HER2/neu IgG3-(GM-CSF) fusion protein.

In the present study, we demonstrate that a physical association between the extracellular domain of human HER2/neu receptor (ECDHER2) plus anti-HER2/neu IgG3-(IL-2) or anti-HER2/neu IgG3-(GM-CSF) was required to elicit the most effective anti-tumor immune response against a syngeneic tumor expressing rat HER2/neu. Immune effectors including CD4+, CD8+, and NK cells contributed to protection against tumor growth. Vaccinated B-cell deficient mice did not elicit tumor protection, suggesting a critical role for B-cells in a protective immune response. These results provide insights into the mechanisms responsible for the protective tumor immunity elicited when antibody-(IL-2 or GM-CSF) are used as enhancers of vaccines targeting tumor antigens.

Animals↗

PubMed2005

Vaccination with novel combinations of anti-HER2/neu cytokines fusion proteins and soluble protein antigen elicits a protective immune response against HER2/neu expressing tumors.

We have previously demonstrated that anti-HER2/neu IgG3-(IL-2), (IL-12)-IgG3, or IgG3-(GM-CSF) antibody fusion proteins (mono-AbFPs) elicit anti-tumor activity against murine tumors expressing HER2/neu when used as adjuvants of extracellular domain of HER2/neu (ECD(HER2)) protein vaccination. We have now studied the effect of combinations of IL-2 and IL-12 or IL-12 and GM-CSF mono-AbFPs during vaccination with ECD(HER2). In addition, we developed two novel anti-HER2/neu IgG3-cytokine fusion proteins in which IL-2 and IL-12 or IL-12 and GM-CSF were fused to the same IgG3 molecule (bi-AbFPs). (IL-12)-IgG3-(IL-2) and (IL-12)-IgG3-(GM-CSF) were properly assembled and retained both cytokine activity and the ability to bind antigen. Vaccination of mice with ECD(HER2) and a combination of cytokines as either bi-AbFPs or two mono-AbFPs activated both Thl and Th2 immune responses and resulted in significant protection against challenge with a HER2/neu expressing tumor. Our results suggest that this approach will be effective in the prevention and/or treatment of HER2/neu expressing tumors.

Adjuvants, Immunologic↗

PubMed2005

Enhanced inhibition of murine tumor and human breast tumor xenografts using targeted delivery of an antibody-endostatin fusion protein.

Endostatin can inhibit angiogenesis and tumor growth in mice. A potential limitation of endostatin as an antitumor agent in humans is the short serum half-life of the protein that may decrease effective concentration at the site of tumor and necessitate frequent dosing. In an effort to improve antitumor activity, endostatin was fused to an antibody specific for the tumor-selective HER2 antigen to create an antibody-endostatin fusion protein (anti-HER2 IgG3-endostatin). Normal endostatin rapidly cleared from serum in mice (T(1/2)(2), = 0.6-3.8 hours), whereas anti-HER2 IgG3-endostatin had a prolonged half-life (90% intact; T(1/2)(2), 40.2-44.0 hours). Antigen-specific targeting of anti-HER2 IgG3-endostatin was evaluated in BALB/c mice implanted with CT26 tumors or CT26 tumors engineered to express the HER2 antigen (CT26-HER2). Radio-iodinated anti-HER2 IgG3-endostatin preferentially localized to CT26-HER2 tumors relative to CT26 tumors. Administration of anti-HER2 IgG3-endostatin to mice showed preferential inhibition of CT26-HER2 tumor growth compared with CT26. Anti-HER2 IgG3-endostatin also markedly inhibited the growth of human breast cancer SK-BR-3 xenografts in severe combined immunodeficient mice. Anti-HER2 IgG3-endostatin inhibited tumor growth significantly more effectively than endostatin, anti-HER2 IgG3 antibody, or the combination of antibody and endostatin. CT26-HER2 tumors treated with the endostatin fusion protein had decreased blood vessel density and branching compared with untreated CT26-HER2 or CT26 treated with the fusion protein. The enhanced effectiveness of anti-HER2 IgG3-endostatin may be due to a longer half-life, improved serum stability, and selective targeting of endostatin to tumors, resulting in decreased angiogenesis. Linking of an antiangiogenic protein, such as endostatin, to a targeting antibody represents a promising and versatile approach to antitumor therapy.

Angiogenesis Inhibitors↗

PubMed2005

Antibody-cytokine fusion proteins for the therapy of cancer.

In recent years the development of tumor-specific recombinant antibodies fused to immuno-stimulatory cytokines such as interleukin-2 (IL-2), interleukin-12 (IL-12), and granulocyte/macrophage colony-stimulating factor (GM-CSF) has provided a promising novel approach to cancer immunotherapy. The combined properties of specific targeting of antibodies and the immune stimulation of cytokines results in high cytokine concentration in the tumor microenvironment, and as a consequence, in an improved tumoricidal activity of the antibody and/or in a secondary effective immune response against the tumor. In the present chapter we describe strategies for the construction, expression, and in vitro characterization of antibody-cytokine fusion proteins, with particular emphasis on antibody/IL-2 fusion proteins.

Antibodies↗

PubMed2004

A human biotin acceptor domain allows site-specific conjugation of an enzyme to an antibody-avidin fusion protein for targeted drug delivery.

We have previously constructed an antibody-avidin (Av) fusion protein, anti-transferrin receptor (TfR) IgG3-Av, which can deliver biotinylated molecules to cells expressing the TfR. We now describe the use of the fusion protein for antibody-directed enzyme prodrug therapy (ADEPT). The 67 amino acid carboxyl-terminal domain (P67) of human propionyl-CoA carboxylase alpha subunit can be metabolically biotinylated at a fixed lysine residue. We genetically fused P67 to the carboxyl terminus of the yeast enzyme FCU1, a derivative of cytosine deaminase that can convert the non-toxic prodrug 5-fluorocytosine to the cytotoxic agent 5-fluorouracil. When produced in Escherichia coli cells overexpressing a biotin protein ligase, the FCU1-P67 fusion protein was efficiently mono-biotinylated. In the presence of 5-fluorocytosine, the biotinylated fusion protein conjugated to anti-rat TfR IgG3-Av efficiently killed rat Y3-Ag1.2.3 myeloma cells in vitro, while the same protein conjugated to an irrelevant (anti-dansyl) antibody fused to Av showed no cytotoxic effect. Efficient tumor cell killing was also observed when E. coli purine nucleoside phosphorylase was similarly targeted to the tumor cells in the presence of the prodrug 2-fluoro-2'-deoxyadenosine. These results suggest that when combined with P67-based biotinylation, anti-TfR IgG3-Av could serve as a universal delivery vector for targeted chemotherapy of cancer.

Animals↗

PubMed2003

Protein vaccination with the HER2/neu extracellular domain plus anti-HER2/neu antibody-cytokine fusion proteins induces a protective anti-HER2/neu immune response in mice.

Previously protein vaccines consisting of the extracellular domain of HER2/neu (ECD(HER2)) were shown to elicit an immune response that does not provide protection against transplantable tumors expressing HER2/neu. Here, we showed that when mice were vaccinated with a mixture of human ECD(HER2) and anti-human HER2/neu IL-12, IL-2 or GM-CSF fusion proteins, significant retardation of the growth of a syngeneic carcinoma expressing rat HER2/neu, and long-term survivors were observed. Immune sera inhibited the in vitro growth of SK-BR-3, a human breast cancer overexpressing HER2/neu. Transfer of immune sera into mice challenged with TUBO also led to partial inhibition of tumor growth. Splenocytes from mice vaccinated with ECD(HER2) plus IgG3-(GM-CSF) incubated with ECD(HER2) demonstrated significant proliferation and IFN-gamma secretion. Taken together these results suggest that vaccines including ECD(HER2) and Ab-cytokine fusion proteins may be used to elicit both humoral and cell-mediated responses against HER2/neu.

Animals↗

PubMed2002

An anti-transferrin receptor-avidin fusion protein exhibits both strong proapoptotic activity and the ability to deliver various molecules into cancer cells.

We have developed an antibody fusion protein (anti-rat TfR IgG3-Av) with the ability to deliver different molecules into cancer cells. It consists of avidin genetically fused to the C(H)3 region of a human IgG3 specific for the rat transferrin receptor. It forms strong, noncovalent interactions with biotinylated molecules such as glucose oxidase and beta-galactosidase, and delivers them into the rat myeloma cell line Y3-Ag1.2.3 through receptor-mediated endocytosis. Importantly, the beta-galactosidase retains activity after internalization. Furthermore, we have unexpectedly discovered that anti-rat TfR IgG3-Av, but not a recombinant anti-rat TfR IgG3 or a nonspecific IgG3-Av, possesses proapoptotic activities against Y3-Ag1.2.3 and the rat T cell lymphoma cell line C58 (NT) D.1.G.OVAR.1. These activities were not observed in two rat cell lines of nonhematopoietic lineage (bladder carcinoma BC47 and gliosarcoma 9L). Anti-human TfR IgG3-Av also demonstrated proapoptotic activity against the human erythroleukemia cell line K562. Studies showed that anti-rat TfR IgG3-Av exists as a dimer, suggesting that cross-linking of the surface transferrin receptor may be responsible for the cytotoxic activity. These findings demonstrate that it is possible to transform an antibody specific for a growth factor receptor that does not exhibit inhibitory activity into a drug with significant intrinsic cytotoxic activity against selected cells by fusing it with avidin. The antitumor activity may be enhanced by delivering biotinylated therapeutics into cancer cells. Further development of this technology may lead to effective therapeutics for in vivo eradication of hematological malignancies, and ex vivo purging of cancer cells in autologous transplantation.

Animals↗

PubMed2002

Myeloma expression systems.

Myeloma expression systems have been utilized successfully for the production of various recombinant proteins. In particular, myeloma cell lines have been exploited to express a variety of different antibodies for diagnostic applications as well as in the treatment of various human diseases. The use of myeloma cells for antibody production is advantageous because they are professional immunoglobulin-secreting cells and are able to make proper post-translational modifications. Proper glycosylation has been shown to be important for antibody function. Advances in genetic engineering and molecular biology techniques have made it possible to isolate murine and human variable regions of almost any desired specificity. Antibodies and antibody variants produced in myeloma cells have been extremely helpful in elucidating the amino acid residues and structural motifs that contribute to antibody function. Because of their domain nature, immunoglobulin genes can be easily manipulated to produce chimeric or humanized antibodies. These antibodies are less immunogenic in humans and also retain their specificity for antigen and biologic properties. In addition, novel proteins in which antibodies are fused to non-immunoglobulin sequences as well as secretory IgA have been produced in myeloma cells.

Animals↗