Paulo Maciag

Cancer Immunotherapy Targeting the High Molecular Weight Melanoma-Associated Antigen Protein Results in a Broad Antitumor Response and Reduction of Pericytes in the Tumor Vasculature

The high molecular weight melanoma-associated antigen (HMW-MAA), also known as melanoma chondroitin sulfate proteoglycan, has been used as a target for the immunotherapy of melanoma. This antigen is expressed on the cell surface and has a restricted distribution in normal tissues. Besides its expression in a broad range of transformed cells, this antigen is also found in pericytes, which are important for tumor angiogenesis. We generated a recombinant Listeria monocytogenes (Lm-LLO-HMW-MAA-C) that expresses and secretes a fragment of HMW-MAA (residues 2,160-2,258) fused to the first 441 residues of the listeriolysin O (LLO) protein. Immunization with Lm-LLO-HMW-MAA-C was able to impede the tumor growth of early established B16F10-HMW-MAA tumors in mice and both CD4(+) and CD8(+) T cells were required for therapeutic efficacy. Immune responses to a known HLA-A2 epitope present in the HMW-MAA(2160-2258) fragment was detected in the HLA-A2/K(b) transgenic mice immunized with Lm-LLO-HMW-MAA-C. Surprisingly, this vaccine also significantly impaired the in vivo growth of other tumorigenic cell lines, such as melanoma, renal carcinoma, and breast tumors, which were not engineered to express HMW-MAA. One hypothesis is that the vaccine could be targeting pericytes, which are important for tumor angiogenesis. In a breast tumor model, immunization with Lm-LLO-HMW-MAA-C caused CD8(+) T-cell infiltration in the tumor stroma and a significant decrease in the number of pericytes in the tumor blood vessels. In conclusion, a Lm-based vaccine against HMW-MAA can trigger cell-mediated immune responses to this antigen that can target not only tumor cells but also pericytes in the tumor vasculature.

Construction and Characterization of an Attenuated Listeria monocytogenes Strain for Clinical Use in Cancer Immunotherapy

ABSTRACT Listeria monocytogenes has been exploited previously as a vaccine vector for the delivery of heterologous proteins such as tumor-specific antigens for active cancer immunotherapy. However, for effective use of live vector in clinics, safety is a major concern. In the present study, we describe an irreversibly attenuated and highly immunogenic L. monocytogenes platform, the L. monocytogenes dal-, dat-, and actA-deleted strain that expresses the human prostate-specific antigen (PSA) using an antibiotic resistance marker-free plasmid (the dal dat ΔactA 142 strain expressing PSA). Despite limited in vivo survival, the dal dat ΔactA 142 strain was able to elicit efficient immune responses required for tumor clearance. Our results showed that immunization of mice with the dal dat ΔactA 142 strain caused the regression of the tumors established by the prostate adenocarcinoma cell line expressing PSA. An evaluation of immunologic potency indicated that the dal dat ΔactA 142 strain elicits a high frequency of PSA-specific immune responses. Interestingly, immunization with the dal dat ΔactA 142 strain induced significant infiltration of PSA-specific T cells in the intratumoral milieu. Collectively, our data suggest that the dal dat ΔactA 142 strain is a safe and potent vector for clinical use and that this platform may be further exploited as a potential candidate to express other single or multiple antigens for cancer immunotherapy.

A Novel Human Her-2/neu Chimeric Molecule Expressed by Listeria monocytogenes Can Elicit Potent HLA-A2 Restricted CD8-positive T cell Responses and Impact the Growth and Spread of Her-2/neu-positive Breast Tumors

Purpose: The aim of this study was to efficiently design a novel vaccine for human Her-2/neu-positive (hHer-2/neu) breast cancer using the live, attenuated bacterial vector Listeria monocytogenes. Experimental Design: Three recombinant L. monocytogenes–based vaccines were generated that could express and secrete extracellular and intracellular fragments of the hHer-2/neu protein. In addition, we generated a fourth construct fusing selected portions of each individual fragment that contained most of the human leukocyte antigen (HLA) epitopes as a combination vaccine (L. monocytogenes–hHer-2/neu chimera). Results: Each individual vaccine was able to either fully regress or slow tumor growth in a mouse model for Her-2/neu-positive tumors. All three vaccines could elicit immune responses directed toward human leukocyte antigen-A2 epitopes of hHer-2/neu. The L. monocytogenes–hHer-2/neu chimera was able to mimic responses generated by the three separate vaccines and prevent spontaneous outgrowth of tumors in an autochthonous model for Her-2/neu-positive breast cancer, induce tumor regression in transplantable models, and prevent seeding of experimental lung metastases in a murine model for metastatic breast cancer. Conclusion: This novel L. monocytogenes–hHer-2/neu chimera vaccine proves to be just as effective as the individual vaccines but combines the strength of all three in a single vaccination. These encouraging results support future clinical trials using this chimera vaccine and may be applicable to other cancer types expressing the Her-2/neu molecule such as colorectal and pancreatic cancer.

An Anti-Vascular Endothelial Growth Factor Receptor 2/Fetal Liver Kinase-1 Listeria monocytogenes Anti-Angiogenesis Cancer Vaccine for the Treatment of Primary and Metastatic Her-2/neu+ Breast Tumors in a Mouse Model

Thirty years after angiogenesis was shown to play an enabling role in cancer, modern medicine is still trying to develop novel compounds and therapeutics to target the tumor vasculature. However, most therapeutics require multiple rounds of administration and can have toxic side effects. In this study, we use anti-angiogenesis immunotherapy to target cells actively involved in forming new blood vessels that support the growth and spread of breast cancer. Targeting a central cell type involved in angiogenesis, endothelial cells, we immunized against host vascular endothelial growth factor receptor 2 to fight the growth of Her-2/neu+ breast tumors. Using the bacterial vector, Listeria monocytogenes (Lm), we fused polypeptides from the mouse vascular endothelial growth factor receptor 2 molecule (fetal liver kinase-1) to the microbial adjuvant, listeriolysin-O, and used Lm to deliver the Ags and elicit potent antitumor CTL responses. Lm-listeriolysin-O-fetal liver kinase-1 was able to eradicate some established breast tumors, reduce microvascular density in the remaining tumors, protect against tumor rechallenge and experimental metastases, and induce epitope spreading to various regions of the tumor-associated Ag Her-2/neu. Tumor eradication was found to be dependent on epitope spreading to HER-2/neu and was not solely due to the reduction of tumor vasculature. However, vaccine efficacy did not affect normal wound healing nor have toxic side effects on pregnancy. We show that an anti-angiogenesis vaccine can overcome tolerance to the host vasculature driving epitope spreading to an endogenous tumor protein and drive active tumor regression.

Listeria monocytogenes Delivery of HPV-16 Major Capsid Protein L1 Induces Systemic and Mucosal Cell-Mediated CD4+ and CD8+ T-Cell Responses After Oral Immunization

Neutralizing antibodies are thought to be required at mucosal surfaces to prevent human papillomavirus (HPV) transmission. However, the potential for cell-mediated immunity in mediating protection against HPV infection has not been well explored. We generated recombinant Listeria monocytogenes (Lm) constructs that secrete listeriolysin O (LLO) fused with overlapping N-terminal (LLO-L1(1-258)) or C-terminal (LLO-L1(238-474)) fragments of HPV type 16 major capsid protein L1 (HPV-16-L1). Oral immunization of mice with either construct induced IFN-gamma-producing CD8+ and CD4+ T cells in the spleen and in the Peyers patches with the C-terminal construct. Oral immunization with both constructs resulted in diminished viral titers in the cervix and uterus of mice after intravaginal challenge with vaccinia virus expressing HPV-16-L1.

Listeria monocytogenes-derived listeriolysin O has pathogen-associated molecular pattern-like properties independent of its hemolytic ability.

ABSTRACT There is a constant need for improved adjuvants to augment the induction of immune responses against tumor-associated antigens (TAA) during immunotherapy. Previous studies have established that listeriolysin O (LLO), a cholesterol-dependent cytolysin derived from Listeria monocytogenes, exhibits multifaceted effects to boost the stimulation of immune responses to a variety of antigens. However, the direct ability of LLO as an adjuvant and whether it acts as a pathogen-associated molecular pattern (PAMP) have not been demonstrated. In this paper, we show that a detoxified, nonhemolytic form of LLO (dtLLO) is an effective adjuvant in tumor immunotherapy and may activate innate and cellular immune responses by acting as a PAMP. Our investigation of the adjuvant activity demonstrates that dtLLO, either fused to or administered as a mixture with a human papillomavirus type 16 (HPV-16) E7 recombinant protein, can augment antitumor immune responses and facilitate tumor eradication. Further mechanistic studies using bone marrow-derived dendritic cells suggest that dtLLO acts as a PAMP by stimulating production of proinflammatory cytokines and inducing maturation of antigen-presenting cells (APC). We propose that dtLLO is an effective adjuvant for tumor immunotherapy, and likely for other therapeutic settings.

Effect of a Novel DNA Vaccine on Angiogenesis and Tumor Growth In Vivo

OBJECTIVE To develop a DNA cancer vaccine that targets the vascular endothelial growth factor receptor. DESIGN Mice were vaccinated intramuscularly with listeriolysin O-fetal liver kinase 1 (LLO-Flk1) or controls. Mice were also challenged subcutaneously with the tumor cell line TC-1. Tumor sizes were measured after vaccination. At the conclusion of the experiments, the tumors were harvested for immunohistochemical analysis and determination of hemoglobin content. SETTING Research laboratory. SUBJECTS Six- to 8-week-old C57BL/6 mice. INTERVENTION Fifty micrograms of each vector was administered 3 times at weekly intervals. MAIN OUTCOME MEASURES Tumor size, mean vessel density of tumors, hemoglobin content of tumor. RESULTS Mice treated with the LLO-Flk1 vaccine experienced slower tumor growth relative to the other treatment groups. Complete tumor regression was observed in several cases. Immunohistochemical staining of tumors revealed fewer blood vessels in the mice vaccinated with the LLO-Flk1 vaccine relative to the other treatment groups. Finally, colorimetric assessment for hemoglobin suggested decreased vasculature in the tumor bed of these mice relative to the control groups. CONCLUSION The novel DNA cancer vaccine LLO-Flk1 can slow tumor growth in vivo.

Targeting Bcl-2 for the treatment of multiple myeloma

Despite advances in the treatment of multiple myeloma, the disease still remains incurable for the majority of patients. The overexpression of anti-apoptotic proteins (i.e., Bcl-2, Bcl-XL or Mcl-1) is a hallmark of cancer and favors tumor cell survival and resistance to therapy. The oral drug venetoclax is the first-in-class Bcl-2-specific BH3 mimetic. In myeloma, in vitro sensitivity to venetoclax is mainly observed in plasma cells harboring the t(11;14) translocation, a molecular subgroup associated with high Bcl-2 and low Mcl-1/Bcl-XL expression. In addition with Bcl-2 members expression profile, functional tests as BH3 profiling or in vitro BH3 mimetic drug testing also predict sensitivity to the drug. Phase 1 clinical trials recently confirmed the efficacy of venetoclax monotherapy in heavily pretreated myeloma patients, mostly in patients with t(11;14). In combination with the proteasome inhibitor bortezomib, venetoclax therapy was found to be feasible and allowed promising response rate in relapsed myeloma patients, independent of t(11;14) status. The present review summarizes the current knowledge, “from bench to bedside”, about venetoclax for the treatment of multiple myeloma.

Abstract CT122:BCL2expression is a potential predictive biomarker of response to venetoclax in combination with bortezomib and dexamethasone in patients with relapsed/refractory multiple myeloma

The anti-apoptotic proteins BCL-2 and MCL-1 have been shown to promote multiple myeloma (MM) cell survival. Venetoclax (VEN) is a potent, selective, and orally bioavailable small-molecule inhibitor of BCL-2. Bortezomib (BTZ) is a proteasome inhibitor that can inhibit MCL-1 activity by increasing the MCL-1 antagonist, NOXA. Results presented herein describe correlative biomarker analyses in the ongoing phase 1b study of VEN in combination with BTZ and dexamethasone in patients with relapsed/refractory (R/R) MM (NCT01794507). As of 19 Aug 2016, 66 patients were enrolled on study. Baseline bone marrow aspirate samples were available from 52 patients, of which 45 were evaluable for BCL-2 family gene expression by droplet digital PCR in CD138-selected tumor cells. Correlation between BCL2 (BCL-2), BCL2L1 (BCL-XL) and MCL1 (MCL-1) mRNA expression (log2-transformed copies/ul normalized to housekeeping gene) and preliminary efficacy [overall response rate (ORR), time to disease progression (TTP) and duration of response (DoR)] were examined by Log-rank and Wilcoxon tests for binary biomarkers, and by risk ratio from Cox proportional hazard model for continuous biomarkers. The ORR was 68% (44/65) for all evaluable patients and 89% (31/35) in patients who had 1-3 prior therapies (31/35). A broad range of BCL2, BCL2L1 and MCL1 expression was observed, however higher BCL2 levels were detected in patients who achieved a partial response (PR) or better (median: 3.01 vs 0.87, p Citation Format: Jeremy Ross, Brenda Chyla, Rasna Goswami, Lisa Roberts-Rapp, Yan Sun, Yanwen Jiang, Elizabeth Punnoose, Maria Verdugo, Anahita Bhathena, Paulo Maciag. BCL2 expression is a potential predictive biomarker of response to venetoclax in combination with bortezomib and dexamethasone in patients with relapsed/refractory multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT122. doi:10.1158/1538-7445.AM2017-CT122