Efficacy and pharmacodynamic effect of anti-CD73 and anti-PD-L1 monoclonal antibodies in combination with cytotoxic therapy: observations from mouse tumor models.

CD73 is a cell surface 5'nucleotidase (NT5E) and key node in the catabolic process generating immunosuppressive adenosine in cancer. Using a murine monoclonal antibody surrogate of Oleclumab, we investigated the effect of CD73 inhibition in concert with cytotoxic therapies (chemotherapies as well as fractionated radiotherapy) and PD-L1 blockade. Our results highlight improved survival in syngeneic tumor models of colorectal cancer (CT26 and MC38) and sarcoma (MCA205). This therapeutic outcome was in part driven by cytotoxic CD8 T-cells, as evidenced by the detrimental effect of CD8 depleting antibody treatment of MCA205 tumor bearing mice treated with anti-CD73, anti-PD-L1 and 5-Fluorouracil+Oxaliplatin (5FU+OHP). We hypothesize that the improved responses are tumor microenvironment (TME)-driven, as suggested by the lack of anti-CD73 enhanced cytopathic effects mediated by 5FU+OHP on cell lines in vitro. Pharmacodynamic analysis, using imaging mass cytometry and RNA-sequencing, revealed noteworthy changes in specific cell populations like cytotoxic T cells, B cells and NK cells in the CT26 TME. Transcriptomic analysis highlighted treatment-related modulation of gene profiles associated with an immune response, NK and T-cell activation, T cell receptor signaling and interferon (types 1 & 2) pathways. Inclusion of comparator groups representing the various components of the combination allowed deconvolution of contribution of the individual therapeutic elements; highlighting specific effects mediated by the anti-CD73 antibody with respect to immune-cell representation, chemotaxis and myeloid biology. These pre-clinical data reflect complementarity of adenosine blockade with cytotoxic therapy, and T-cell checkpoint inhibition, and provides new mechanistic insights in support of combination therapy.

CD73 controls Myosin II-driven invasion, metastasis, and immunosuppression in amoeboid pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) has a very poor prognosis because of its high propensity to metastasize and its immunosuppressive microenvironment. Using a panel of pancreatic cancer cell lines, three-dimensional (3D) invasion systems, microarray gene signatures, microfluidic devices, mouse models, and intravital imaging, we demonstrate that ROCK-Myosin II activity in PDAC cells supports a transcriptional program conferring amoeboid invasive and immunosuppressive traits and in vivo metastatic abilities. Moreover, we find that immune checkpoint CD73 is highly expressed in amoeboid PDAC cells and drives their invasive, metastatic, and immunomodulatory traits. Mechanistically, CD73 activates RhoA-ROCK-Myosin II downstream of PI3K. Tissue microarrays of human PDAC biopsies combined with bioinformatic analysis reveal that rounded-amoeboid invasive cells with high CD73-ROCK-Myosin II activity and their immunosuppressive microenvironment confer poor prognosis to patients. We propose targeting amoeboid PDAC cells as a therapeutic strategy.

Defining the spatial distribution of extracellular adenosine revealed a myeloid-dependent immunosuppressive microenvironment in pancreatic ductal adenocarcinoma.

BACKGROUND: The prognosis for patients with pancreatic ductal adenocarcinoma (PDAC) remains extremely poor. It has been suggested that the adenosine pathway contributes to the ability of PDAC to evade the immune system and hence, its resistance to immuno-oncology therapies (IOT), by generating extracellular adenosine (eAdo). METHODS: Using genetically engineered allograft models of PDAC in syngeneic mice with defined and different immune infiltration and response to IOT and autochthonous tumors in KPC mice we investigated the impact of the adenosine pathway on the PDAC tumor microenvironment (TME). Flow cytometry and imaging mass cytometry (IMC) were used to characterize the subpopulation frequency and spatial distribution of tumor-infiltrating immune cells. Mass spectrometry imaging (MSI) was used to visualize adenosine compartmentalization in the PDAC tumors. RNA sequencing was used to evaluate the influence of the adenosine pathway on the shaping of the immune milieu and correlate our findings to published data sets in human PDAC. RESULTS: We demonstrated high expression of adenosine pathway components in tumor-infiltrating immune cells (particularly myeloid populations) in the murine models. MSI demonstrated that extracellular adenosine distribution is heterogeneous in tumors, with high concentrations in peri-necrotic, hypoxic regions, associated with rich myeloid infiltration, demonstrated using IMC. Protumorigenic M2 macrophages express high levels of the Adora2a receptor; particularly in the IOT resistant model. Blocking the in vivo formation and function of eAdo (Adoi), using a combination of anti-CD73 antibody and an Adora2a inhibitor slowed tumor growth and reduced metastatic burden. Additionally, blocking the adenosine pathway improved the efficacy of combinations of cytotoxic agents or immunotherapy. Adoi remodeled the TME, by reducing the infiltration of M2 macrophages and regulatory T cells. RNA sequencing analysis showed that genes related to immune modulation, hypoxia and tumor stroma were downregulated following Adoi and a specific adenosine signature derived from this is associated with a poorer prognosis in patients with PDAC. CONCLUSIONS: The formation of eAdo promotes the development of the immunosuppressive TME in PDAC, contributing to its resistance to conventional and novel therapies. Therefore, inhibition of the adenosine pathway may represent a strategy to modulate the PDAC immune milieu and improve therapy response in patients with PDAC.

CD73 and PD-L1 dual blockade amplifies antitumor efficacy of SBRT in murine PDAC models.

BACKGROUND: Stereotactic body radiotherapy (SBRT) induces immunogenic cell death, leading to subsequent antitumor immune response that is in part counterbalanced by activation of immune evasive processes, for example, upregulation of programmed cell death-ligand 1 (PD-L1) and adenosine generating enzyme, CD73. CD73 is upregulated in pancreatic ductal adenocarcinoma (PDAC) compared with normal pancreatic tissue and high expression of CD73 in PDACs is associated with increased tumor size, advanced stage, lymph node involvement, metastasis, PD-L1 expression and poor prognosis. Therefore, we hypothesized that blockade of both CD73 and PD-L1 in combination with SBRT might improve antitumor efficacy in an orthotopic murine PDAC model. METHODS: We assessed the combination of systemic blockade of CD73/PD-L1 and local SBRT on tumor growth in primary pancreatic tumors, and investigated systemic antitumor immunity using a metastatic murine model bearing both orthotopic primary pancreatic tumor and distal hepatic metastases. Immune response was quantified by flow cytometric and Luminex analyses. RESULTS: We demonstrated that blockade of both CD73 and PD-L1 significantly amplified the antitumor effect of SBRT, leading to superior survival. The triple therapy (SBRT+anti-CD73+anti-PD-L1) modulated tumor-infiltrating immune cells with increases of interferon-γ+CD8+ T cells. Additionally, triple therapy reprogramed the profile of cytokines/chemokines in the tumor microenvironment toward a more immunostimulatory phenotype. The beneficial effects of triple therapy are completely abrogated by depletion of CD8+ T cells, and partially reversed by depletion of CD4+ T cells. Triple therapy promoted systemic antitumor responses illustrated by: (1) potent long-term antitumor memory and (2) enhanced both primary and liver metastases control along with prolonged survival.

Recombinant Newcastle Disease Virus Immunotherapy Drives Oncolytic Effects and Durable Systemic Antitumor Immunity.

A recombinant Newcastle Disease Virus (NDV), encoding either a human (NDVhuGM-CSF, MEDI5395) or murine (NDVmuGM-CSF) GM-CSF transgene, combined broad oncolytic activity with the ability to significantly modulate genes related to immune functionality in human tumor cells. Replication in murine tumor lines was significantly diminished relative to human tumor cells. Nonetheless, intratumoral injection of NDVmuGM-CSF conferred antitumor effects in three syngeneic models in vivo; with efficacy further augmented by concomitant treatment with anti-PD-1/PD-L1 or T-cell agonists. Ex vivo immune profiling, including T-cell receptor sequencing, revealed profound immune-contexture changes consistent with priming and potentiation of adaptive immunity and tumor microenvironment (TME) reprogramming toward an immune-permissive state. CRISPR modifications rendered CT26 tumors significantly more permissive to NDV replication, and in this setting, NDVmuGM-CSF confers immune-mediated effects in the noninjected tumor in vivo Taken together, the data support the thesis that MEDI5395 primes and augments cell-mediated antitumor immunity and has significant utility as a combination partner with other immunomodulatory cancer treatments.

In vivo screen of genetically conserved Streptococcus pneumoniae proteins for protective immunogenicity.

We evaluated 52 different E. coli expressed pneumococcal proteins as immunogens in a BALB/c mouse model of S. pneumoniae lung infection. Proteins were selected based on genetic conservation across disease-causing serotypes and bioinformatic prediction of antibody binding to the target antigen. Seven proteins induced protective responses, in terms of reduced lung burdens of the serotype 3 pneumococci. Three of the protective proteins were histidine triad protein family members (PhtB, PhtD and PhtE). Four other proteins, all bearing LPXTG linkage domains, also had activity in this model (PrtA, NanA, PavB and Eng). PrtA, NanA and Eng were also protective in a CBA/N mouse model of lethal pneumococcal infection. Despite data inferring widespread genomic conservation, flow-cytometer based antisera binding studies confirmed variable levels of antigen expression across a panel of pneumococcal serotypes. Finally, BALB/c mice were immunized and intranasally challenged with a viulent serotype 8 strain, to help understand the breadth of protection. Those mouse studies reaffirmed the effectiveness of the histidine triad protein grouping and a single LPXTG protein, PrtA.

Nonreplicating vaccines can protect african green monkeys from the memphis 37 strain of respiratory syncytial virus.

BACKGROUND: We evaluated the immunological responses of African green monkeys immunized with multiple F and G protein-based vaccines and assessed protection against the Memphis 37 strain of respiratory syncytial virus (RSV). METHODS: Monkeys were immunized with F and G proteins adjuvanted with immunostimulatory (CpG) oligodeoxyribonucleotides admixed with either Alhydrogel or ISCOMATRIX adjuvant. Delivery of F and G proteins via replication incompetent recombinant vesicular stomatitis viruses (VSVs) and human adenoviruses was also evaluated. Mucosally or parenterally administered recombinant adenoviruses were used in prime-boost regimens with adjuvanted proteins or recombinant DNA. RESULTS: Animals primed by intranasal delivery of recombinant adenoviruses, and boosted by intramuscular injection of adjuvanted F and G proteins, developed neutralizing antibodies and F/G protein-specific T cells and were protected from RSV infection. Intramuscular injections of Alhydrogel (plus CpG) adjuvanted F and G proteins reduced peak viral loads in the lungs of challenged monkeys. Granulocyte numbers were not significantly elevated, relative to controls, in postchallenge bronchoalveolar lavage samples from vaccinated animals. CONCLUSIONS: This study has validated the use of RSV (Memphis 37) in an African green monkey model of intranasal infection and identified nonreplicating vaccines capable of eliciting protection in this higher species challenge model.

The efficacy of HSV-2 vaccines based on gD and gB is enhanced by the addition of ICP27.

DNA vaccines expressing HSV-2 gD, gB, ICP27, VP22 and VP13/14 were shown to be immunogenic in mice; gD and gB elicited neutralising antibody, and all five antigens induced T cell responses measured by IFNγ ELISPOT. In murine HSV-2 challenge studies, gD and gB provided moderate to high levels of protection while ICP27 provided a lower level of protection depending on the model (intravaginal or intranasal) and the challenge dose. Combining vaccines expressing gB or gD with vaccines expressing ICP27 provided greater protection than any antigen alone. We conclude that the addition of ICP27 to enhance the anti-viral T cell response can improve the efficacy of gD- and gB-based vaccines.

Immunogenicity and efficacy of an anthrax/plague DNA fusion vaccine in a mouse model.

The efficacy of multi-agent DNA vaccines consisting of a truncated gene encoding Bacillus anthracis lethal factor (LFn) fused to either Yersinia pestis V antigen (V) or Y . pestis F1 was evaluated. A/J mice were immunized by gene gun and developed predominantly IgG1 responses that were fully protective against a lethal aerosolized B. anthracis spore challenge but required the presence of an additional DNA vaccine expressing anthrax protective antigen to boost survival against aerosolized Y. pestis.

Peripheral human γδ T cells control growth of both avirulent and highly virulent strains of Francisella tularensis in vitro.

In this paper we evaluate the role of human γδ T cells in control of Francisella tularensis infection. Using an in vitro model of infection, a reduction in bacterial numbers was detected in the presence of human γδ T cells for both attenuated LVS and virulent SCHU S4 strains of F. tularensis. Antibody neutralisation of IFN-γ caused an increase in survival of F. tularensis LVS suggesting that γδ T cell-mediated control of F. tularensis infection is partially mediated by IFN-γ.

Single and combination herpes simplex virus type 2 glycoprotein vaccines adjuvanted with CpG oligodeoxynucleotides or monophosphoryl lipid A exhibit differential immunity that is not correlated to protection in animal models.

Despite several attempts to develop an effective prophylactic vaccine for HSV-2, all have failed to show efficacy in the clinic. The most recent of these failures was the GlaxoSmithKline (GSK) subunit vaccine based on the glycoprotein gD with the adjuvant monophosphoryl lipid A (MPL). In a phase 3 clinical trial, this vaccine failed to protect from HSV-2 disease, even though good neutralizing antibody responses were elicited. We aimed to develop a superior, novel HSV-2 vaccine containing either gD or gB alone or in combination, together with the potent adjuvant CpG oligodeoxynucleotides (CPG). The immunogenic properties of these vaccines were compared in mice. We show that gB/CPG/alum elicited a neutralizing antibody response similar to that elicited by gD/CPG/alum vaccine but a significantly greater gamma interferon (IFN-γ) T cell response. Furthermore, the combined gB-gD/CPG/alum vaccine elicited significantly greater neutralizing antibody and T cell responses than gD/MPL/alum. The efficacies of these candidate vaccines were compared in the mouse and guinea pig disease models, including a novel male guinea pig genital disease model. These studies demonstrated that increased immune response did not correlate to improved protection. First, despite a lower IFN-γ T cell response, the gD/CPG/alum vaccine was more effective than gB/CPG/alum in mice. Furthermore, the gB-gD/CPG/alum vaccine was no more effective than gD/MPL/alum in mice or male guinea pigs. We conclude that difficulties in correlating immune responses to efficacy in animal models will act as a deterrent to researchers attempting to develop effective HSV vaccines.

Deletion of the Bacillus anthracis capB homologue in Francisella tularensis subspecies tularensis generates an attenuated strain that protects mice against virulent tularaemia.

As there is currently no licensed vaccine against Francisella tularensis, the causative agent of tularaemia, the bacterium is an agent of concern as a potential bioweapon. Although F. tularensis has a low infectious dose and high associated mortality, it possesses few classical virulence factors. An analysis of the F. tularensis subspecies tularensis genome sequence has revealed the presence of a region containing genes with low sequence homology to part of the capBCADE operon of Bacillus anthracis. We have generated an isogenic capB mutant of F. tularensis subspecies tularensis SchuS4 and shown it to be attenuated. Furthermore, using BALB/c mice, we have demonstrated that this capB strain affords protection against significant homologous challenge with the wild-type strain. These data have important implications for the development of a defined and efficacious tularaemia vaccine.

Concomitant administration of Yersinia pestis specific monoclonal antibodies with plague vaccine has a detrimental effect on vaccine mediated immunity.

Antibodies can be used to confer rapid immunity against infectious agents for short periods of time. By comparison, vaccine induced immunity is more protective, but takes a relatively long time to develop. Concomitant administration of antibody and vaccine by different routes was evaluated as a means of providing both rapid and long-term protection against plague. BALB/c mice were treated intraperitoneally with monoclonal antibodies, with specificities for Yersinia pestis LcrV and F1 antigens. A cohort of these mice was simultaneously vaccinated with rF1 and rLcrV by the intramuscular route. Antibody co-administration with vaccine reduced the level of vaccine mediated protection afforded against a high level Y. pestis challenge. Conversely, antibody-mediated protection was unaffected by vaccine co-administration and lasted for at least 8 weeks post administration. We also evaluated the effect of administering vaccine intradermally and antibody intratracheally and observed that, irrespective of administration route, concomitant administration of antibody reduced the effectiveness of vaccine mediated immunity. The results of passive transfer experiments supported the thesis that the development of protective antibody responses following vaccination is impaired by the presence of circulating monoclonal antibodies with specificities for important B-cell epitopes in the vaccine. We also noted that intradermal injection of LcrV antigen and cholera toxin adjuvant afforded good levels of protection against systemic and aerosol challenge with Y. pestis: intradermal injection might therefore be considered as a potential minimally invasive method of plague vaccine administration. These data have implications for the design of therapeutic strategies against plague infection.

Therapeutic use of molecules that mimic pathogen danger signals.

Major advances in our understanding of how the mammalian immune system recognises pathogens have evolved from initial observations of fruit-fly developmental mutants. Through this work it has been established that vertebrates possess a specialised 'early warning system' in the form of a panel of detectors to rapidly sense and trigger responses to the presence of microbial invaders through detection of so called pathogen-associated molecular patterns (PAMPs). This discovery has led to the search for new agonists and antagonists that can be used, therapeutically, for rational manipulation of the immune response. These efforts have already yielded several exciting new strategies to treat autoimmune, atopic, malignant and infectious disease. This review article provides an overview of the potential impact of these drugs on human medicine.

Immunodominant Francisella tularensis antigens identified using proteome microarray.

Stimulation of protective immune responses against intracellular pathogens is difficult to achieve using non-replicating vaccines. BALB/c mice immunized by intramuscular injection with killed Francisella tularensis (live vaccine strain) adjuvanted with preformed immune stimulating complexes admixed with CpG, were protected when systemically challenged with a highly virulent strain of F. tularensis (Schu S4). Serum from immunized mice was used to probe a whole proteome microarray in order to identify immunodominant antigens. Eleven out of the top 12 immunodominant antigens have been previously described as immunoreactive in F. tularensis. However, 31 previously unreported immunoreactive antigens were revealed using this approach. Twenty four (50%) of the ORFs on the immunodominant hit list belonged to the category of surface or membrane associated proteins compared to only 22% of the entire proteome. There were eight hypothetical protein hits and eight hits from proteins associated with different aspects of metabolism. The chip also allowed us to readily determine the IgG subclass bias, towards individual or multiple antigens, in protected and unprotected animals. These data give insight into the protective immune response and have potentially important implications for the rational design of non-living vaccines for tularemia and other intracellular pathogens.

Administration of antibody to the lung protects mice against pneumonic plague.

Intratracheal delivery of aerosolized monoclonal antibodies with specificity for Yersinia pestis LcrV and F1 antigens protected mice in a model of pneumonic plague. These data support the utility of inhaled antibodies as a fast-acting postexposure treatment for plague.

Immunological responses after immunisation of mice with microparticles containing antigen and single stranded RNA (polyuridylic acid).

Certain toll-like receptor (TLR) agonists, e.g. CpG DNA, can be used as potent vaccine 'adjuvants'. It is known that some sequences of single stranded (ss) RNA stimulate proinflammatory and antiviral responses following interaction with TLR 7 and 8. We have encapsulated ovalbumin (OVA) in the presence and absence of polyuridylic acid (poly-U) inside polylactide microparticles. In comparison to microparticles containing only OVA, bulk cultures of bone marrow-derived plasmacytoid and myeloid dendritic cells produced more (P<0.05) IL-12 and interferon (IFN)-alpha when stimulated with microparticles containing OVA and poly-U. Subcutaneous injection of comicroencapsulated OVA and poly-U resulted in statistically elevated levels of serum anti-OVA IgG1 (P<0.05 versus naïve mice). Conversely, anti-OVA IgG1 levels in C57 BL6 mice immunised with OVA loaded microparticles (without RNA) were statistically indifferent to naïve animals. Furthermore, injection of coencapsulated OVA and poly-U resulted in (P<0.05) greater numbers of OVA specific IFN-gamma secreting T-cells as compared with mice injected with OVA loaded microparticles. A similar trend was seen in mice immunised with OVA loaded microparticles decorated with CpG or solutions of admixed OVA and CpG (P<0.05). These data demonstrate, for the first time, that appropriately formulated ssRNA can act as a potent adjuvant and modulator of adaptive immunological responses.

Particulate delivery systems for biodefense subunit vaccines.

Expanding identification of potentially protective subunit antigens and correlates of protection has provided a basis for the introduction of safer vaccines. Despite encouraging results in animal models, the significant potential of particulate delivery systems in vaccine design has not yet translated into effective vaccines available for use in humans. This review article will focus on the current status of the development of particulate vaccines, mainly liposomes and bio-degradable polymers, against potential agents for biowarfare: plague, anthrax, botulinum, and smallpox; and filoviruses: Marburg and Ebola.

Activation of dendritic cells by microparticles containing Bacillus anthracis protective antigen.

We have carried out an in vitro investigation into the mechanism by which microencapsulation enhances the immunogenicity of recombinant protective antigen (rPA) from Bacillus anthracis. Murine bone marrow derived dendritic cells (DC) were cocultured with soluble and microencapsulated rPA and the activation status of the cells monitored using FACS. As compared with soluble rPA, it was found that coculture of DC with rPA-loaded microparticles stimulated higher levels of MHC II, CD54, CD80 and CD86 expression (p<0.05). To investigate the longevity of antigen presentation, splenocytes from naïve mice were pulsed overnight with (3)H-thymidine following 1, 3 or 6 days coculture with DC transiently exposed to soluble or microencapsulated rPA. Splenocyte proliferation was more pronounced, and continued for a more protracted period, if the 'feeder' cells were exposed to microencapsulated antigen as compared with soluble antigen or 'empty' microspheres. To this end, our findings indicate that microsphere uptake increases the surface expression of MHC and co-stimulatory molecules on DC and can facilitate prolonged presentation of antigen to T-cells, possibly by acting as an intracellular depot.

Mucosal delivery of microparticle encapsulated ESAT-6 induces robust cell-mediated responses in the lung milieu.

ESAT-6 from Mycobacterium tuberculosis is an important T-cell antigen for cell-mediated immunity in the early phase of tuberculosis infection. Since the lung is the organ in which infection is initiated, immune responses in the lung play a significant role in restricting the initial infection with M. tuberculosis. The aim of the present study was to assess whether efficient cell-mediated immune responses in the lung and draining mediastinal lymph nodes could be stimulated by pulmonary administration of ESAT-6 encapsulated in poly(lactide) (PLA) microspheres. BALB/c mice were immunised intranasally on days 1, 28 and 56 with 2 microg microencapsulated ESAT-6. Cellular responses in the lungs, spleen and mediastinal lymph nodes (MLN) were characterised using ELISPOT and proliferation assays. Fluorescence activated cell sorting (FACS) was used to assess the expression of CD44 on CD4+ and CD8+ cells derived from the MLN of immunised animals. For comparison, groups of mice were immunised intranasally with soluble 'free' ESAT-6 or intramuscularly with ESAT-6 in Alhydrogel. Intranasal instillation of microencapsulated ESAT-6 induced greatest numbers of ESAT-6 specific IFN-gamma and IL-4 secreting cells in the lung and MLN (P<0.05). Similarly, ESAT-6 specific recall responses were strongest following intranasal immunisation of mice with microsphere encapsulated antigen (P<0.05). FACS demonstrated a higher proportion of T cells expressing CD44 in the MLN from mice immunised intranasally with microencapsulated ESAT-6. These data support the notion that the immune system is compartmentalised and responses are often strongest in compartments proximal to the site of vaccine application. Furthermore, our data indicate that, for efficient activation of cell-mediated responses, antigens must be presented to the immune system in an appropriate formulation.