Journal Articles

Immunotherapy design

Novel approaches and designs of biologicals used for cancer immunotherapy as well as changes in the timing, combination sequence, adjuvant choice or route of immunization in immunotherapy regimens; biomarkers of response to therapy

Reprogramming the murine colon cancer microenvironment using lentivectors encoding shRNA against IL-10 as a component of a potent DC-based chemoimmunotherapy

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BACKGROUND: The excessive amounts of immunosuppressive factors present in a tumor microenvironment (TME) reduce the effectiveness of cancer vaccines. The main objective of our research was to improve the effectiveness of dendritic cell (DC)-based immunotherapy or chemoimmunotherapy composed of cyclophosphamide (CY) and DCs by application of lentivectors encoding shRNA specific to IL-10 (shIL10 LVs) in murine colon carcinoma MC38 model. METHODS: The efficacy of shIL10 LVs in silencing of IL-10 expression was measured both in vitro and in vivo using Real-Time PCR and ELISA assays. In addition, the influence of intratumorally inoculated lentivectors on MC38 tumor microenvironment was examined using flow cytometry method. The effect of applied therapeutic schemes was determined by measurement of tumor growth inhibition and activation state of local and systemic immune response. RESULTS: We observed that intratumorally inoculated shIL10 LVs transduced tumor and TME-infiltrating cells and reduced the secretion of IL-10. Application of shIL10 LVs for three consecutive weeks initiated tumor growth inhibition, whereas treatment with shIL10 LVs and BMDC/TAg did not enhance the antitumor effect. However, when pretreatment with CY was introduced to the proposed scheme, we noticed high MC38 tumor growth inhibition accompanied by reduction of MDSCs and Tregs in TME, as well as activation of potent local and systemic Th1-type antitumor response. CONCLUSIONS: The obtained data shows that remodeling of TME by shIL10 LVs and CY enhances DC activity and supports them during regeneration and actuation of a potent antitumor response. Therefore, therapeutic strategies aimed at local IL-10 elimination using lentiviral vectors should be further investigated in context of combined chemoimmunotherapies.

Author Info: (1) Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12, 53-114, Wroclaw, Poland. joanna@iitd.pan.wroc.pl. (2) Hirszfeld Institute of Immunology

Author Info: (1) Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12, 53-114, Wroclaw, Poland. joanna@iitd.pan.wroc.pl. (2) Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12, 53-114, Wroclaw, Poland. (3) Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12, 53-114, Wroclaw, Poland. (4) Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12, 53-114, Wroclaw, Poland. (5) Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12, 53-114, Wroclaw, Poland.

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An Anti-CLL-1 Antibody-Drug Conjugate for the Treatment of Acute Myeloid Leukemia

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PURPOSE: The treatment of acute myeloid leukemia (AML) has not significantly changed in 40 years. Cytarabine and anthracyclinebased chemotherapy induction regimens (7 + 3) remain the standard of care, and most patients have poor long-term survival. The re-approval of Mylotarg, an anti-CD33-calicheamicin antibody-drug conjugate (ADC), has demonstrated ADCs as a clinically validated option to enhance the effectiveness of induction therapy. We are interested in developing a next generation ADC for AML to improve upon the initial success of Mylotarg. EXPERIMENTAL DESIGN: The expression pattern of CLL-1 and its hematopoietic potential were investigated. A novel anti-CLL-1-ADC, with a highly potent pyrrolobenzodiazepine (PBD) dimer conjugated through a self-immolative disulfide linker, was developed. The efficacy and safety profiles of this ADC were evaluated in mouse xenograft models and in cynomolgus monkeys. RESULTS: We demonstrate that CLL-1 shares similar prevalence and trafficking properties that make CD33 an excellent ADC target for AML, but lacks expression on hematopoietic stem cells that hampers current CD33 targeted ADCs. Our anti-CLL-1-ADC is highly effective at depleting tumor cells in AML xenograft models and lacks target independent toxicities at doses that depleted target monocytes and neutrophils in cynomolgus monkeys. CONCLUSIONS: Collectively, our data suggest that an anti-CLL-1-ADC has the potential to become an effective and safer treatment for AML in humans, by reducing and allowing for faster recovery from initial cytopenias than the current generation of ADCs for AML.

Author Info: (1) Translational Oncology & Cancer Signaling, Genentech Inc. (2) Translational Oncology, Genentech, Inc. (3) In Vivo Pharmacology, Genentech. (4) Research and Early Development, Genentech, Inc

Author Info: (1) Translational Oncology & Cancer Signaling, Genentech Inc. (2) Translational Oncology, Genentech, Inc. (3) In Vivo Pharmacology, Genentech. (4) Research and Early Development, Genentech, Inc. (5) Translational Oncology, Genentech, Inc. (6) Research and Early Development, Genentech, Inc. (7) Research and Early Development, Genentech, Inc. (8) Antibody Engineering, Genentech, Inc. (9) Antibody Engineering, Genentech, Inc. (10) Pathology, Genentech. (11) Genentech, Inc. (12) Safety Assessment, Genentech, Inc. (13) Research and Early Development, Genentech, Inc. (14) Safety Assessment, Amgen, Inc. (15) Oncology Clinical Science, Genentech Inc. (16) Genentech, Inc. (17) Safety Assessment, Genentech. (18) Genentech, Inc. (19) Research and Early Development, Genentech, Inc. polson@gene.com.

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Glycogen Synthase Kinase 3 Inhibition Lowers PD-1 Expression, Promotes Long-term Survival and Memory Generation in Antigen-specific CAR-T cells

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Successful remission in hematological cancers by CAR-T cell immunotherapy has yet to be replicated in solid tumors like GBM. A significant impediment of CAR-T immunotherapy in solid tumors is poor exposure of T cells to tumor antigens resulting in suboptimal CAR-T cell activation, which ultimately fails to induce a robust anti-tumor immune response. Costimulatory moieties in advanced-generation CARs, along with additional IL2 therapy has been shown to be insufficient to overcome this hurdle and have its cytotoxic limitations. GSK3 is constitutively active in naive T cells and is transiently inactivated during T cell activation resulting in rapid T cell proliferation. Pharmacologic inhibition of GSK3 in GBM-specific CAR-T cells reduced FasL expression, increased T cell proliferation and reduced exhaustion by lowering PD-1 levels resulting in the development of CAR-T effector memory phenotype. Treatment with GSK3-inhibited CAR-T cells resulted in 100% tumor elimination during the tumor-rechallenge experiment in GBM-bearing animals and increased accumulation of memory CAR-T cells in secondary lymphoid organs. These adjuvant-like effects of GSK3 inhibition on activated CAR-T cells may be a valuable adjunct to a successful implementation of CAR-T immunotherapy against GBM and other solid tumors.

Author Info: (1) Brain Tumor Laboratory, Roger Williams Medical Center, Providence, RI, USA; Department of Neurosurgery, Alpert School of Medicine, Brown University, Providence, RI, USA. Electronic address

Author Info: (1) Brain Tumor Laboratory, Roger Williams Medical Center, Providence, RI, USA; Department of Neurosurgery, Alpert School of Medicine, Brown University, Providence, RI, USA. Electronic address: sadhak_sengupta@brown.edu. (2) Department of Surgery, Roger Williams Medical Center, Providence, RI, USA; Department of Surgery, Boston University School of Medicine, Boston, MA, USA. (3) Brain Tumor Laboratory, Roger Williams Medical Center, Providence, RI, USA. (4) Brain Tumor Laboratory, Roger Williams Medical Center, Providence, RI, USA; Department of Neurosurgery, Alpert School of Medicine, Brown University, Providence, RI, USA.

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Therapeutic vaccines and immune checkpoints inhibition options for gynecological cancers

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Treatments for gynecological cancer include surgery, chemotherapy, and radiation. However, overall survival is not improved, and novel approaches are needed. Immunotherapy has been proven efficacious in various types of cancers and multiple approaches have been recently developed. Since numerous gynecological cancers are associated to human papilloma virus (HPV) infections, therapeutic vaccines, targeting HPV epitopes, have been developed. The advancing understanding of the immune system, regulatory pathways and tumor microenvironment have produced a major interest in immune checkpoint blockade, Indeed, immune checkpoint molecules are important clinical targets in a wide variety of tumors, including gynecological. In this review, we will describe the immunotherapeutic targets and modalities available and review the most recent immunotherapeutic clinical trials in the context of gynecological cancers. The synergic results obtained from the combination of HPV therapeutic vaccines with radiotherapy, chemotherapy, or immune checkpoint inhibitors, may underlie the potential for a novel therapeutic scenario for these tumors.

Author Info: (1) Department of Gynecological and Obstetric Sciences, and Urological Sciences, University of Rome "Sapienza", Umberto I Hospital, Rome, Italy. Electronic address: chiara.ditucci@uniroma1.it. (2) Department of

Author Info: (1) Department of Gynecological and Obstetric Sciences, and Urological Sciences, University of Rome "Sapienza", Umberto I Hospital, Rome, Italy. Electronic address: chiara.ditucci@uniroma1.it. (2) Department of Gynecological and Obstetric Sciences, and Urological Sciences, University of Rome "Sapienza", Umberto I Hospital, Rome, Italy. Electronic address: michelecarlo.schiavi@uniroma1.it. (3) Department of Gynecological and Obstetric Sciences, and Urological Sciences, University of Rome "Sapienza", Umberto I Hospital, Rome, Italy. Electronic address: pierangelo.faiano@uniroma1.it. (4) Department of Gynecological and Obstetric Sciences, and Urological Sciences, University of Rome "Sapienza", Umberto I Hospital, Rome, Italy. Electronic address: ottavia.doria@uniroma1.it. (5) Department of Gynecological and Obstetric Sciences, and Urological Sciences, University of Rome "Sapienza", Umberto I Hospital, Rome, Italy. Electronic address: giovanni.prata@uniroma1.it. (6) Department of Gynecological and Obstetric Sciences, and Urological Sciences, University of Rome "Sapienza", Umberto I Hospital, Rome, Italy. Electronic address: valentina.sciuga@uniroma1.it. (7) Department of Gynecological and Obstetric Sciences, and Urological Sciences, University of Rome "Sapienza", Umberto I Hospital, Rome, Italy. (8) Department of Gynecological and Obstetric Sciences, and Urological Sciences, University of Rome "Sapienza", Umberto I Hospital, Rome, Italy. (9) Department of Gynecological and Obstetric Sciences, and Urological Sciences, University of Rome "Sapienza", Umberto I Hospital, Rome, Italy. (10) Department of Gynecological and Obstetric Sciences, and Urological Sciences, University of Rome "Sapienza", Umberto I Hospital, Rome, Italy.

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TLS11a Aptamer/CD3 Antibody Anti-Tumor System for Liver Cancer

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New therapeutic approaches are needed for hepatocellular carcinoma (HCC), which is the most common primary malignancy of the liver. Bispecific T-cell engagers (BiTE) can effectively redirect T cells against tumors and show a strong anti-tumor effect. However, the potential immunogenicity, complexity, and high cost significantly limit their clinical application. In this paper, we used the hepatoma cells-specific aptamer TLS11a and anti-CD3 for to establish an aptamer/antibody bispecific system (AAbs), TLS11a/CD3, which showed advantages over BiTE and can specifically redirect T cells to lyse tumor cells. TLS11a-SH and anti-CD3-NH2 were crosslinked with sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC). T cell activation, proliferation, and cytotoxicity of TLS11a/CD3 were analyzed by flow cytometry. Cytokine array was used to detect cytokine released from activated T cells. Hepatoma xenograft model was used to monitor the tumor volume and survival. TLS11a/CD3 could specifically bind hepatoma cells (H22) and T cells, activated T cells to mediate antigen-specific lysis of H22 cells in vitro, and effectively inhibited the growth of implanted H22 tumors as well as prolonged mice survival. TLS11a/CD3 could simultaneously target hepatoma cells and T cells, specifically guide T cells to kill tumor cells, and enhance the anti-tumor effect of T cells both in vitro and in vivo.

Author Info: (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13)

Author Info: (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13)

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Functionalized Multi-Walled Carbon Nanotubes for Targeting Delivery of Immunostimulatory CpG Oligonucleotides Against Prostate Cancer

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Immuno-based oncotherapy has been successfully implemented for cancer treatment. In the present study, we developed a Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs (CpG ODNs) nano-delivery system based on Multi-walled carbon nanotubes (MWCNTs) conjugated with H3R6 polypeptide (MHR-CpG) for prostate cancer immunotherapy. The in vitro and in vivo toxicity data revealed that the prepared MHR showed high biocompatibility. Confocal laser scanning microscopy confirmed that MHR-CpG could specifically target the endosomal TLR9. In addition, the use of MHR enhanced the immunogenicity of CpG in both humoral and cellular immune pathways, as evidenced by the increased expression of CD4+ T-cells, CD8+ T-cells, TNF-alpha, and IL-6. The in vivo anti-cancer efficacy study on RM-1 tumor-bearing mice demonstrated that MHR-CpG could deliver the immunotherapeutics to the tumor site and the tumor-draining lymph node to suppress tumor growth. These results suggested that MHR-CpG was a promising multifunctional nano system for prostate cancer immunotherapy.

Author Info: (1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

Author Info: (1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

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Size-Dependent Segregation Controls Macrophage Phagocytosis of Antibody-Opsonized Targets

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Macrophages protect the body from damage and disease by targeting antibody-opsonized cells for phagocytosis. Though antibodies can be raised against antigens with diverse structures, shapes, and sizes, it is unclear why some are more effective at triggering immune responses than others. Here, we define an antigen height threshold that regulates phagocytosis of both engineered and cancer-specific antigens by macrophages. Using a reconstituted model of antibody-opsonized target cells, we find that phagocytosis is dramatically impaired for antigens that position antibodies >10 nm from the target surface. Decreasing antigen height drives segregation of antibody-bound Fc receptors from the inhibitory phosphatase CD45 in an integrin-independent manner, triggering Fc receptor phosphorylation and promoting phagocytosis. Our work shows that close contact between macrophage and target is a requirement for efficient phagocytosis, suggesting that therapeutic antibodies should target short antigens in order to trigger Fc receptor activation through size-dependent physical segregation.

Author Info: (1) Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA; UC Berkeley/UC San Francisco Graduate Group in Bioengineering, Berkeley, CA 94720, USA. (2)

Author Info: (1) Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA; UC Berkeley/UC San Francisco Graduate Group in Bioengineering, Berkeley, CA 94720, USA. (2) Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA; UC Berkeley/UC San Francisco Graduate Group in Bioengineering, Berkeley, CA 94720, USA. (3) Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA. (4) Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA. (5) Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA. (6) Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA; UC Berkeley/UC San Francisco Graduate Group in Bioengineering, Berkeley, CA 94720, USA; Division of Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA. Electronic address: fletch@berkeley.edu.

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Modulation of the tumor microenvironment by intratumoral administration of IMO-2125, a novel TLR9 agonist, for cancer immunotherapy

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The objective of cancer immunotherapy is to prime the host's immune system to recognize and attack malignant tumor cells. IMO2125, a Tolllike receptor 9 (TLR9) agonist, exhibited potent antitumor effects in the murine syngeneic A20 lymphoma and the CT26 colon carcinoma models. IMO2125 exhibited superior A20 antitumor activity when injected intratumorally (i.t.) compared with equivalent subcutaneous doses. In mice bearing dual CT26 grafts, the i.t. injection of right flank tumors elicited infiltration of cluster of differentiation (CD)3+ T lymphocytes into tumors, resulting in the regression of injected and uninjected left flank tumors. Depletion of CD8+, but not CD4+, Tcells abrogated the IMO2125mediated antitumor response, suggesting that CD8+ lymphocytes are required for the antitumor activity. In mice harboring right flank CT26 and left flank betagalactosidase (betagal)expressing CT26.CL25 grafts, the i.t. administration of IMO2125 to the CT26 graft resulted in potent and dosedependent antitumor activity against the two grafts. Splenic Tcells isolated from these mice responded to AH1 antigen (present in the two tumors) and betagal antigen (present only in CT26.CL25) in an interferon gamma enzymelinked immunospot assay, suggesting the clonal expansion of Tcells directed against antigens from the two tumors. Mice with ablated CT26 tumors by previous IMO2125 treatment rejected reimplanted CT26 tumor cells, but not A20 tumor cells, demonstrating that the initial IMO2125 treatment created a longlived tumorspecific immune memory of CT26 antigens. A quantitative increase in CD3+ T lymphocytes in injected A20 tumors and an upregulation of selected checkpoint genes, including indoleamine 2,3dioxygenase (IDO)1, IDO2, programmed cell death protein-1 (PD-1); programmed cell death protein ligand 1 (PD-L1), carcinoembryonic antigenrelated cell adhesion molecule 1, tumor necrosis factor receptor superfamily member 4 (OX40), OX40 ligand, Tcell immunoglobulin and mucindomaincontaining 3 protein, lymphocyteactivation gene 3, cytotoxic Tlymphocyteassociated protein 4, were observed following IMO2125 treatment. IMO2125 also increased immune checkpoint gene expression in injected and uninjected contralateral CT26 tumors, suggesting that the coadministration of antiCTLA4, antiPD1 or antiPDL1 therapies with IMO2125 may provide additional therapeutic efficacy.

Author Info: (1) Idera Pharmaceuticals, Inc., Cambridge, MA 02139, USA. (2) Idera Pharmaceuticals, Inc., Cambridge, MA 02139, USA. (3) Idera Pharmaceuticals, Inc., Cambridge, MA 02139, USA. (4)

Author Info: (1) Idera Pharmaceuticals, Inc., Cambridge, MA 02139, USA. (2) Idera Pharmaceuticals, Inc., Cambridge, MA 02139, USA. (3) Idera Pharmaceuticals, Inc., Cambridge, MA 02139, USA. (4) Idera Pharmaceuticals, Inc., Cambridge, MA 02139, USA. (5) Idera Pharmaceuticals, Inc., Cambridge, MA 02139, USA.

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A Consistent Method to Identify and Isolate Mononuclear Phagocytes from Human Lung and Lymph Nodes

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Mononuclear phagocytes (MP) consist of macrophages, dendritic cells (DCs), and monocytes. In all organs, including the lung, there are multiple subtypes within these categories. The existence of all these cell types suggest that there is a clear division of labor and delicate balance between the MPs under steady state and inflammatory conditions. Although great strides have been made to understand MPs in the mouse lung, and human blood, little is known about the MPs that exist in the human lung and lung-draining lymph nodes (LNs), and even less is known about their functional roles, studies of which will require a large number of sorted cells. We have comprehensively examined cell surface markers previously used in a variety of organs to identify human pulmonary MPs. In the lung, we consistently identify five extravascular pulmonary MPs and three LN MPs. These MPs were present in over 100 lungs regardless of age or gender. Notably, the human blood CD141(+) DCs, as described in the literature, were not observed in non-diseased lungs or their draining LNs. In the lung and draining LNs, expression of CD141 was only observed on HLADR(+) CD11c(+) CD14(+) extravascular monocytes (often confused in the LN as resident DCs based on the level of HLADR expression and mouse LN data). In the human lung and LNs there are at least two DC subtypes expressing HLADR, DEC205 and CD1c, along with circulating monocytes that behave as either antigen-presenting cells or macrophages. Furthermore, we demonstrate how to distinguish between alveolar macrophages and interstitial macrophage subtypes. It still remains unclear how the human pulmonary MPs identified here align with mouse MPs. Clearly, we are now past the stage of cell surface marker characterization, and future studies will need to move toward understanding what these cell types are and how they function. Our hope is that the strategy described here can help the pulmonary community take this next step.

Author Info: (1) Department of Pediatrics, National Jewish Health, Denver, CO, USA. (2) Department of Pediatrics, National Jewish Health, Denver, CO, USA. jakubzickc@njhealth.org. Department of Microbiology and

Author Info: (1) Department of Pediatrics, National Jewish Health, Denver, CO, USA. (2) Department of Pediatrics, National Jewish Health, Denver, CO, USA. jakubzickc@njhealth.org. Department of Microbiology and Immunology, University of Colorado, Denver, CO, USA. jakubzickc@njhealth.org.

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Glutamic acid-valine-citrulline linkers ensure stability and efficacy of antibody-drug conjugates in mice

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Valine-citrulline linkers are commonly used as enzymatically cleavable linkers for antibody-drug conjugates. While stable in human plasma, these linkers are unstable in mouse plasma due to susceptibility to an extracellular carboxylesterase. This instability often triggers premature release of drugs in mouse circulation, presenting a molecular design challenge. Here, we report that an antibody-drug conjugate with glutamic acid-valine-citrulline linkers is responsive to enzymatic drug release but undergoes almost no premature cleavage in mice. We demonstrate that this construct exhibits greater treatment efficacy in mouse tumor models than does a valine-citrulline-based variant. Notably, our antibody-drug conjugate contains long spacers facilitating the protease access to the linker moiety, indicating that our linker assures high in vivo stability despite a high degree of exposure. This technology could add flexibility to antibody-drug conjugate design and help minimize failure rates in pre-clinical studies caused by linker instability.

Author Info: (1) Texas Therapeutics Institute, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 1881 East Road, Houston, TX

Author Info: (1) Texas Therapeutics Institute, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 1881 East Road, Houston, TX, 77054, USA. (2) Texas Therapeutics Institute, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 1881 East Road, Houston, TX, 77054, USA. (3) Texas Therapeutics Institute, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 1881 East Road, Houston, TX, 77054, USA. (4) Texas Therapeutics Institute, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 1881 East Road, Houston, TX, 77054, USA. (5) Texas Therapeutics Institute, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 1881 East Road, Houston, TX, 77054, USA. (6) Texas Therapeutics Institute, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 1881 East Road, Houston, TX, 77054, USA. (7) Texas Therapeutics Institute, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 1881 East Road, Houston, TX, 77054, USA. Kyoji.Tsuchikama@uth.tmc.edu.

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