Additive toxicity arising from combined use of immune checkpoint inhibitors and tyrosine kinase inhibitors in patients with renal or endometrial carcinoma: Protocol for a rapid systematic review.

The combined use of immune checkpoint inhibitors and tyrosine kinase inhibitors (ICI/TKI) is an effective treatment strategy for some cancers. A better understanding of the potential additive toxicity for ICI/TKI combinations is needed to inform patient and provider treatment decisions. We aim to evaluate the safety of ICI/TKI combinations for individuals with renal cell or endometrial carcinoma. This rapid systematic review (SR) protocol follows PRISMA guidelines. A systematic search will be designed, peer reviewed and executed by experienced information specialists (Cochrane Central, MEDLINE, Embase) to identify published SRs and primary studies published since the most recent SR search. Randomized, quasi- or non-randomized controlled trials and comparative cohort studies are eligible if they compare ICI/TKI combinations to monotherapy or standard of care in participants with renal cell or endometrial carcinoma. The primary outcome is grade ≥ 3 treatment-related adverse-effects. Studies will be screened, selected, extracted and assessed for risk of bias by a single reviewer and checked completely by a second. Where feasible and appropriate, we will pool studies separately by design and indication using meta-analysis and test robustness of effects using prespecified subgroup and sensitivity analyses. Results will be summarized descriptively and presented in tables and figures. (PROSPERO ID: CRD42023416388).•This will be a comprehensive systematic review of the additive toxicity arising from the combined use of ICI/TKIs in patients with renal-cell or endometrial carcinoma.•We will consider treatment-related, treatment-emergent adverse events (Grade 3 or higher).•Identified safety profile may be used to inform patient or provider treatment decisions.

Serum-derived carcinoembryonic antigen (CEA) activates fibroblasts to induce a local re-modeling of the extracellular matrix that favors the engraftment of CEA-expressing tumor cells.

Elevated levels of the carcinoembryonic antigen (CEA; CEACAM5) in the serum of colorectal cancer (CRC) patients represent a clinical biomarker that correlates with disease recurrence. However, a mechanistic role for soluble CEA (sCEA) in tumor progression and metastasis remains to be established. In our study, we report that sCEA acts as a paracrine factor, activating human fibroblasts by signaling through both the STAT3 and AKT1-mTORC1 pathways, promoting their transition to a cancer-associated fibroblast (CaF) phenotype. sCEA-activated fibroblasts express and secrete higher levels of fibronectin, including cellular EDA+ -fibronectin (Fn-EDA) that selectively promote the implantation and adherence of CEA-expressing cancer cells. Immunohistochemical analyses of liver tissues derived from CRC patients with elevated levels of sCEA reveal that the expression of cellular Fn-EDA co-registers with CEA-expressing liver metastases. Taken together, these findings indicate a direct role for sCEA as a human fibroblast activation factor, in priming target tissues for the engraftment of CEA-expressing cancer cells, through the differentiation of tissue-resident fibroblasts, resulting in a local change in composition of the extracellular matrix.

VISTA.COMP - an engineered checkpoint receptor agonist that potently suppresses T cell-mediated immune responses.

V-domain immunoglobulin suppressor of T cell activation (VISTA) is a recently discovered immune checkpoint ligand that functions to suppress T cell activity. The therapeutic potential of activating this immune checkpoint pathway to reduce inflammatory responses remains untapped, largely due to the inability to derive agonists targeting its unknown receptor. A dimeric construct of the IgV domain of VISTA (VISTA-Fc) was shown to suppress the activation of T cells in vitro. However, this effect required its immobilization on a solid surface, suggesting that VISTA-Fc may display limited efficacy as a VISTA-receptor agonist in vivo. Herein, we have designed a stable pentameric VISTA construct (VISTA.COMP) by genetically fusing its IgV domain to the pentamerization domain from the cartilage oligomeric matrix protein (COMP). In contrast to VISTA-Fc, VISTA.COMP does not require immobilization to inhibit the proliferation of CD4+ T cells undergoing polyclonal activation. Furthermore, we show that VISTA.COMP, but not VISTA-Fc, functions as an immunosuppressive agonist in vivo capable of prolonging the survival of skin allografts in a mouse transplant model as well as rescuing mice from acute concanavalin-A-induced hepatitis. Collectively, we believe our data demonstrate that VISTA.COMP is a checkpoint receptor agonist and the first agent to our knowledge targeting the putative VISTA-receptor to suppress T cell-mediated immune responses.

Induction of antigen-specific TH 9 immunity accompanied by mast cell activation blocks tumor cell engraftment.

The engraftment of circulating cancer cells at distal sites represents a key step in the metastatic cascade, yet remains an unexplored target for therapeutic intervention. In this study, we establish that a vaccination strategy yielding an antigen-specific TH 9 response induces long term host surveillance and prevents the engraftment of circulating cancer cells. Specifically, we show that vaccination with a recombinant CEA IgV-like N domain, formulated with the TLR3 ligand poly I:C, elicits a CEA-specific TH 9 response, wherein IL-9 secreting TH cells act in concert with CEA N domain-specific antibodies as well as activated mast cells in preventing tumor cell engraftment. The development of this immune response was dependent on TLR3, since interference with the TLR3-dsRNA complex formation led to a reduction in vaccine-imparted protection and a shift in the resulting immune response toward a TH 2 response. These findings point to the existence of an alternate tumor targeting immune mechanism that can be exploited for the purpose of developing vaccine therapies targeting tumor dissemination and engraftment.

Slow binding kinetics of secreted protein, acidic, rich in cysteine-VEGF interaction limit VEGF activation of VEGF receptor 2 and attenuate angiogenesis.

VEGF-A (VEGF) drives angiogenesis through activation of downstream effectors to promote endothelial cell proliferation and migration. Although VEGF binds both VEGF receptor 1 (R1) and receptor 2 (R2), its proangiogenic effects are attributed to R2. Secreted protein, acidic, rich in cysteine (SPARC) is a matricellular glycoprotein thought to inhibit angiogenesis by preventing VEGF from activating R1, but not R2. Because R2 rather than R1 mediates proangiogenic activities of VEGF, the role of human SPARC in angiogenesis was reevaluated. We confirm that association of SPARC with VEGF inhibits VEGF-induced HUVEC adherence, motility, and proliferation in vitro and blocks VEGF-induced blood vessel formation ex vivo. SPARC decreases VEGF-induced phosphorylation of R2 and downstream effectors ERK, Akt, and p38 MAPK as shown by Western blot and/or phosphoflow analysis. Surface plasmon resonance indicates that SPARC binds slowly to VEGF (0.865 ± 0.02 × 10(4) M(-1) s(-1)) with a Kd of 150 nM, forming a stable complex that dissociates slowly (1.26 ± 0.003 × 10(-3) s(-1)). Only domain III of SPARC binds VEGF, exhibiting a 15-fold higher affinity than full-length SPARC. These findings support a model whereby SPARC regulates angiogenesis by sequestering VEGF, thus restricting the activation of R2 and the subsequent activation of downstream targets critical for endothelial cell functions.

Targeting the PD-1/PD-L1 Immune Evasion Axis With DNA Aptamers as a Novel Therapeutic Strategy for the Treatment of Disseminated Cancers.

Blocking the immunoinhibitory PD-1:PD-L1 pathway using monoclonal antibodies has led to dramatic clinical responses by reversing tumor immune evasion and provoking robust and durable antitumor responses. Anti-PD-1 antibodies have now been approved for the treatment of melanoma, and are being clinically tested in a number of other tumor types as both a monotherapy and as part of combination regimens. Here, we report the development of DNA aptamers as synthetic, nonimmunogenic antibody mimics, which bind specifically to the murine extracellular domain of PD-1 and block the PD-1:PD-L1 interaction. One such aptamer, MP7, functionally inhibits the PD-L1-mediated suppression of IL-2 secretion in primary T-cells. A PEGylated form of MP7 retains the ability to block the PD-1:PD-L1 interaction, and significantly suppresses the growth of PD-L1+ colon carcinoma cells in vivo with a potency equivalent to an antagonistic anti-PD-1 antibody. Importantly, the anti-PD-1 DNA aptamer treatment was not associated with off-target TLR-9-related immune responses. Due to the inherent advantages of aptamers including their lack of immunogenicity, low cost, long shelf life, and ease of synthesis, PD-1 antagonistic aptamers may represent an attractive alternative over antibody-based anti PD-1 therapeutics.

Agonistic CD200R1 DNA Aptamers Are Potent Immunosuppressants That Prolong Allogeneic Skin Graft Survival.

CD200R1 expressed on the surface of myeloid and lymphoid cells delivers immune inhibitory signals to modulate inflammation when engaged with its ligand CD200. Signalling through CD200/CD200R1 has been implicated in a number of immune-related diseases including allergy, infection, cancer and transplantation, as well as several autoimmune disorders including arthritis, systemic lupus erythematosus, and multiple sclerosis. We report the development and characterization of DNA aptamers, which bind to murine CD200R1 and act as potent signalling molecules in the absence of exogenous CD200. These agonistic aptamers suppress cytotoxic T-lymphocyte induction in 5-day allogeneic mixed leukocyte culture and induce rapid phosphorylation of the CD200R1 cytoplasmic tail thereby initiating immune inhibitory signalling. PEGylated conjugates of these aptamers show significant in vivo immunosuppression and enhance survival of allogeneic skin grafts as effectively as soluble CD200Fc. As DNA aptamers exhibit inherent advantages over conventional protein-based therapeutics including low immunogenicity, ease of synthesis, low cost, and long shelf life, such CD200R1 agonistic aptamers may emerge as useful and safe nonsteroidal anti-inflammatory therapeutic agents.

The carcinoembryonic antigen IgV-like N domain plays a critical role in the implantation of metastatic tumor cells.

The human carcinoembryonic antigen (CEA) is a cell adhesion molecule involved in both homotypic and heterotypic interactions. The aberrant overexpression of CEA on adenocarcinoma cells correlates with their increased metastatic potential. Yet, the mechanism(s) by which its adhesive properties can lead to the implantation of circulating tumor cells and expansion of metastatic foci remains to be established. In this study, we demonstrate that the IgV-like N terminal domain of CEA directly participates in the implantation of cancer cells through its homotypic and heterotypic binding properties. Specifically, we determined that the recombinant N terminal domain of CEA directly binds to fibronectin (Fn) with a dissociation constant in the nanomolar range (K(D) 16 ± 3 nM) and interacts with itself (K(D) 100 ± 17 nM) and more tightly to the IgC-like A(3) domain (K(D) 18 ± 3 nM). Disruption of these molecular associations through the addition of antibodies specific to the CEA N or A(3)B(3) domains, or by adding soluble recombinant forms of the CEA N, A(3) or A(3)B(3) domains or a peptide corresponding to residues 108-115 of CEA resulted in the inhibition of CEA-mediated intercellular aggregation and adherence events in vitro. Finally, pretreating CEA-expressing murine colonic carcinoma cells (MC38.CEA) with rCEA N, A3 or A(3)B(3) modules blocked their implantation and the establishment of tumor foci in vivo. Together, these results suggest a new mechanistic insight into how the CEA IgV-like N domain participates in cellular events that can have a macroscopic impact in terms of cancer progression and metastasis.

Blocking the attachment of cancer cells in vivo with DNA aptamers displaying anti-adhesive properties against the carcinoembryonic antigen.

The formation of metastatic foci occurs through a series of cellular events, initiated by the attachment and aggregation of cancer cells leading to the establishment of micrometastases. We report the derivation of synthetic DNA aptamers bearing anti-adhesive properties directed at cancer cells expressing the carcinoembryonic antigen (CEA). Two DNA aptamers targeting the homotypic and heterotypic IgV-like binding domain of CEA were shown to block the cell adhesion properties of CEA, while not recognizing other IgV-like domains of CEACAM family members that share strong sequence and structural homologies. More importantly, the pre-treatment of CEA-expressing tumour cells with these aptamers prior to their intraperitoneal implantation resulted in the prevention of peritoneal tumour foci formation. Taken together, these results highlight the effectiveness of targeting the cell adhesion properties of cancer cells with aptamers in preventing tumour implantation.

The Pseudomonas aeruginosa exotoxin A translocation domain facilitates the routing of CPP-protein cargos to the cytosol of eukaryotic cells.

The use of cell-penetrating peptides (CPPs), such as polyarginine, has been shown to facilitate the import of drugs and other cargos into cells. However, a major obstacle limiting their use as delivery agents is their entrapment following internalization into endocytic vesicles, leading to either their recycling out of cells or their degradation in lysosomes. To address this challenge, we fused a CPP sequence to the translocation domain of Pseudomonas aeruginosa exotoxin A (ETA) to facilitate the endosomal escape of imported CPP-containing protein constructs. Specifically, a fusion protein incorporating ten arginines linked to residues 253 to 412 of ETA (ETA(253-412)) was tested for its ability to effectively route a protein cargo (enhanced green fluorescent protein, eGFP) to the cytosol of cells. Using flow cytometry and fluorescence live-cell imaging, we observed a 5-fold improvement of cellular uptake as well as a 40-fold increase in cytosolic delivery of the CPP-ETA(253-412)-eGFP construct in relation to CPP-eGFP. Furthermore, analysis of intracellular routing events indicated that the incorporation of ETA(253-412) within the CPP-containing protein fusion construct avoided lysosomal degradation by re-directing the construct from early endosomes to the ER lumen and finally to the cytosol. Studies using inhibitors of vesicular transport confirmed that the ER lumen is a key compartment reached by the CPP-ETA(253-412)-eGFP construct before accessing the cytosol. Together, these findings suggest that incorporating a CPP motif and the ETA translocation domain into protein constructs can facilitate their cytosolic delivery.

A focused immune response targeting the homotypic binding domain of the carcinoembryonic antigen blocks the establishment of tumor foci in vivo.

Metastatic forms of cancers remain the main cause of death in cancer patients. In this study, we demonstrate that directing a sustained antibody response towards the homotypic binding function of CEA interferes with the implantation and development of tumor foci in CEA-expressing transgenic (CEA.Tg) mice. Specifically, vaccinating CEA.Tg mice with a recombinant, altered self-form of the CEA Ig V-like N domain led to the production of circulating IgG1 and IgG2a antibodies that inhibited CEA-mediated adhesion of murine carcinoma expressing CEA (MC38.CEA) and mediated antibody-dependent lysis of tumor cells. Moreover, vaccinated CEA.Tg mice were resistant to the development of tumor nodules in the lungs and the peritoneal cavity, suggesting that mounting a focused antibody response to the CEA N domain may represent a simple therapeutic strategy to control the establishment of metastatic foci in cancer patients.

An evolved ribosome-inactivating protein targets and kills human melanoma cells in vitro and in vivo.

BACKGROUND: Few treatment options exist for patients with metastatic melanoma, resulting in poor prognosis. One standard treatment, dacarbazine (DTIC), shows low response rates ranging from 15 to 25 percent with an 8-month median survival time. The development of targeted therapeutics with novel mechanisms of action may improve patient outcome. Ribosome-inactivating proteins (RIPs) such as Shiga-like Toxin 1 (SLT-1) represent powerful scaffolds for developing selective anticancer agents. Here we report the discovery and properties of a single chain ribosome-inactivating protein (scRIP) derived from the cytotoxic A subunit of SLT-1 (SLT-1A), harboring the 7-amino acid peptide insertion IYSNKLM (termed SLT-1A IYSNKLM) allowing the toxin variant to selectively target and kill human melanoma cells. RESULTS: SLT-1A IYSNKLM was able to kill 7 of 8 human melanoma cell lines. This scRIP binds to 518-A2 human melanoma cells with a dissociation constant of 18 nM, resulting in the blockage of protein synthesis and apoptosis in such cells. Biodistribution and imaging studies of radiolabeled SLT-1A IYSNKLM administered intravenously into SCID mice bearing a human melanoma xenograft indicate that SLT-1AI YSNKLM readily accumulates at the tumor site as opposed to non-target tissues. Furthermore, the co-administration of SLT-1A IYSNKLM with DTIC resulted in tumor regression and greatly increased survival in this mouse xenograft model in comparison to DTIC or SLT-1A IYSNKLM treatment alone (115 day median survival versus 46 and 47 days respectively; P values < 0.001). SLT-1A IYSNKLM is stable in serum and its intravenous administration resulted in modest immune responses following repeated injections in CD1 mice. CONCLUSIONS: These results demonstrate that the evolution of a scRIP template can lead to the discovery of novel cancer cell-targeted compounds and in the case of SLT-1A IYSNKLM can specifically kill human melanoma cells in vitro and in vivo.

Comparison of the local immune response against Giardia lamblia cyst wall protein 2 induced by recombinant Lactococcus lactis and Streptococcus gordonii.

Lactococcus lactis and Streptococcus gordonii are lactic acid bacteria (LAB) currently being advocated for use as live antigen delivery vehicles to mucosal sites. Since both vehicles differ in their capability to persist within the small intestine and in their mode of antigen delivery, we sought to compare them to determine which one was superior. In this study, we compared the efficacy of recombinant L. lactis and S. gordonii to stimulate intestinal immune responses against Giardia lamblia cyst wall protein-2 in BALB/c mice. Oral administration of either vector significantly increased the number of CD4(+) T helper and B-cells in the mesenteric lymph nodes (MLN) and Peyer's patches (PP) of immunized animals. Delivery of recombinant CWP2 (rCWP2) by L. lactis stimulated a balanced IFN-gamma/IL-4 response (MLN and PP cells) and a CWP2-specific intestinal IgA antibody response. Alternatively, delivery of rCWP2 by S. gordonii stimulated a higher frequency of IFN-gamma secreting MLN and PP cells, as well as doubling the amount of CWP2-specific intestinal IgA. In challenge studies, L. lactis and S. gordonii reduced cyst output by 71 and 90%, respectively. When compared to each other, S. gordonii-immunized animals shed 65% fewer cysts than their L. lactis-immunized counterparts. Based on these findings, we concluded that S. gordonii was superior to L. lactis as an intestinal vaccine delivery vehicle.

Characterization of the local immune response to cyst antigens during the acute and elimination phases of primary murine giardiasis.

During the course of a giardial infection, the host's immune system is presented with a variety of Giardia antigens as trophozoites differentiate, through encysting cells, to form the infective cysts. Previous studies examining the host's immune response during giardial infections have focused on trophozoite-derived antigens (Ags). In this study, we were interested to determine if the host's immune system reacts to cyst Ags during the acute and elimination phases, when there is cyst shedding. For this purpose, we used antigenic extracts from trophozoites (Troph), encysting cells (ENC), and purified giardial cyst walls (PCW), as well as purified recombinant cyst wall protein 2 (rCWP2). Comparative analysis of the parasite extracts using SDS-PAGE analysis and surface-enhanced laser desorption/ionization time of flight mass spectrometry resulted in the detection of 175 protein entities, of which 26 were Troph-specific proteins, 17 ENC-specific proteins, and 31 were PCW-specific proteins. On the other hand, we detected 34 proteins shared between Troph and ENC, 19 proteins that were shared between ENC and PCW, and 29 proteins that were common to Troph and PCW. Finally, we detected 19 proteins that were shared by all three extract samples. BALB/c mice were infected with 10(5)Giardia muris cysts and sacrificed either at the acute or elimination phases of infection (days 12 and 40, respectively), and lymphocytes were isolated from the Peyer's patches (PP). Using flow cytometry, we detected significant increases in the number of PP-derived CD4(+) and CD19(+), but not CD8(+) lymphocytes. Quantification of the number of mucosal IL-4 and IFN-gamma secreting T-lymphocytes by enzyme-linked immunosorbent spot assay showed that these cells reacted by secreting similar levels of IL-4 and IFN-gamma, regardless of the Ag or the phase of infection. Analysis of intestinal humoral immune responses by ELISA resulted in the detection of Ag-specific IgA and IgG intestinal antibodies. Regardless of the Ag tested, a trend was consistently observed where the concentration of local antibodies was found to be slightly increased by the acute phase, where we detected approximately 200microg/mg of specific IgA and approximately 300ng/ml of specific IgG in intestinal lavage of infected mice. By the elimination phase, the amount of specific antibodies was found to increase to approximately 600microg/mg of specific IgA and approximately 1300ng/ml of specific IgG antibodies. Finally, we tested the biological activity of these antibodies and found that they were able to reduce the ability of trophozoites to differentiate into cysts in vitro. Collectively, we believe these results demonstrate for the first time the existence of significant cellular and humoral immune responses against Giardia cyst Ags that may contribute to the reduction of cyst shedding in infected animals.

Mucosal delivery of a transmission-blocking DNA vaccine encoding Giardia lamblia CWP2 by Salmonella typhimurium bactofection vehicle.

In this study, we investigated the use of Salmonella typhimurium (STM1 strain) as a bactofection vehicle to deliver a transmission-blocking DNA vaccine (TBDV) plasmid to the intestinal immune system. The gene encoding the full length cyst wall protein-2 (CWP2) from Giardia lamblia was subcloned into the pCDNA3 mammalian expression vector and stably introduced into S. typhimurium STM1. Eight-week-old female BALB/c mice were orally immunized every 2 weeks, for a total of three immunizations. Vaccinated and control mice were sacrificed 1 week following the last injection. Administration of the DNA vaccine led to the production of CWP2-specific cellular immune responses characterized by a mixed Th1/Th2 response. Using ELISA, antigen-specific IgA and IgG antibodies were detected in intestinal secretions. Moreover, analysis of sera demonstrated that the DNA immunization also stimulated the production of CWP2-specific IgG antibodies that were mainly of the IgG2a isotype. Finally, challenge infection with live Giardia muris cysts revealed that mice receiving the CWP2-encoding DNA vaccine were able to reduce cyst shedding by approximately 60% compared to control mice. These results demonstrate, for the first time, the development of parasite transmission-blocking immunity at the intestinal level following the administration of a mucosal DNA vaccine delivered by S. typhimurium STM1.