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Supplementary Figure from HER2 Mediates PSMA/mGluR1-Driven Resistance to the DS-7423 Dual PI3K/mTOR Inhibitor in PTEN Wild-type Prostate Cancer Models

Prostate cancer remains a major cause of male mortality. Genetic alteration of the PI3K/AKT/mTOR pathway is one of the key events in tumor development and progression in prostate cancer, with inactivation of the PTEN tumor suppressor being very common in this cancer type. Extensive evaluation has been performed on the therapeutic potential of PI3K/AKT/mTOR inhibitors and the resistance mechanisms arising in patients with PTEN-mutant background. However, in patients with a PTEN wild-type phenotype, PI3K/AKT/mTOR inhibitors have not demonstrated efficacy, and this remains an area of clinical unmet need. In this study, we have investigated the response of PTEN wild-type prostate cancer cell lines to the dual PI3K/mTOR inhibitor DS-7423 alone or in combination with HER2 inhibitors or mGluR1 inhibitors. Upon treatment with the dual PI3K/mTOR inhibitor DS-7423, PTEN wild-type prostate cancer CWR22/22RV1 cells upregulate expression of the proteins PSMA, mGluR1, and the tyrosine kinase receptor HER2, while PTEN-mutant LNCaP cells upregulate androgen receptor and HER3. PSMA, mGluR1, and HER2 exert control over one another in a positive feedback loop that allows cells to overcome treatment with DS-7423. Concomitant targeting of PI3K/mTOR with either HER2 or mGluR1 inhibitors results in decreased cell survival and tumor growth in xenograft studies. Our results suggest a novel therapeutic possibility for patients with PTEN wild-type PI3K/AKT-mutant prostate cancer based in the combination of PI3K/mTOR blockade with HER2 or mGluR1 inhibitors.

A subset of tumor associated macrophages (TAMs) identified by their expression of the lymphatic vessel endothelial hyaluronan receptor-1 (Lyve-1) reside proximal to blood vasculature and contribute to disease progression. Using a spontaneous murine model of mammary adenocarcinoma (MMTV-PyMT), we show that Lyve-1+ TAMs, which co-express heme oxygenase-1, form coordinated multi-cellular ‘nest’ structures in the perivascular niche. We show that TAM nest formation is dependent on IL-6 and a communication axis involving CCR5 and its cognate ligands CCL3/4. We demonstrate that Lyve-1+ TAM nests are associated with CD8+ T-cell exclusion from the tumor and the resistance to immune-stimulating chemotherapeutics. This study highlights an unappreciated collaboration between TAMs and uncovers a spatially driven therapeutic resistance mechanism of these cells in cancer which can be therapeutically targeted.

Prostate cancer remains a major cause of male mortality. Genetic alteration of the PI3K/AKT/mTOR pathway is one of the key events in tumor development and progression in prostate cancer, with inactivation of the PTEN tumor suppressor being very common in this cancer type. Extensive evaluation has been performed on the therapeutic potential of PI3K/AKT/mTOR inhibitors and the resistance mechanisms arising in patients with PTEN-mutant background. However, in patients with a PTEN wild-type phenotype, PI3K/AKT/mTOR inhibitors have not demonstrated efficacy, and this remains an area of clinical unmet need. In this study, we have investigated the response of PTEN wild-type prostate cancer cell lines to the dual PI3K/mTOR inhibitor DS-7423 alone or in combination with HER2 inhibitors or mGluR1 inhibitors. Upon treatment with the dual PI3K/mTOR inhibitor DS-7423, PTEN wild-type prostate cancer CWR22/22RV1 cells upregulate expression of the proteins PSMA, mGluR1, and the tyrosine kinase receptor HER2, while PTEN-mutant LNCaP cells upregulate androgen receptor and HER3. PSMA, mGluR1, and HER2 exert control over one another in a positive feedback loop that allows cells to overcome treatment with DS-7423. Concomitant targeting of PI3K/mTOR with either HER2 or mGluR1 inhibitors results in decreased cell survival and tumor growth in xenograft studies. Our results suggest a novel therapeutic possibility for patients with PTEN wild-type PI3K/AKT-mutant prostate cancer based in the combination of PI3K/mTOR blockade with HER2 or mGluR1 inhibitors.

Despite their role in cancer surveillance, adoptive immunotherapy using γδ T-cells has achieved limited efficacy. To enhance trafficking to bone marrow, circulating γδ T-cells were expanded in serum-free medium containing TGF-β1 and IL-2 (γδ[T2] cells).Unexpectedly, the yield and viability of γδ[T2] cells were also increased by TGF-β1,when compared to γδ[2] controls (IL-2 alone). γδ[T2] cells were less differentiated and yet displayed increased cytolytic activity, cytokine release and anti-tumor activity in several leukemic and solid tumor models. Efficacy was further enhanced by cancer cell sensitization using amino bisphosphonates or cytarabine, or by CAR re-programming. A number of contributory effects of TGF-β1 were identified, including prostaglandinE2 receptor downmodulation, upregulation of CD103 and enhanced IL-9 production by γδ[T2] cells. Biological relevance is supported by the identification of a favorable γδ[T2] signature in acute myeloid leukemia. Given their enhanced therapeutic activity and compatibility with allogeneic use, γδ[T2] cells warrant evaluation in cancer immunotherapy.

ObjectiveCD3+CD8+CD28− cells are increased in the periphery and tissues of rheumatoid arthritis (RA) patients. The aim of this study was to characterise CD3+CD8+CD28− cells for the presence of cell surface receptors that regulate immune activation and function and to track their presence in a disease model of RA. Cell surface receptors expressed by CD3+CD8+CD28− cells were then related to serological and clinical disease parameters to establish whether these cells are prognostic of a clinical response to conventional DMARDs.MethodUsing healthy donor peripheral blood mononuclear (PBMC) cell surface expression of > 50 candidate markers were tested using flow cytometry and compared against CD28 expression. The prevalence of cells expressing the most suitable candidate was investigated in the collagen induced arthritis (CIA) and the antigen-induced arthritis (AIA) models. Fifty RA patients were recruited from University Hospital of Wales (UHW) rheumatology outpatient clinic. Clinical and serological markers of inflammation were noted, and PBMC were analysed using flow cytometry +/− in vitro stimulation.ResultsCD3+CD8+CD28− T cells express CD244, CD57, CX3CR1 and KLRG1. The strongest inverse correlate of CD28 expression was KLRG1. CD3+CD8+CD28−KLRG1+ cells were elevated in experimental models of RA. Notably, Il-10-deficiency was linked with exacerbated arthritis and an increase in the number of CD3+CD8+CD28−KLRG1+ cells, suggesting a regulatory role for Il-10 in their development or survival. In RA patients, CD3+CD8+CD28−KLRG1+ cells correlate with ACPA, RF and ESR, and produce more IL-10 than controls. Finally, these cells are higher in early arthritis patients that do not respond to treatment with synthetic DMARDs at six months.ConclusionKLRG1 is a marker for regulatory CD3+CD8+CD28− cells. The presence of CD3+CD8+CD28-KLRG1+ cells increases with certain measures of disease, and is indicative of poor treatment response to DMARDs in early arthritis.Key MessagesKLRG1 is a marker of CD28 negativity on CD8 T cellsCD3+CD8+CD28−KLRG1+ cells are increased in CIA mice and correlate with disease severity.CD3+CD8+CD28−KLRG1+ cells positively correlate with ESR / ACPA and RF in patients.CD3+CD8+CD28−KLRG1+ cells are increased in Il-10-deficient mice with inflammatory arthritis.CD3+CD8+CD28−KLRG1+ cells produce more Il-10 than CD3+CD8+CD28+KLRG1−cells in RA patients.CD3+CD8+CD28−KLRG1+ cells are higher in patients who do not respond to treatment with synthetic DMARDs after six months.

The discovery of compounds and proteins from plants has greatly contributed to modern medicine, especially in malaria where both quinine and artemisinin have been the cornerstone of therapeutics. Here we describe the first known plant-derived cytokine binding agent. Chlorophyll a-b binding protein AB96, present to varying levels in the leaves of all chlorophyll-containing plants, binds to active TGFβ1. Active TGFβ1 contributes to the pathology of many infectious and neoplastic diseases and therefore, by inhibiting these processes, chlorophyll a-b binding protein opens up new approaches with therapeutic potential.

Aberrant mucin-type O-linked glycosylation is a common occurrence in cancer where the upregulation of sialyltransferases is often seen leading to the early termination of O-glycan chains. Mucin-type O-linked glycosylation is not limited to mucins and occurs on many cell surface glycoproteins including EGFR, where the number of sites can be limited. Upon EGF ligation, EGFR induces a signaling cascade and may also translocate to the nucleus where it directly regulates gene transcription, a process modulated by Galectin-3 and MUC1 in some cancers. Here, we show that upon EGF binding, breast cancer cells carrying different O-glycans respond by transcribing different gene expression signatures. MMP10, the principal gene upregulated when cells carrying sialylated core 1 glycans were stimulated with EGF, is also upregulated in ER-positive breast carcinoma reported to express high levels of ST3Gal1 and hence mainly core 1 sialylated O-glycans. In contrast, isogenic cells engineered to carry core 2 glycans upregulate CX3CL1 and FGFBP1 and these genes are upregulated in ER-negative breast carcinomas, also known to express longer core 2 O-glycans. Changes in O-glycosylation did not significantly alter signal transduction downstream of EGFR in core 1 or core 2 O-glycan expressing cells. However, striking changes were observed in the formation of an EGFR/galectin-3/MUC1/β-catenin complex at the cell surface that is present in cells carrying short core 1-based O-glycans but absent in core 2 carrying cells.

The tumour microenvironment plays a crucial role in the growth and progression of cancer and the presence of tumour-associated macrophages (TAMs) is associated with poor prognosis. Recent studies show that TAMs show transcriptomic, phenotypic, functional and geographical diversity. Here we show that a sialylated tumour-associated glycoform of the mucin MUC1, MUC1-ST, through the engagement of Siglec-9 can specifically and independently induce the differentiation of monocytes into TAMs with a unique phenotype. These TAMs can recruit and maintain neutrophils, inhibit the function of T cells, degrade basement membrane allowing for invasion, are inefficient at phagocytosis, and can induce plasma clotting. This novel macrophage phenotype is enriched in the stroma at the edge of breast cancer nests and their presence is associated with poor prognosis in breast cancer patients.

Myelodysplastic syndromes (MDS) represent a spectrum of clonal bone marrow neoplasms from low risk disease through to those transforming into acute myeloid leukaemia. The COVID-19 pandemic has presented a great risk to those with hematological malignancies who are at higher risk of severe disease and death than the general population. Previous studies looking at the immune response to influenza vaccination in those with MDS had shown promising results, with immune responses not differing from those of healthy family members. Whilst some data exist to reassure the MDS community that majority of patients show seroconversion following Covid-19 vaccination, little data exists on their neutralizing capacity or post vaccination T-cell responses in this cohort. In addition, the majority of patients in these studies received BNT162b2 and there is little published data on vaccine response to the ChAdOx1 nCoV-19 vaccine. We have investigated the humoral and T-cell response of 39 patients with MDS two to four weeks following Covid-19 booster vaccination with BNT162b2 or ChAdOx1 nCoV-19 through the SOAP study (Sars-cov-2 fOr cAncer Patients, IRAS project ID:282337). Plasma and PBMCs from MDS cases and healthy controls have been collected, and are being assessed for both humoral and cellular responses to SARS_CoV_2, the alpha (B.1.1.7) and delta (B.1.617.2) variants. Humoral responses will be assessed using ELISA (peptide binding) and functional viral neutralization assays. Cellular responses will be assessed using IFNy ELISPOT and flow cytometry (CD25 and CD69 expression) after 24h peptide stimulation. All data at time point 1 (2 - 4 weeks following booster vaccination) have been collected and will subsequently be collected at 6 months and 12 months post-vaccination. We also report on the safety data for these vaccines within this patient population. Of this cohort 64% were male with a median age of 65 years (range 21-84). 54% received vaccination with ChAdOx1 nCoV-19 and 44% received BNT162b2 (2% unrecorded). The vaccines were well tolerated with no serious adverse events to date. The mean interval between doses was 70.7 days (range 50 - 90 days). 71% of the cohort were receiving no disease modifying therapy at the time of vaccination, half of whom were receiving supportive therapy and the other half no intervention for their MDS. Of those receiving disease modifying therapy; 5 were receiving azacitidine, (1 in conjunction with low-dose cytarabine) and 3 ciclosporin. We will report the largest study of the humoral and T-cell mediated response to the Covid-19 vaccine in MDS patients to date. This will include cellular response to the delta variant and immunogenicity of both the BNT162b2 and ChAdOx1 nCoV-19 vaccines. Given the vulnerability of these patients to severe disease, investigating the immune response to the vaccines begins to build an evidence base for advising MDS patients on their ongoing risk of infection during the pandemic and going forward. The SOAP study will reassess the immune response at 6 and 12 months post-vaccination to continue to investigate post-vaccine immunity in this cohort. Disclosures Kulasekararaj: F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Speakers Bureau; Apellis: Consultancy; Akari: Consultancy, Honoraria, Speakers Bureau; Biocryst: Consultancy, Honoraria, Speakers Bureau; Achilleon: Consultancy, Honoraria, Speakers Bureau; Alexion: Consultancy, Honoraria, Speakers Bureau; Ra Pharma: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Alexion, AstraZeneca Rare Disease Inc.: Consultancy, Honoraria, Other: Travel support. Patten: JANSSEN: Honoraria; NOVARTIS: Honoraria; GILEAD SCIENCES: Honoraria, Research Funding; ROCHE: Research Funding; ASTRA ZENECA: Honoraria; ABBVIE: Honoraria.

The tumour microenvironment plays a crucial role in the growth and progression of cancer, and the presence of tumour-associated macrophages (TAMs) is associated with poor prognosis. Recent studies have demonstrated that TAMs display transcriptomic, phenotypic, functional and geographical diversity. Here we show that a sialylated tumour-associated glycoform of the mucin MUC1, MUC1-ST, through the engagement of Siglec-9 can specifically and independently induce the differentiation of monocytes into TAMs with a unique phenotype that to the best of our knowledge has not previously been described. These TAMs can recruit and prolong the lifespan of neutrophils, inhibit the function of T cells, degrade basement membrane allowing for invasion, are inefficient at phagocytosis, and can induce plasma clotting. This macrophage phenotype is enriched in the stroma at the edge of breast cancer nests and their presence is associated with poor prognosis in breast cancer patients., Beatson et al. show that a sialylated tumour-associated glycoform of the mucin MUC1 induces the differentiation of monocytes into tumour-associated macrophages. These macrophages are found in breast cancer stroma and their presence is associated with poor prognosis.

Adoptive immunotherapy using γδ T cells harnesses their natural role in tumor immunosurveillance. The efficacy of this approach is enhanced by aminobisphosphonates such as zoledronic acid and alendronic acid, both of which promote the accumulation of stimulatory phosphoantigens in target cells. However, the inefficient and nonselective uptake of these agents by tumor cells compromises the effective clinical exploitation of this principle. To overcome this, we have encapsulated aminobisphosphonates within liposomes. Expanded Vγ9Vδ2 T cells from patients and healthy donors displayed similar phenotype and destroyed autologous and immortalized ovarian tumor cells, following earlier pulsing with either free or liposome-encapsulated aminobisphosphonates. However, liposomal zoledronic acid proved highly toxic to SCID Beige mice. By contrast, the maximum tolerated dose of liposomal alendronic acid was 150-fold higher, rendering it much more suited to in vivo use. When injected into the peritoneal cavity, free and liposomal alendronic acid were both highly effective as sensitizing agents, enabling infused γδ T cells to promote the regression of established ovarian tumors by over one order of magnitude. Importantly however, liposomal alendronic acid proved markedly superior compared with free drug following i.v. delivery, exploiting the “enhanced permeability and retention effect” to render advanced tumors susceptible to γδ T cell–mediated shrinkage. Although folate targeting of liposomes enhanced the sensitization of folate receptor–α+ ovarian tumor cells in vitro, this did not confer further therapeutic advantage in vivo. These findings support the development of an immunotherapeutic approach for ovarian and other tumors in which adoptively infused γδ T cells are targeted using liposomal alendronic acid.

DCs are the most potent APCs and are the focus of many immunotherapeutic approaches for the treatment of cancer, although most of these approaches require the ex vivo generation and pulsing of DCs. We have targeted a subset of DCs in vivo using an Ab to DNGR-1, a C-type lectin dedicated to the cross-presentation of Ag expressed by subsets of DCs. HLA-A2 epitopes from the tumour-associated Ag, MUC1, were coupled to the anti-DNGR-1 Ab, and their efficacy in generating a Th1-cell response and inhibiting tumour growth was evaluated in a clinically relevant double transgenic mouse model expressing human MUC1 and A2K/b. Using this strategy, we demonstrate that an effective immune response to MUC1 can be generated, which results in a significant delay in the growth of MUC1-expressing tumours in both prophylactic and therapeutic settings. In addition, we also show, using PBMCs isolated from healthy volunteer blood, that target an MUC1 HLA-A2 epitope to human DNGR-1 in vitro can induce an MUC1-specific CD8(+) -T-cell response, which confirms the relevance of our in vivo murine results in the human setting.

Carbohydrate–protein interactions govern many crucial processes in biological systems including cell recognition events. We have used the sensitive force probe optical tweezers to quantify the interactions occurring between MGL lectins and MUC1 carrying the cancer-associated glycan antigens mucins Tn and STn. Unbinding forces of 7.6±1.1 pN and 7.1±1.1 pN were determined for the MUC1(Tn)—MGL and MUC1(STn)—MGL interactions, at a force loading rate of ~40 pN/s. The interaction strength increased with increasing force loading rate, to 27.1±4.4 and 36.9±3.6 pN at a force loading rate of ~ 310 pN/s. No interactions were detected between MGL and MUC1(ST), a glycoform of MUC1 also expressed by breast carcinoma cells. Interestingly, this glycan (ST) can be found on proteins expressed by normal cells, although in this case not on MUC1. Additionally, GalNAc decorated polyethylene glycol displayed similar rupture forces as observed for MUC1(Tn) and MUC1(STn) when forced to unbind from MGL, indicating that GalNAc is an essential group in these interactions. Since the STn glycan decoration is more frequently found on the surface of carcinomas than the Tn glycan, the binding of MUC1 carrying STn to MGL may be more physiologically relevant and may be in part responsible for some of the characteristics of STn expressing tumours. © 2017 Hadjialirezaei et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Siglec-9 is a sialic-acid-binding lectin expressed predominantly on myeloid cells. Aberrant glycosylation occurs in essentially all types of cancers and results in increased sialylation. Thus, when the mucin MUC1 is expressed on cancer cells, it is decorated by multiple short, sialylated O-linked glycans (MUC1-ST). Here we found that this cancer-specific MUC1 glycoform, through engagement of Siglec-9, 'educated' myeloid cells to release factors associated with determination of the tumor microenvironment and disease progression. Moreover, MUC1-ST induced macrophages to display a tumor-associated macrophage (TAM)-like phenotype, with increased expression of the checkpoint ligand PD-L1. Binding of MUC1-ST to Siglec-9 did not activate the phosphatases SHP-1 or SHP-2 but, unexpectedly, induced calcium flux that led to activation of the kinases MEK-ERK. This work defines a critical role for aberrantly glycosylated MUC1 and identifies an activating pathway that follows engagement of Siglec-9.

Chinese hamster ovary (CHO) cells are widely used for the production of recombinant proteins for clinical use as well as academic research. They are particularly important for the production of glycoproteins where bacteria cannot be used. TGFβ1 is a potent cytokine highly conserved across species with multiple immunological and non-immunological effects. We have discovered that CHOK1, the CHO clone most commonly used by the pharmaceutical industry, constitutively secretes latent TGFβ1 and that this hamster TGFβ1 is active on human cells inducing profound immunological effects. As far as we are aware, the production of TGFβ1 by CHOK1 cells has not been reported before in the literature. As TGFβ1 exerts powerful and pleiotropic effects on diverse cell types, and as CHO cells are used to produce a large number of clinical and non-clinical products, our findings are highly relevant to studies that rely on recombinant proteins.

Changes in glycosylation are common in malignancy, and as almost all surface proteins are glycosylated, this can dramatically affect the behavior of tumor cells. In breast carcinomas, the O-linked glycans are frequently truncated, often as a result of premature sialylation. The sialyltransferase ST3Gal-I adds sialic acid to the galactose residue of core 1 (Galbeta1,3GalNAc) O-glycans and this enzyme is over-expressed in breast cancer resulting in the expression of sialylated core 1 glycans. In order to study the role of ST3Gal-I in mammary tumor development, we developed transgenic mice that over-express the sialyltransferase under the control of the human membrane-bound mucin 1 promoter. These mice were then crossed with PyMT mice that spontaneously develop mammary tumors. As expected, ST3Gal-I transgenic mice showed increased activity and expression of the enzyme in the pregnant and lactating mammary glands, the stomach, lungs and intestine. Although no obvious defects were observed in the fully developed mammary gland, when these mice were crossed with PyMT mice, a highly significant decrease in tumor latency was observed compared to the PyMT mice on an identical background. These results indicate that ST3Gal-I is acting as a tumor promoter in this model of breast cancer. This, we believe, is the first demonstration that over-expression of a glycosyltransferase involved in mucin-type O-linked glycosylation can promote tumorigenesis.

e15104Background: Many tumors, including breast cancers, contain infiltrating immune cells that are unable to eliminate the tumor and in many cases, promote tumor progression by adding to the permi...

The four members of the JARID1/KDM5 family of proteins, a sub-group of the larger ARID (AT rich DNA binding domain) family, have been shown to demethylate trimethylated lysine 4 on histone 3 (H3K4me3), a chromatin mark associated with actively transcribed genes. In some lower organisms a single homologue of JARID1 is found, and functions of the four proteins found in mice and humans may be specific or overlapping. To investigate the function of the Jarid1B protein we examined the effects of deletion of the gene in mice. Systemic knock out of Jarid1b resulted in early embryonic lethality, whereas mice not expressing the related Jarid1A gene are viable and fertile. A second mouse strain expressing a Jarid1b gene with the ARID domain deleted was viable and fertile but displayed a mammary phenotype, where terminal end bud development and side branching was delayed at puberty and in early pregnancy. Since development of terminal end buds are completely dependent on signalling from the estrogen receptor (ERα), we investigated the expression of a target gene (progesterone receptor) in the ∆ARID mouse and found levels to be reduced as compared to wild-type. JARID1B is widely expressed in ER+ breast cancers and breast cancer cell lines, and interaction with ERα was demonstrated by co-immunoprecipitations in cells transfected with tagged ERα and JARID1B genes. Down-regulation of expression of JARID1B using shRNAi in MCF-7 cells resulted in a dramatic decrease in E2 stimulated tumour growth in nude mice. The data demonstrate a specific role for Jarid1B in early embryonic development, in the development and differentiation of the normal mammary gland, and in estrogen induced growth of ER+ breast cancer.

Disease remission is successfully induced in the majority of patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), but is generally not sustained without stem cell transplantation. Adoptive immunotherapy offers an attractive option to consolidate AML remission. Here, we hypothesized that ex-vivo expanded Vg9Vd2 T-cells will mediate a therapeutically beneficial graft versus leukemia (GvL) effect in this disease. Peripheral blood mononuclear cells from healthy donors and from patients with AML were activated in-vitro using zoledronic acid and a cytokine cocktail, containing IL-2. As a result, we observed an average 200-fold expansion of Vg9Vd2 T-cells from AML patients, over 15 days. Such expansion was only observed in patients without circulating AML blasts. Expanded patient-derived Vg9Vd2 T-cells exhibited a less differentiated phenotype than cells from healthy donors, indicated by higher expression of CD62L, CCR7 and CD27. A four hour Annexin V cytotoxicity assay using KG-1 and U937 leukemic cells showed that these Vg9Vd2 T-cells were cytotoxic and produced nanogram amounts of interferon-g; (IFN-g). In a parallel approach, we investigated feasibility of developing a universal allogeneic Vg9Vd2 T-cell therapy. Feasibility of this approach has been demonstrated in a small trial in which haploidentical donor-derived Vg9Vd2 T-cells were safely infused and achieved an efficacy signal in patients with advanced haematological diseases. We have recently developed a novel method to expand Vg9Vd2 T-cells from healthy donors ex-vivo (“Method 2 (M2)” cells – patent protected), which improves their expansion over 2 weeks to 1700 fold. Cytotoxicity data shows that M2 cells destroy luciferase-expressing U937 and KG-1 target cells at 2-3 fold enhanced efficiency, accompanied by 2-3.5-fold increased IFN-g; release when compared to cells expanded using conventional approaches. Furthermore, M2 cells can achieve serial killing of AML cell lines through up to 4 cycles of re-stimulation. Treatment of SCID-Beige mice with an established U937 leukemic burden is also more effective using M2 expanded Vg9Vd2 T-cells, particularly when combined with zoledronic acid. Together these data highlight the promise of Vg9Vd2 T-cell immunotherapy of AML. Citation Format: Ana C. Parente-Pereira, Lynsey M. Whilding, Pramila Krishnamurthy, Richard Beatson, Tomasz Zabinski, Linda Barber, Farzin Farzaneh, Ghulam Mufti, John Maher. Immunotherapy of acute myeloid leukemia using Vg9Vd2 T-cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B124.

This paper describes the results in a series of 38 patients who had received injections of colloidal yttrium-90 silicate into rheumatoid joints, mostly knees. 73 joints were injected and followed up f

Background Antibody-fluorophore conjugates are invaluable reagents used in contemporary molecular cell biology for imaging, cell sorting and tracking intracellular events. However they suffer in some cases from batch to batch variation, partial loss of binding and susceptibility to photo-bleaching. In theory, these issues can all be addressed by using recombinant antibody fused directly to genetically encoded fluorescent reporters. However, single-chain fragment variable domains linked by long flexible linkers are themselves prone to disassociation and aggregation, and in some cases with isoelectric points incompatible with use in physiologically relevant milieu. Here we describe a general approach that permits fully functional intracellular production of a range of coloured fluorescent recombinant antibodies with optimally orientated VH/VL interfaces and isoelectric points compatible for use in physiological solutions at pH 7.4 with a binding site to fluorophore stoichiometry of 1:1. Results Here we report the design, assembly, intracellular bacterial production and purification of a panel of novel antibody fluorescent protein fusion constructs. The insertion of monomeric fluorescent protein derived from either Discosoma or Aequorea in-between the variable regions of anti-p185HER2-ECD antibody 4D5-8 resulted in optimal VH/VL interface interactions to create soluble coloured antibodies each with a single binding site, with isoelectric points of 6.5- 6. The fluorescent antibodies used in cell staining studies with SK-BR-3 cells retained the fluorophore properties and antibody specificity functions, whereas the conventional 4D5-8 single chain antibody with a (Gly4Ser)3 linker precipitated at physiological pH 7.4. Conclusions This modular monomeric recombinant fluorescent antibody platform may be used to create a range of recombinant coloured antibody molecules for quantitative in situ, in vivo and ex vivo imaging, cell sorting and cell trafficking studies. Assembling the single chain antibody with monomeric fluorescent protein linker facilitates optimal variable domain pairing and alters the isoelectric point of the recombinant 4D5-8 protein conferring solubility at physiological pH 7.4. The efficient intracellular expression of these functional molecules opens up the possibility of developing an alternative approach for tagging intracellular targets with fluorescent proteins for a range of molecular cell biology imaging studies.