Macrophage colony-stimulating factor receptor (M-CSFR) is found in cells of the mononuclear phago... more Macrophage colony-stimulating factor receptor (M-CSFR) is found in cells of the mononuclear phagocyte lineage and is aberrantly expressed in a range of tumours, in addition to tumour-associated macrophages. Consequently, a variety of cancer therapies directed against M-CSFR are under development. We set out to engineer chimeric antigen receptors (CARs) that employ the natural ligands of this receptor, namely M-CSF or interleukin (IL)-34, to achieve specificity for M-CSFR-expressing target cells. Both M-CSF and IL-34 bind to overlapping regions of M-CSFR, although affinity of IL-34 is significantly greater than that of M-CSF. Matched second- and third-generation CARs targeted using M-CSF or IL-34 were expressed in human T-cells using the SFG retroviral vector. We found that both M-CSF- and IL-34-containing CARs enable T-cells to mediate selective destruction of tumour cells that express enforced or endogenous M-CSFR, accompanied by production of both IL-2 and interferon (IFN)-γ. Alth...
Co-stimulation is critical to the function of chimeric antigen receptor (CAR) T-cells. Previously... more Co-stimulation is critical to the function of chimeric antigen receptor (CAR) T-cells. Previously, we demonstrated that dual co-stimulation can be effectively harnessed by a parallel (p)CAR architecture in which a CD28-containing second generation CAR is co-expressed with a 4-1BB containing chimeric co-stimulatory receptor (CCR). When compared to linear CARs, pCAR-engineered T-cells elicit superior anti-tumor activity in a range of pre-clinical models. Since CD19 is the best validated clinical target for cellular immunotherapy, we evaluated a panel of CD19-specific CAR and pCAR T-cells in this study. First, we generated a panel of single chain antibody fragments (scFvs) by alanine scanning mutagenesis of the CD19-specific FMC63 scFv (VH domain) and these were incorporated into second generation CD28+CD3ζ CARs. The resulting panel of CAR T-cells demonstrated a broad range of CD19 binding ability and avidity for CD19-expressing tumor cells. Each scFv-modified CAR was then converted in...
C himeric antigen receptors are fusion molecules that couple the direct (antibody-like) binding o... more C himeric antigen receptors are fusion molecules that couple the direct (antibody-like) binding of a cell surface target to the delivery of a tailored T-cell activating signal. These molecules are delivered to peripheral blood T or NK cells, using retroviral, lentiviral or a number of nonviral vector systems. Three generations of CARs have been described in which signalling is provided by CD3ζ alone (first generation), with either CD28 or 4-1BB co-stimulation (second generation) or with a combination of both of these co-stimulatory signals (third generation). Immunotherapy using autologous CAR T-cells has made a profound impact on the management of refractory B-cell malignancy. In that setting, second generation CAR T-cells are re-targeted against the ubiquitous B-cell antigen, CD19, enabling them to eliminate both malignant and healthy B-cells. Complete response rates approaching 90% have been achieved in patients with refractory acute lymphoblastic leukaemia. Emphasizing robustness, these data have been achieved in a number of distinct centres, using CARs that vary in their antibody targeting moiety or co-stimulatory domain (either CD28 or 4-1BB). However, successful implementation of CAR T-cell immunotherapy for solid tumours remains an elusive goal. Mindful of the role played by ErbB dysre-gulation in many solid tumours, we set out to target this family using a CAR-based approach. The ErbB family of receptor tyrosine kinases comprises four members (ErbB1-4) which form a series of 9 possible homo-or heterodimeric pairs. Several highly successful anti-cancer pharmaceuticals have been developed to target one or more members of the family, including herceptin, cetuximab and erlotinib. To achieve broad targeting specificity across the network, we used a chimeric peptide named T1E. T1E is derived from transforming growth factor-α (TGF-α) and epidermal growth factor (EGF). While TGF-α and EGF are both unique ErbB1 ligands, the T1E chimera binds to all ErbB1 and ErbB4 homo-and heterodimers, in addition to the ErbB2/3 heterodimer. A second generation CAR named T1E28z was engineered by fusing T1E to a chimeric endodomain comprising CD28 and CD3ζ. Pre-clinical evaluation of T1E28z-engineered human T-cells demonstrated antitumour activity against diverse solid tumour cell lines, provided that one or more of the indicated ErbB dimer species was present. Furthermore, it was anticipated that evasion of T1E28z + CAR T-cells by tumour cells through antigen loss was unlikely since the CAR recognized multiple targets, coupled with the fact that ErbB dysregulation is an intrinsic driver of transformation. However, the major risk anticipated with clinical development of this CAR was potential for on-target toxicity. Underlining this, a fatal SUSAR (suspected unexpected severe adverse reaction) was described recently following the intravenous administration of 10 billion ErbB2 re-targeted CAR T-cells to a patient with metastatic ErbB2+ colorectal cancer. To mitigate risk, we identified a clinical niche whereby localized rather than systemic delivery of CAR T-cells could be justified. Refractory head and neck squamous cell cancer (HNSCC) provides such an indication since locally advanced or recurrent tumour formation represents
Summary Immunotherapy of cancer using chimeric antigen receptor-engineered T-cells has transforme... more Summary Immunotherapy of cancer using chimeric antigen receptor-engineered T-cells has transformed the management of selected haematological malignancies, triggering intense clinical trial activity in this arena. This article summarises trial activity that has been published to date across the spectrum of haematological and solid tumour types.
CAR-engineered T cell immunotherapy has proven transformative in selected hematological malignanc... more CAR-engineered T cell immunotherapy has proven transformative in selected hematological malignancies. However, solid tumors largely remain impervious to these approaches. In addressing this challenge, Srivastava et al. in this issue demonstrate that oxaliplatin-based lymphodepleting chemotherapy promotes enhanced CAR T cell recruitment to lung tumors, boosting therapeutic impact in combination with anti-PD-L1.
If citing, it is advised that you check and use the publisher's definitive version for pagination... more If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you are again advised to check the publisher's website for any subsequent corrections.
Immunotherapy using CAR-T-cells is acquiring an expanding role in the treatment of malignant dise... more Immunotherapy using CAR-T-cells is acquiring an expanding role in the treatment of malignant disease. However, efficacy of solid tumour CAR-T-immunotherapy has proven inconsistent. Limitations include poor T-cell trafficking to tumour deposits, insufficient T-cell longevity in-vivo and the occurrence of both predicted and unanticipated on-target and off-target toxicity. To refine this therapeutic approach, it is desirable to develop systems that allow the monitoring of T-cell location and persistence in-vivo. A retroviral vector named PiN-4 was constructed, which co-expresses: (i) an interleukin (IL)-4-responsive chimeric cytokine receptor (4αβ); (ii) a prostate-specific membrane antigen (PSMA)-targeted CAR (P28z) and (iii) hNIS, which promotes the uptake of technetium-99m pertechnetate (99mTcO4−) in viable cells. IL-4 enriched human 4P28zN+ T-cells were administered intravenously to Nod Scid Gamma (NSG) mice bearing prostate tumour xenografts. Measurement of the tumour xenografts by bioluminescent imaging (BLI) found that only PSMA-expressing tumours responded to 4P28zN+ T-cell treatment. Longitudinal SPECT-CT imaging further confirmed this with the preferential accumulation of 4P28zN+ T-cells in PSMA-expressing tumours compared to PSMA-negative tumours. Use of NIS as a T-cell imaging reporter brings several potential advantages. It promotes receptor-mediated uptake of the inexpensive, low toxicity and clinically useful SPECT tracer, technetium-99m pertechnetate (99mTcO4−). In addition, the ectopic expression of NIS is well tolerated by host cells and hNIS functions only in viable cells. These data demonstrate proof of concept for the utility of hNIS as an imaging reporter in genetically engineered T-cells. Citation Format: Nia Emami-Shahri, Julie Foster, Jane Sosabowski, John Maher, Sophie Papa. Dynamic SPECT imaging of PSMA-specific CAR T cells in mice bearing prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2315.
-9 is a sialic acid binding lectin predominantly expressed on myeloid 19 cells. Aberrant glycosyl... more -9 is a sialic acid binding lectin predominantly expressed on myeloid 19 cells. Aberrant glycosylation occurs in essentially all types of cancers 20 resulting in increased sialylation. Thus when MUC1 is expressed on cancer 21 cells it is decorated by multiple short, sialylated O-linked glycans (MUC1-ST). 22 Here we show that this cancer-specific MUC1 glycoform could, through the 23 engagement of Siglec-9, educate myeloid cells to release factors associated 24 with tumor microenvironment determination and disease progression. 25 Moreover MUC1-ST induced macrophages to display a TAM-like phenotype 26 with increased expression of PD-L1. MUC1-ST binding to Siglec-9 did not 27 activate SHP-1/2 but surprisingly induced calcium flux leading to MEK-ERK 28 activation. This work defines a critical role for aberrantly glycosylated MUC1 29 and identifies an activating pathway following Siglec-9 engagement. 30 31 Cancers have developed a plethora of mechanisms to evade the immune response 32 including initiating a permissive local environment. For cancer cells to remodel their 33 microenvironment they need to acquire changes that include the recruitment and 34 education of monocytes, and the repolarization of resident macrophages 1 . 35 Macrophages are phenotypically plastic and factors produced by cancer cells can 36 polarize macrophages to become tumor-promoting. These tumor-educated 37 macrophages promote the growth and invasion of cancer cells by contributing to all 38 the stages involved in cancer dissemination, cumulating in metastasis 2 39 40 Changes in glycosylation occur in essentially all types of cancers and changes in 41 mucin-type O-linked glycans are the most prevalent aberrant glycophenotype when 42 increased sialylation often occurs 3,4 . The transmembrane mucin MUC1 is 43 upregulated in breast and the majority of adenocarcinomas and, due to the presence 44 of a variable number of tandem repeats that contain the O-linked glycosylation sites, 45 can carry from 100 to over 750 O-glycans 5 . The aberrant glycosylation seen in 46 cancer results in the multiple O-linked glycans carried by MUC1 being mainly short 47 and sialylated 3,6 , in contrast to the long, branched chains seen on MUC1 expressed 48 by normal epithelial cells 7 . In carcinomas the aberrant O-linked glycosylation of 49 MUC1 can alter the interaction of MUC1 with lectins of the immune system 8 and 50 thereby influence tumor-immune interplay. While it is clear that expression of MUC1 51 carrying short, sialylated core 1 glycans (NeuAcα2,3Galβ1-3GalNAc; MUC1-ST) 52 enhances tumor growth 9,10 , the mechanisms underlying this increased growth are ill-53 defined. However, the immune system appears to play a role as syngeneic mouse 54 tumor cells expressing MUC1-ST grow significantly faster in MUC1-transgenic mice 55 than the same cells expressing MUC1 carrying branched core 2 glycans associated 56 with normal glycosylation, while this differential growth is not seen in 57 immunosuppressed mice 9 . 58 59 Siglecs (sialic acid-binding immunoglobulin-like lectins) are a family of sialic acid 60 binding lectins, which, with the exception of Siglec-4, are expressed on various cells 61 of the immune system 11 . The cytoplasmic domains of most Siglecs contain 62 immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which recruit the tyrosine 63 phosphatases, SHP-1 and/or SHP-2 (ref. 12 ) and so regulate the cells of the innate 64 and adaptive immune response 13 . It has recently become clear that Siglecs play a 65 role in cancer immune suppression, the hypersialylation seen in cancers inducing 66 binding to these lectins 14-16 . MUC1 expressed by cancer cells has been shown to 67 bind to Siglec-9 resulting in the recruitment of β-catenin to the cytoplasmic tail of 68 MUC1 inducing its translocation to the nucleus and increased tumor cell growth 17 . This work focused on the effect of the interaction with Siglec-9 on the MUC1 70 expressing cancer cells. In contrast we have investigated the effect of the interaction 71 on the Siglec-9 expressing immune cells using a defined glycoform of MUC1 (ref. ). Siglec-9 is predominantly expressed on myeloid cells and has a preference for sialic 73 acid α2,3 linked to galactose . Here we show that MUC1 carrying the sialylated core 74 1 glycan (MUC1-ST) a glycan not found on this mucin expressed by normal epithelial 75 cells, binds to Siglec-9 on primary human monocytes and macrophages, and induces 76 a unique secretome signature from each cell type. Moreover, when MUC1-ST binds 77 to Siglec-9 expressed by primary macrophages a tumor-associated macrophage 78 (TAM) phenotype is actively induced shown by the inhibition of CD8 + T cell 79 proliferation and the upregulation of IDO (indoleamine 2,3-dioxygenase), CD163, 80 CD206 and of the checkpoint ligand PD-L1 (programmed death ligand 1). To investigate the interaction of MUC1-ST with cells of the immune system, immune 86 cell subsets were isolated from donor blood and incubated with biotinylated purified 87 recombinant tumor-associated MUC1 glycoforms 18 (Fig. and Supplementary Fig. ). MUC1 carrying sialylated core-1 glycans (NeuAcα2,3Galβ1-3GalNAc; MUC1-89 ST), bound to primary monocytes and macrophages and acute myeloid leukemia 90 (AML) lines (Fig. ). This interaction was lost upon neuraminidase treatment of 91 MUC1-ST to give MUC1-T, demonstrating that the binding was dependent upon 92 sialic acid (Fig. ). The binding also increased with time, maximum binding 93 occurring at 5 hours, and with increased the concentration of MUC1-ST 94 (Supplementary Fig. ) but was calcium independent (Fig. ). Moreover, the 95 binding was enhanced when cells were pre-treated with neuraminidase 96 (Supplementary Fig. ), which removes competing cis-binding sialic acid sites 97 from the surface of the cells. This pattern is characteristic of binding to Siglec 98 molecules 11 and indeed MUC1-ST bound recombinant Siglecs-3, 7, 9 and 10; with 99 the greatest binding seen for Siglec-9 (Fig. ). Although Siglecs-3, 7 and 9 are 100 expressed by monocytes and macrophages (Supplementary Fig. ), a blocking 101 antibody to Siglec-9 inhibited 80-95% of the MUC1-ST binding to these cells (Fig. 1h,i; Supplementary Fig. ) indicating this is the 103 dominant binding Siglec. It should be noted that this anti-Siglec-9 antibody can also 104 act as an activating antibody as it recognizes the sialic acid binding site on Siglec-9 21 , 105 Importantly, Siglec-9 bound to the breast cancer cell line T47D that expresses MUC1 106 carrying sialylated core-1 glycans (Fig. ) . However, a multivalent polymer of 107 sialylated core 1 glycans bound only weakly to monocytes and this interaction could 108 not be inhibited with anti-Siglec-9 (Supplementary Fig. ). This finding suggests a 109 contribution of the protein backbone to the binding specificity of Siglec-9, possibly by 110 defining a specific spacing of the sialic acids. Thus MUC1-ST specifically binds to 111 Siglec-9 on primary monocytes and macrophages. 112 113 MUC1-ST binding induces the secretion of various factors 114 To determine whether MUC1-ST binding induced a cellular response, we assayed for 115 soluble factors released from cultured primary monocytes upon binding recombinant 116 MUC1-ST. MUC1-ST induced monocytes to secrete several factors associated with 117 inflammation and tumor progression (Fig. and Supplementary Fig. ). We
Immunotherapy of cancer using chimeric antigen receptor (CAR) T-cells is a rapidly expanding fiel... more Immunotherapy of cancer using chimeric antigen receptor (CAR) T-cells is a rapidly expanding field. CARs are fusion molecules that couple the binding of a tumour-associated cell surface target to the delivery of a tailored T-cell activating signal. Re-infusion of such genetically engineered T-cells to patients with haematological disease has demonstrated unprecedented response rates in Phase I clinical trials. However, such successes have not yet been observed using CAR T-cells against solid malignancies and this is, in part, due to a lack of safe tumour-specific targets. The αvβ6 integrin is strongly up-regulated in multiple solid tumours including those derived from colon, lung, breast, cervix, ovaries/fallopian tube, pancreas and head and neck. It is associated with poorer prognosis in several cancers and exerts pro-tumorigenic activities including promotion of tumour growth, migration and invasion. By contrast, physiologic expression of αvβ6 is largely restricted to wound healin...
Chimeric antigen receptor (CAR) based immunotherapy has been under development for the last 25 ye... more Chimeric antigen receptor (CAR) based immunotherapy has been under development for the last 25 years and is now a promising new treatment modality in the field of cancer immunotherapy. The approach involves genetically engineering T cells to target malignant cells through expression of a bespoke fusion receptor that couples an HLA-independent antigen recognition domain to one or more intracellular T-cell activating modules. Multiple clinical trials are now underway in several centers to investigate CAR T-cell immunotherapy of diverse hematologic and solid tumor types. The most successful results have been achieved in the treatment of patients with B-cell malignancies, in whom several complete and durable responses have been achieved. This review focuses on the preclinical and clinical development of CAR T-cell immunotherapy of solid cancers, targeted against members of the ErbB family.
Chimeric antigen receptor- (CAR-) based immunotherapy has been under development for almost 25 ye... more Chimeric antigen receptor- (CAR-) based immunotherapy has been under development for almost 25 years, over which period it has progressed from a new but cumbersome technology to an emerging therapeutic modality for malignant disease. The approach involves the genetic engineering of fusion receptors (CARs) that couple the HLA-independent binding of cell surface target molecules to the delivery of a tailored activating signal to host immune cells. Engineered CARs are delivered most commonly to peripheral blood T cells using a range of vector systems, most commonly integrating viral vectors. Preclinical refinement of this approach has proceeded over several years to the point that clinical testing is now being undertaken at several centres, using increasingly sophisticated and therapeutically successful genetic payloads. This paper considers several aspects of the pre-clinical and clinical development of CAR-based immunotherapy and how this technology is acquiring an increasing niche i...
Adoptive immunotherapy using gd T cells harnesses their natural role in tumor immunosurveillance.... more Adoptive immunotherapy using gd 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 Vg9Vd2 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 gd 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 gd T cell-mediated shrinkage. Although folate targeting of liposomes enhanced the sensitization of folate receptor-a + 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 gd T cells are targeted using liposomal alendronic acid.
Sensitive and specific detection of nodal status, sites of metastases and low-volume recurrent di... more Sensitive and specific detection of nodal status, sites of metastases and low-volume recurrent disease could greatly improve management of patients with advanced prostate cancer. Prostate-specific membrane antigen (PSMA) is a well-established marker for prostate carcinoma with increased levels of expression in high-grade, hormone-refractory and metastatic disease. The monoclonal antibody (mAb) J591 is directed against an extracellular epitope of PSMA and has been shown to efficiently target disseminated disease including metastases in lymph nodes and bone. Its use as a diagnostic imaging agent however is limited due to its slow pharmacokinetics. In this study a diabody derived from mAb J591 was developed as a single photon emission computed tomography (SPECT) tracer with improved pharmacokinetics for the detection of PSMA expression in prostate cancer. A diabody in VH-VL orientation and with a C-terminal cysteine was expressed in HEK293T cells and purified by a combination of metal ...
MUC1 is a highly attractive immunotherapeutic target owing to increased expression, altered glyco... more MUC1 is a highly attractive immunotherapeutic target owing to increased expression, altered glycosylation, and loss of polarity in >80% of human cancers. To exploit this, we have constructed a panel of chimeric Ag receptors (CAR) that bind selectively to tumor-associated MUC1. Two parameters proved crucial in optimizing the CAR ectodomain. First, we observed that the binding of CAR-grafted T cells to anchored MUC1 is subject to steric hindrance, independent of glycosylation status. This was overcome by insertion of the flexible and elongated hinge found in immunoglobulins of the IgD isotype. Second, CAR function was highly dependent upon strong binding capacity across a broad range of tumor-associated MUC1 glycoforms. This was realized by using an Ab-derived single-chain variable fragment (scFv) cloned from the HMFG2 hybridoma. To optimize CAR signaling, tripartite endodomains were constructed. Ultimately, this iterative design process yielded a potent receptor termed HOX that contains a fused CD28/OX40/CD3 endodomain. HOX-expressing T cells proliferate vigorously upon repeated encounter with soluble or membrane-associated MUC1, mediate production of proinflammatory cytokines (IFN-␥ and IL-17), and elicit brisk killing of MUC1 ؉ tumor cells. To test function in vivo, a tumor xenograft model was derived using MDA-MB-435 cells engineered to coexpress MUC1 and luciferase. Mice bearing an established tumor were treated i.p. with a single dose of engineered T cells. Compared with control mice, this treatment resulted in a significant delay in tumor growth as measured by serial bioluminescence imaging. Together, these data demonstrate for the first time that the near-ubiquitous MUC1 tumor Ag can be targeted using CAR-grafted T cells.
Background Epidemiological studies have shown that only about 20% of the familial clustering of b... more Background Epidemiological studies have shown that only about 20% of the familial clustering of breast cancer is explained by the known highly penetrant mutations in BRCA1 and BCRA2. We have set out to search for the genes for the remaining 80%. Twin studies indicate a predominant role of shared genes rather than a shared environment; the patterns of occurrence of breast cancer in families are consistent with a major polygenic component. Methods We have assembled a population based set of 5,000 breast cancer cases and 5,000 controls from the East Anglian population. We have simple clinical and epidemiological information, including family history, and samples of blood and paraffin embedded tumour. We have used association studies based on single nucleotide polymorphisms, first with candidate genes and then in a genome-wide scan of 266,000 single nucleotide polymorphisms, to search for the putative predisposing genes. We have as yet searched only for common variants (frequency >5%). We have modelled the effects of polygenic predisposition in the East Anglian population, and have shown that the model predicts a wide distribution of individual risk in the population, such that half of all breast cancers may occur in the 12% of women at greatest risk. Both the candidate gene-based and genome-wide scans have provided provisional identification of a number of novel susceptibility genes, and these are currently being confirmed by a world-wide consortium of independent laboratories totalling 20,000-plus cases and controls. No single gene so far identified contributes more than 2% of the total inherited component, consistent with a model in which susceptibility is the result of a large number of individually small genetic effects.
Chinese hamster ovary (CHO) cells are widely used for the production of recombinant proteins for ... more 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.
www.jbmethods.org 1 PROTOCOLJournal of Biological Methods | 2014 | Vol. 1(2) | e7 DOI: 10.14440/j... more www.jbmethods.org 1 PROTOCOLJournal of Biological Methods | 2014 | Vol. 1(2) | e7 DOI: 10.14440/jbm.2014.30 POL Scientific Use of retroviral-mediated gene transfer to deliver and test function of chimeric antigen receptors in human
Prostate cancer is the most common cancer in men, both in the USA and Europe. Although incurable,... more Prostate cancer is the most common cancer in men, both in the USA and Europe. Although incurable, metastatic disease can often be controlled for years with anti-androgen therapy. Once the disease becomes castrate resistant, the median survival is 18 months. There is growing evidence that the immune system, and in particular cytokines, play an important role in prostate cancer immunosurveillance and progression. Here, we have undertaken a clinical investigation of the role of two closely related cytokines, IL-4 and IL-13 in prostate cancer. In the largest series studied to date, we show that serum IL-4, but not IL-13 is significantly elevated in castrate resistant, compared to androgen sensitive disease. Notably however, serum IL-4 levels are also raised in patients with benign prostatic OPEN ACCESS Cancers 2011, 3 4282 disease. Analysis of benign and malignant prostate tissue demonstrates that the source of IL-4 is epithelial cells rather than infiltrating leukocytes. Together, our data are consistent with a dual role for IL-4 in prostate cancer development. In benign disease, our data add to the evidence that IL-4 serves a protective role. By contrast, the data support a direct role for IL-4 in the progression of prostate cancer from androgen responsive, to advanced castrate-resistant disease.
Macrophage colony-stimulating factor receptor (M-CSFR) is found in cells of the mononuclear phago... more Macrophage colony-stimulating factor receptor (M-CSFR) is found in cells of the mononuclear phagocyte lineage and is aberrantly expressed in a range of tumours, in addition to tumour-associated macrophages. Consequently, a variety of cancer therapies directed against M-CSFR are under development. We set out to engineer chimeric antigen receptors (CARs) that employ the natural ligands of this receptor, namely M-CSF or interleukin (IL)-34, to achieve specificity for M-CSFR-expressing target cells. Both M-CSF and IL-34 bind to overlapping regions of M-CSFR, although affinity of IL-34 is significantly greater than that of M-CSF. Matched second- and third-generation CARs targeted using M-CSF or IL-34 were expressed in human T-cells using the SFG retroviral vector. We found that both M-CSF- and IL-34-containing CARs enable T-cells to mediate selective destruction of tumour cells that express enforced or endogenous M-CSFR, accompanied by production of both IL-2 and interferon (IFN)-γ. Alth...
Co-stimulation is critical to the function of chimeric antigen receptor (CAR) T-cells. Previously... more Co-stimulation is critical to the function of chimeric antigen receptor (CAR) T-cells. Previously, we demonstrated that dual co-stimulation can be effectively harnessed by a parallel (p)CAR architecture in which a CD28-containing second generation CAR is co-expressed with a 4-1BB containing chimeric co-stimulatory receptor (CCR). When compared to linear CARs, pCAR-engineered T-cells elicit superior anti-tumor activity in a range of pre-clinical models. Since CD19 is the best validated clinical target for cellular immunotherapy, we evaluated a panel of CD19-specific CAR and pCAR T-cells in this study. First, we generated a panel of single chain antibody fragments (scFvs) by alanine scanning mutagenesis of the CD19-specific FMC63 scFv (VH domain) and these were incorporated into second generation CD28+CD3ζ CARs. The resulting panel of CAR T-cells demonstrated a broad range of CD19 binding ability and avidity for CD19-expressing tumor cells. Each scFv-modified CAR was then converted in...
C himeric antigen receptors are fusion molecules that couple the direct (antibody-like) binding o... more C himeric antigen receptors are fusion molecules that couple the direct (antibody-like) binding of a cell surface target to the delivery of a tailored T-cell activating signal. These molecules are delivered to peripheral blood T or NK cells, using retroviral, lentiviral or a number of nonviral vector systems. Three generations of CARs have been described in which signalling is provided by CD3ζ alone (first generation), with either CD28 or 4-1BB co-stimulation (second generation) or with a combination of both of these co-stimulatory signals (third generation). Immunotherapy using autologous CAR T-cells has made a profound impact on the management of refractory B-cell malignancy. In that setting, second generation CAR T-cells are re-targeted against the ubiquitous B-cell antigen, CD19, enabling them to eliminate both malignant and healthy B-cells. Complete response rates approaching 90% have been achieved in patients with refractory acute lymphoblastic leukaemia. Emphasizing robustness, these data have been achieved in a number of distinct centres, using CARs that vary in their antibody targeting moiety or co-stimulatory domain (either CD28 or 4-1BB). However, successful implementation of CAR T-cell immunotherapy for solid tumours remains an elusive goal. Mindful of the role played by ErbB dysre-gulation in many solid tumours, we set out to target this family using a CAR-based approach. The ErbB family of receptor tyrosine kinases comprises four members (ErbB1-4) which form a series of 9 possible homo-or heterodimeric pairs. Several highly successful anti-cancer pharmaceuticals have been developed to target one or more members of the family, including herceptin, cetuximab and erlotinib. To achieve broad targeting specificity across the network, we used a chimeric peptide named T1E. T1E is derived from transforming growth factor-α (TGF-α) and epidermal growth factor (EGF). While TGF-α and EGF are both unique ErbB1 ligands, the T1E chimera binds to all ErbB1 and ErbB4 homo-and heterodimers, in addition to the ErbB2/3 heterodimer. A second generation CAR named T1E28z was engineered by fusing T1E to a chimeric endodomain comprising CD28 and CD3ζ. Pre-clinical evaluation of T1E28z-engineered human T-cells demonstrated antitumour activity against diverse solid tumour cell lines, provided that one or more of the indicated ErbB dimer species was present. Furthermore, it was anticipated that evasion of T1E28z + CAR T-cells by tumour cells through antigen loss was unlikely since the CAR recognized multiple targets, coupled with the fact that ErbB dysregulation is an intrinsic driver of transformation. However, the major risk anticipated with clinical development of this CAR was potential for on-target toxicity. Underlining this, a fatal SUSAR (suspected unexpected severe adverse reaction) was described recently following the intravenous administration of 10 billion ErbB2 re-targeted CAR T-cells to a patient with metastatic ErbB2+ colorectal cancer. To mitigate risk, we identified a clinical niche whereby localized rather than systemic delivery of CAR T-cells could be justified. Refractory head and neck squamous cell cancer (HNSCC) provides such an indication since locally advanced or recurrent tumour formation represents
Summary Immunotherapy of cancer using chimeric antigen receptor-engineered T-cells has transforme... more Summary Immunotherapy of cancer using chimeric antigen receptor-engineered T-cells has transformed the management of selected haematological malignancies, triggering intense clinical trial activity in this arena. This article summarises trial activity that has been published to date across the spectrum of haematological and solid tumour types.
CAR-engineered T cell immunotherapy has proven transformative in selected hematological malignanc... more CAR-engineered T cell immunotherapy has proven transformative in selected hematological malignancies. However, solid tumors largely remain impervious to these approaches. In addressing this challenge, Srivastava et al. in this issue demonstrate that oxaliplatin-based lymphodepleting chemotherapy promotes enhanced CAR T cell recruitment to lung tumors, boosting therapeutic impact in combination with anti-PD-L1.
If citing, it is advised that you check and use the publisher's definitive version for pagination... more If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you are again advised to check the publisher's website for any subsequent corrections.
Immunotherapy using CAR-T-cells is acquiring an expanding role in the treatment of malignant dise... more Immunotherapy using CAR-T-cells is acquiring an expanding role in the treatment of malignant disease. However, efficacy of solid tumour CAR-T-immunotherapy has proven inconsistent. Limitations include poor T-cell trafficking to tumour deposits, insufficient T-cell longevity in-vivo and the occurrence of both predicted and unanticipated on-target and off-target toxicity. To refine this therapeutic approach, it is desirable to develop systems that allow the monitoring of T-cell location and persistence in-vivo. A retroviral vector named PiN-4 was constructed, which co-expresses: (i) an interleukin (IL)-4-responsive chimeric cytokine receptor (4αβ); (ii) a prostate-specific membrane antigen (PSMA)-targeted CAR (P28z) and (iii) hNIS, which promotes the uptake of technetium-99m pertechnetate (99mTcO4−) in viable cells. IL-4 enriched human 4P28zN+ T-cells were administered intravenously to Nod Scid Gamma (NSG) mice bearing prostate tumour xenografts. Measurement of the tumour xenografts by bioluminescent imaging (BLI) found that only PSMA-expressing tumours responded to 4P28zN+ T-cell treatment. Longitudinal SPECT-CT imaging further confirmed this with the preferential accumulation of 4P28zN+ T-cells in PSMA-expressing tumours compared to PSMA-negative tumours. Use of NIS as a T-cell imaging reporter brings several potential advantages. It promotes receptor-mediated uptake of the inexpensive, low toxicity and clinically useful SPECT tracer, technetium-99m pertechnetate (99mTcO4−). In addition, the ectopic expression of NIS is well tolerated by host cells and hNIS functions only in viable cells. These data demonstrate proof of concept for the utility of hNIS as an imaging reporter in genetically engineered T-cells. Citation Format: Nia Emami-Shahri, Julie Foster, Jane Sosabowski, John Maher, Sophie Papa. Dynamic SPECT imaging of PSMA-specific CAR T cells in mice bearing prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2315.
-9 is a sialic acid binding lectin predominantly expressed on myeloid 19 cells. Aberrant glycosyl... more -9 is a sialic acid binding lectin predominantly expressed on myeloid 19 cells. Aberrant glycosylation occurs in essentially all types of cancers 20 resulting in increased sialylation. Thus when MUC1 is expressed on cancer 21 cells it is decorated by multiple short, sialylated O-linked glycans (MUC1-ST). 22 Here we show that this cancer-specific MUC1 glycoform could, through the 23 engagement of Siglec-9, educate myeloid cells to release factors associated 24 with tumor microenvironment determination and disease progression. 25 Moreover MUC1-ST induced macrophages to display a TAM-like phenotype 26 with increased expression of PD-L1. MUC1-ST binding to Siglec-9 did not 27 activate SHP-1/2 but surprisingly induced calcium flux leading to MEK-ERK 28 activation. This work defines a critical role for aberrantly glycosylated MUC1 29 and identifies an activating pathway following Siglec-9 engagement. 30 31 Cancers have developed a plethora of mechanisms to evade the immune response 32 including initiating a permissive local environment. For cancer cells to remodel their 33 microenvironment they need to acquire changes that include the recruitment and 34 education of monocytes, and the repolarization of resident macrophages 1 . 35 Macrophages are phenotypically plastic and factors produced by cancer cells can 36 polarize macrophages to become tumor-promoting. These tumor-educated 37 macrophages promote the growth and invasion of cancer cells by contributing to all 38 the stages involved in cancer dissemination, cumulating in metastasis 2 39 40 Changes in glycosylation occur in essentially all types of cancers and changes in 41 mucin-type O-linked glycans are the most prevalent aberrant glycophenotype when 42 increased sialylation often occurs 3,4 . The transmembrane mucin MUC1 is 43 upregulated in breast and the majority of adenocarcinomas and, due to the presence 44 of a variable number of tandem repeats that contain the O-linked glycosylation sites, 45 can carry from 100 to over 750 O-glycans 5 . The aberrant glycosylation seen in 46 cancer results in the multiple O-linked glycans carried by MUC1 being mainly short 47 and sialylated 3,6 , in contrast to the long, branched chains seen on MUC1 expressed 48 by normal epithelial cells 7 . In carcinomas the aberrant O-linked glycosylation of 49 MUC1 can alter the interaction of MUC1 with lectins of the immune system 8 and 50 thereby influence tumor-immune interplay. While it is clear that expression of MUC1 51 carrying short, sialylated core 1 glycans (NeuAcα2,3Galβ1-3GalNAc; MUC1-ST) 52 enhances tumor growth 9,10 , the mechanisms underlying this increased growth are ill-53 defined. However, the immune system appears to play a role as syngeneic mouse 54 tumor cells expressing MUC1-ST grow significantly faster in MUC1-transgenic mice 55 than the same cells expressing MUC1 carrying branched core 2 glycans associated 56 with normal glycosylation, while this differential growth is not seen in 57 immunosuppressed mice 9 . 58 59 Siglecs (sialic acid-binding immunoglobulin-like lectins) are a family of sialic acid 60 binding lectins, which, with the exception of Siglec-4, are expressed on various cells 61 of the immune system 11 . The cytoplasmic domains of most Siglecs contain 62 immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which recruit the tyrosine 63 phosphatases, SHP-1 and/or SHP-2 (ref. 12 ) and so regulate the cells of the innate 64 and adaptive immune response 13 . It has recently become clear that Siglecs play a 65 role in cancer immune suppression, the hypersialylation seen in cancers inducing 66 binding to these lectins 14-16 . MUC1 expressed by cancer cells has been shown to 67 bind to Siglec-9 resulting in the recruitment of β-catenin to the cytoplasmic tail of 68 MUC1 inducing its translocation to the nucleus and increased tumor cell growth 17 . This work focused on the effect of the interaction with Siglec-9 on the MUC1 70 expressing cancer cells. In contrast we have investigated the effect of the interaction 71 on the Siglec-9 expressing immune cells using a defined glycoform of MUC1 (ref. ). Siglec-9 is predominantly expressed on myeloid cells and has a preference for sialic 73 acid α2,3 linked to galactose . Here we show that MUC1 carrying the sialylated core 74 1 glycan (MUC1-ST) a glycan not found on this mucin expressed by normal epithelial 75 cells, binds to Siglec-9 on primary human monocytes and macrophages, and induces 76 a unique secretome signature from each cell type. Moreover, when MUC1-ST binds 77 to Siglec-9 expressed by primary macrophages a tumor-associated macrophage 78 (TAM) phenotype is actively induced shown by the inhibition of CD8 + T cell 79 proliferation and the upregulation of IDO (indoleamine 2,3-dioxygenase), CD163, 80 CD206 and of the checkpoint ligand PD-L1 (programmed death ligand 1). To investigate the interaction of MUC1-ST with cells of the immune system, immune 86 cell subsets were isolated from donor blood and incubated with biotinylated purified 87 recombinant tumor-associated MUC1 glycoforms 18 (Fig. and Supplementary Fig. ). MUC1 carrying sialylated core-1 glycans (NeuAcα2,3Galβ1-3GalNAc; MUC1-89 ST), bound to primary monocytes and macrophages and acute myeloid leukemia 90 (AML) lines (Fig. ). This interaction was lost upon neuraminidase treatment of 91 MUC1-ST to give MUC1-T, demonstrating that the binding was dependent upon 92 sialic acid (Fig. ). The binding also increased with time, maximum binding 93 occurring at 5 hours, and with increased the concentration of MUC1-ST 94 (Supplementary Fig. ) but was calcium independent (Fig. ). Moreover, the 95 binding was enhanced when cells were pre-treated with neuraminidase 96 (Supplementary Fig. ), which removes competing cis-binding sialic acid sites 97 from the surface of the cells. This pattern is characteristic of binding to Siglec 98 molecules 11 and indeed MUC1-ST bound recombinant Siglecs-3, 7, 9 and 10; with 99 the greatest binding seen for Siglec-9 (Fig. ). Although Siglecs-3, 7 and 9 are 100 expressed by monocytes and macrophages (Supplementary Fig. ), a blocking 101 antibody to Siglec-9 inhibited 80-95% of the MUC1-ST binding to these cells (Fig. 1h,i; Supplementary Fig. ) indicating this is the 103 dominant binding Siglec. It should be noted that this anti-Siglec-9 antibody can also 104 act as an activating antibody as it recognizes the sialic acid binding site on Siglec-9 21 , 105 Importantly, Siglec-9 bound to the breast cancer cell line T47D that expresses MUC1 106 carrying sialylated core-1 glycans (Fig. ) . However, a multivalent polymer of 107 sialylated core 1 glycans bound only weakly to monocytes and this interaction could 108 not be inhibited with anti-Siglec-9 (Supplementary Fig. ). This finding suggests a 109 contribution of the protein backbone to the binding specificity of Siglec-9, possibly by 110 defining a specific spacing of the sialic acids. Thus MUC1-ST specifically binds to 111 Siglec-9 on primary monocytes and macrophages. 112 113 MUC1-ST binding induces the secretion of various factors 114 To determine whether MUC1-ST binding induced a cellular response, we assayed for 115 soluble factors released from cultured primary monocytes upon binding recombinant 116 MUC1-ST. MUC1-ST induced monocytes to secrete several factors associated with 117 inflammation and tumor progression (Fig. and Supplementary Fig. ). We
Immunotherapy of cancer using chimeric antigen receptor (CAR) T-cells is a rapidly expanding fiel... more Immunotherapy of cancer using chimeric antigen receptor (CAR) T-cells is a rapidly expanding field. CARs are fusion molecules that couple the binding of a tumour-associated cell surface target to the delivery of a tailored T-cell activating signal. Re-infusion of such genetically engineered T-cells to patients with haematological disease has demonstrated unprecedented response rates in Phase I clinical trials. However, such successes have not yet been observed using CAR T-cells against solid malignancies and this is, in part, due to a lack of safe tumour-specific targets. The αvβ6 integrin is strongly up-regulated in multiple solid tumours including those derived from colon, lung, breast, cervix, ovaries/fallopian tube, pancreas and head and neck. It is associated with poorer prognosis in several cancers and exerts pro-tumorigenic activities including promotion of tumour growth, migration and invasion. By contrast, physiologic expression of αvβ6 is largely restricted to wound healin...
Chimeric antigen receptor (CAR) based immunotherapy has been under development for the last 25 ye... more Chimeric antigen receptor (CAR) based immunotherapy has been under development for the last 25 years and is now a promising new treatment modality in the field of cancer immunotherapy. The approach involves genetically engineering T cells to target malignant cells through expression of a bespoke fusion receptor that couples an HLA-independent antigen recognition domain to one or more intracellular T-cell activating modules. Multiple clinical trials are now underway in several centers to investigate CAR T-cell immunotherapy of diverse hematologic and solid tumor types. The most successful results have been achieved in the treatment of patients with B-cell malignancies, in whom several complete and durable responses have been achieved. This review focuses on the preclinical and clinical development of CAR T-cell immunotherapy of solid cancers, targeted against members of the ErbB family.
Chimeric antigen receptor- (CAR-) based immunotherapy has been under development for almost 25 ye... more Chimeric antigen receptor- (CAR-) based immunotherapy has been under development for almost 25 years, over which period it has progressed from a new but cumbersome technology to an emerging therapeutic modality for malignant disease. The approach involves the genetic engineering of fusion receptors (CARs) that couple the HLA-independent binding of cell surface target molecules to the delivery of a tailored activating signal to host immune cells. Engineered CARs are delivered most commonly to peripheral blood T cells using a range of vector systems, most commonly integrating viral vectors. Preclinical refinement of this approach has proceeded over several years to the point that clinical testing is now being undertaken at several centres, using increasingly sophisticated and therapeutically successful genetic payloads. This paper considers several aspects of the pre-clinical and clinical development of CAR-based immunotherapy and how this technology is acquiring an increasing niche i...
Adoptive immunotherapy using gd T cells harnesses their natural role in tumor immunosurveillance.... more Adoptive immunotherapy using gd 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 Vg9Vd2 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 gd 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 gd T cell-mediated shrinkage. Although folate targeting of liposomes enhanced the sensitization of folate receptor-a + 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 gd T cells are targeted using liposomal alendronic acid.
Sensitive and specific detection of nodal status, sites of metastases and low-volume recurrent di... more Sensitive and specific detection of nodal status, sites of metastases and low-volume recurrent disease could greatly improve management of patients with advanced prostate cancer. Prostate-specific membrane antigen (PSMA) is a well-established marker for prostate carcinoma with increased levels of expression in high-grade, hormone-refractory and metastatic disease. The monoclonal antibody (mAb) J591 is directed against an extracellular epitope of PSMA and has been shown to efficiently target disseminated disease including metastases in lymph nodes and bone. Its use as a diagnostic imaging agent however is limited due to its slow pharmacokinetics. In this study a diabody derived from mAb J591 was developed as a single photon emission computed tomography (SPECT) tracer with improved pharmacokinetics for the detection of PSMA expression in prostate cancer. A diabody in VH-VL orientation and with a C-terminal cysteine was expressed in HEK293T cells and purified by a combination of metal ...
MUC1 is a highly attractive immunotherapeutic target owing to increased expression, altered glyco... more MUC1 is a highly attractive immunotherapeutic target owing to increased expression, altered glycosylation, and loss of polarity in >80% of human cancers. To exploit this, we have constructed a panel of chimeric Ag receptors (CAR) that bind selectively to tumor-associated MUC1. Two parameters proved crucial in optimizing the CAR ectodomain. First, we observed that the binding of CAR-grafted T cells to anchored MUC1 is subject to steric hindrance, independent of glycosylation status. This was overcome by insertion of the flexible and elongated hinge found in immunoglobulins of the IgD isotype. Second, CAR function was highly dependent upon strong binding capacity across a broad range of tumor-associated MUC1 glycoforms. This was realized by using an Ab-derived single-chain variable fragment (scFv) cloned from the HMFG2 hybridoma. To optimize CAR signaling, tripartite endodomains were constructed. Ultimately, this iterative design process yielded a potent receptor termed HOX that contains a fused CD28/OX40/CD3 endodomain. HOX-expressing T cells proliferate vigorously upon repeated encounter with soluble or membrane-associated MUC1, mediate production of proinflammatory cytokines (IFN-␥ and IL-17), and elicit brisk killing of MUC1 ؉ tumor cells. To test function in vivo, a tumor xenograft model was derived using MDA-MB-435 cells engineered to coexpress MUC1 and luciferase. Mice bearing an established tumor were treated i.p. with a single dose of engineered T cells. Compared with control mice, this treatment resulted in a significant delay in tumor growth as measured by serial bioluminescence imaging. Together, these data demonstrate for the first time that the near-ubiquitous MUC1 tumor Ag can be targeted using CAR-grafted T cells.
Background Epidemiological studies have shown that only about 20% of the familial clustering of b... more Background Epidemiological studies have shown that only about 20% of the familial clustering of breast cancer is explained by the known highly penetrant mutations in BRCA1 and BCRA2. We have set out to search for the genes for the remaining 80%. Twin studies indicate a predominant role of shared genes rather than a shared environment; the patterns of occurrence of breast cancer in families are consistent with a major polygenic component. Methods We have assembled a population based set of 5,000 breast cancer cases and 5,000 controls from the East Anglian population. We have simple clinical and epidemiological information, including family history, and samples of blood and paraffin embedded tumour. We have used association studies based on single nucleotide polymorphisms, first with candidate genes and then in a genome-wide scan of 266,000 single nucleotide polymorphisms, to search for the putative predisposing genes. We have as yet searched only for common variants (frequency >5%). We have modelled the effects of polygenic predisposition in the East Anglian population, and have shown that the model predicts a wide distribution of individual risk in the population, such that half of all breast cancers may occur in the 12% of women at greatest risk. Both the candidate gene-based and genome-wide scans have provided provisional identification of a number of novel susceptibility genes, and these are currently being confirmed by a world-wide consortium of independent laboratories totalling 20,000-plus cases and controls. No single gene so far identified contributes more than 2% of the total inherited component, consistent with a model in which susceptibility is the result of a large number of individually small genetic effects.
Chinese hamster ovary (CHO) cells are widely used for the production of recombinant proteins for ... more 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.
www.jbmethods.org 1 PROTOCOLJournal of Biological Methods | 2014 | Vol. 1(2) | e7 DOI: 10.14440/j... more www.jbmethods.org 1 PROTOCOLJournal of Biological Methods | 2014 | Vol. 1(2) | e7 DOI: 10.14440/jbm.2014.30 POL Scientific Use of retroviral-mediated gene transfer to deliver and test function of chimeric antigen receptors in human
Prostate cancer is the most common cancer in men, both in the USA and Europe. Although incurable,... more Prostate cancer is the most common cancer in men, both in the USA and Europe. Although incurable, metastatic disease can often be controlled for years with anti-androgen therapy. Once the disease becomes castrate resistant, the median survival is 18 months. There is growing evidence that the immune system, and in particular cytokines, play an important role in prostate cancer immunosurveillance and progression. Here, we have undertaken a clinical investigation of the role of two closely related cytokines, IL-4 and IL-13 in prostate cancer. In the largest series studied to date, we show that serum IL-4, but not IL-13 is significantly elevated in castrate resistant, compared to androgen sensitive disease. Notably however, serum IL-4 levels are also raised in patients with benign prostatic OPEN ACCESS Cancers 2011, 3 4282 disease. Analysis of benign and malignant prostate tissue demonstrates that the source of IL-4 is epithelial cells rather than infiltrating leukocytes. Together, our data are consistent with a dual role for IL-4 in prostate cancer development. In benign disease, our data add to the evidence that IL-4 serves a protective role. By contrast, the data support a direct role for IL-4 in the progression of prostate cancer from androgen responsive, to advanced castrate-resistant disease.
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