Rosendo Luria-Pérez

Polymalic acid nanobioconjugate for simultaneous immunostimulation and inhibition of tumor growth in HER2/neu-positive breast cancer.

Breast cancer remains the second leading cause of cancer death among women in the United States. Breast cancer prognosis is particularly poor in case of tumors overexpressing the oncoprotein HER2/neu. A new nanobioconjugate of the Polycefin(TM) family of anti-cancer drugs based on biodegradable and non-toxic polymalic acid (PMLA) was engineered for a multi-pronged attack on HER2/neu-positive breast cancer cells. An antibody-cytokine fusion protein consisting of the immunostimulatory cytokine interleukin-2 (IL-2) genetically fused to an antibody specific for human HER2/neu [anti-HER2/neu IgG3-(IL-2)] was covalently attached to the PMLA backbone to target HER2/neu expressing tumors and ensure the delivery of IL-2 to the tumor microenvironment. Antisense oligonucleotides (AON) were conjugated to the nanodrug to inhibit the expression of vascular tumor protein laminin-411 in order to block tumor angiogenesis. It is shown that the nanobioconjugate was capable of specifically binding human HER2/neu and retained the biological activity of IL-2. We also showed the uptake of the nanobioconjugate into HER2/neu-positive breast cancer cells and enhanced tumor targeting in vivo. The nanobioconjugate exhibited marked anti-tumor activity manifested by significantly longer animal survival and significantly increased anti-HER2/neu immune response in immunocompetent mice bearing D2F2/E2 murine mammary tumors that express human HER2/neu. The combination of laminin-411 AON and antibody-cytokine fusion protein on a single polymeric platform results in a new nanobioconjugate that can act against cancer cells through inhibition of tumor growth and angiogenesis and the orchestration of an immune response against the tumor. The present Polycefin(TM) variant may be a promising agent for treating HER2/neu expressing tumors and demonstrates the versatility of the Polycefin(TM) nanobioconjugate platform.

An antibody-based multifaceted approach targeting the human transferrin receptor for the treatment of B-cell malignancies.

We previously developed an antibody-avidin fusion protein (ch128.1Av) targeting the human transferrin receptor 1 (TfR1, also known as CD71), which demonstrates direct in vitro cytotoxicity against malignant hematopoietic cells. This cytotoxicity is attributed to its ability to decrease the level of TfR1 leading to lethal iron deprivation. We now report that ch128.1Av shows the ability to bind the Fc&ggr; receptors and the complement component C1q, suggesting that it is capable of eliciting Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-mediated cytotoxicity. In addition, in 2 disseminated multiple myeloma xenograft mouse models, we show that a single dose of ch128.1Av results in significant antitumor activity, including long-term survival. It is interesting to note that the parental antibody without avidin (ch128.1) also shows remarkable in vivo anticancer activity despite its limited in vitro cytotoxicity. Finally, we demonstrate that ch128.1Av is not toxic to pluripotent hematopoietic progenitor cells using the long-term cell-initiating culture assay suggesting that these important progenitors would be preserved in different therapeutic approaches, including the in vitro purging of cancer cells for autologous transplantation and in vivo passive immunotherapy. Our results suggest that ch128.1Av and ch128.1 may be effective in the therapy of human multiple myeloma and potentially other hematopoietic malignancies.

Lethal iron deprivation induced by non-neutralizing antibodies targeting transferrin receptor 1 in malignant B cells

Abstract A number of antibodies have been developed that induce lethal iron deprivation (LID) by targeting the transferrin receptor 1 (TfR1/CD71) and either neutralizing transferrin (Tf) binding, blocking internalization of the receptor and/or inducing its degradation. We have developed recombinant antibodies targeting human TfR1 (ch128.1 and ch128.1Av), which induce receptor degradation and are cytotoxic to certain malignant B-cells. We now show that internalization of TfR1 bound to these antibodies can lead to its sequestration and degradation, as well as reduced Tf uptake, and the induction of a transcriptional response consistent with iron deprivation, which is mediated in part by downstream targets of p53. Cells resistant to these antibodies do not sequester and degrade TfR1 after internalization of the antibody/receptor complex, and accordingly maintain their ability to internalize Tf. These findings are expected to facilitate the rational design and clinical use of therapeutic agents targeting iron import via TfR1 in hematopoietic malignancies.

Visualization and quantification of cytotoxicity mediated by antibodies using imaging flow cytometry

Conventional approaches for the detection of antibody dependent cell-mediated cytotoxicity (ADCC) activity rely on quantification of the release of traceable compounds from target cells or flow cytometry analysis of population-wide phenomena. We report a new method for the direct imaging and quantification of ADCC of cancer cells. The proposed method using imaging flow cytometry combines the statistical power of flow cytometry with the analytical advantages of cell imaging, providing a novel and more comprehensive perspective of effector/target cell interactions during ADCC events. With this method we can quantify and show in detail the morphological changes in target and effector cells, their apoptotic index, the physical interaction between effector and target cells, and a directional transfer of cytosolic contents from effector to target cells. As a model system we used the therapeutic anti-CD20 antibody rituximab to target CFSE labeled Ramos human Burkitts lymphoma cells, to CMTPX-labeled human monocytic U-937 effector cells. We expect that similar studies using different effector and target cell populations may contribute to the pre-clinical evaluation of therapeutic antibodies and help to identify mechanisms that could be beneficial in the immunotherapy of cancer.

Antibody-mediated targeting of the transferrin receptor in cancer cells

Iron is essential for cell growth and is imported into cells in part through the action of transferrin (Tf), a protein that binds its receptor (TfR1 or CD71) on the surface of a cell, and then releases iron into endosomes. TfR1 is a single pass type-II transmembrane protein expressed at basal levels in most tissues. High expression of TfR1 is typically associated with rapidly proliferating cells, including various types of cancer. TfR1 is targeted by experimental therapeutics for several reasons: its cell surface accessibility, constitutive endocytosis into cells, essential role in cell growth and proliferation, and its overexpression by cancer cells. Among the therapeutic agents used to target TfR1, antibodies stand out due to their remarkable specificity and affinity. Clinical trials are being conducted to evaluate the safety and efficacy of agents targeting TfR1 in cancer patients with promising results. These observations suggest that therapies targeting TfR1 as direct therapeutics or delivery conduits remain an attractive alternative for the treatment of cancers that overexpress the receptor.

Salmonella enterica: un aliado en la terapia contra el cáncer

Salmonella enterica, a species of facultative anaerobic bacteria, has demonstrated success as a live-attenuated bacterial vector for vaccination. S. enterica has also demonstrated promise as a therapeutic agent against cancer. Pre-clinical and clinical trials have shown that S. enterica is localized in both solid and semi-solid tumors as well as in metastatic tumors. Moreover, S. enterica reduces resistance to treatment with other agents. In this review we present the novel therapeutic anti-cancer approaches that use S. enterica both for its ability as a delivery system for heterologous moieties against cancer and for its direct anti-cancer properties.

Abstract 3622: An antibody-based multifaceted approach targeting the human transferrin receptor for the treatment of multiple myeloma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL We previously developed an antibody-avidin fusion protein (ch128.1Av) targeting the human transferrin receptor 1 (TfR1, CD71) that demonstrates direct in vitro cytotoxicity against malignant hematopoietic cells. This cytotoxicity is due to its ability to decrease TfR1 levels leading to lethal iron deprivation. We now report that ch128.1Av shows the ability to bind the Fc gamma receptors and the complement component C1q, suggesting that is capable of eliciting Fc-mediated effector functions such as antibody-dependent cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC). Additionally, in two disseminated multiple myeloma xenograft mouse models, we show that a single dose of ch128.1Av results in significant anti-tumor activity including long-term survival. Interestingly, the parental antibody without avidin (ch128.1) also shows remarkable in vivo anti-cancer activity despite its lack of in vitro cytotoxicity. Finally, we demonstrate that ch128.1Av is not toxic to pluripotent hematopoietic progenitor cells using the Long-Term Cell-Initiating Culture (LTC-IC) assay suggesting that these important progenitors would be preserved in different therapeutic approaches, including the in vitro purging of cancer cells for autologous transplantation and in vivo passive immunotherapy. Our results suggest that ch128.1Av and ch128.1 may be effective in the therapy of human multiple myeloma and potentially other hematopoietic malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3622. doi:10.1158/1538-7445.AM2011-3622

Abstract 4456: Molecular events required for the induction of lethal iron deprivation in malignant hematopoietic cells via an antibody-avidin fusion protein specific for human transferrin receptor 1

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Iron is an essential nutrient for cell growth and division. The transferrin receptor 1 (TfR1 or CD71) is a major gateway for iron import, and as such is expressed on a variety of cells; it is over-expressed in a number of tumors including hematopoietic malignancies. We have previously shown that an antibody-avidin fusion protein (ch128.1Av) specific for TfR1 is toxic to cancer cells, capable of inducing lethal iron deprivation in some malignant B-cells such as IM-9, but not in others such as U266. Importantly, ch128.1Av induces iron deprivation without directly preventing the binding of transferrin (Tf) to TfR1. In an attempt to identify the molecular events behind the iron deprivation induced by ch128.1Av, we have found that like Tf, internalization of ch128.1Av bound to TfR1 is clathrin, dynamin, and temperature dependent. Additionally, recycling of TfR1-bound ch128.1Av at short timescales (30min) is also similar to that of Tf and does not significantly alter the rate of Tf uptake. Long-term incubation (>6h) of IM-9 cells with ch128.1Av precludes their uptake of Tf and induces degradation of TfR1. This is not the case with U266 cells, in which Tf uptake remains unaffected and degradation of TfR1 is negligible in the presence of ch128.1Av. Clustering of cell surface TfR1 by ch128.1Av, given its multivalent nature, was previously thought to cause degradation of the receptor in sensitive cells such as IM-9. We now show that clustering of TfR1 occurs both in cells that are sensitive and resistant to treatment with the antibody (IM-9 and U266), indicating that clustering of TfR1 induced by ch128.1Av is not sufficient to induce receptor degradation and subsequent iron deprivation. Instead, resistance to treatment with ch128.1Av may be attributed to elements downstream of receptor binding/clustering; possibly related to those involved with TfR1 recycling and degradation. Further studies are needed to identify these resistance factors and potentially enhance the efficacy of ch128.1Av as well as other antibodies and/or small molecules that similarly target TfR1 for the treatment of hematologic malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4456.