The correlation between immune subtypes and consensus molecular subtypes in colorectal cancer identifies novel tumour microenvironment profiles, with prognostic and therapeutic implications.

BACKGROUND: Solid tumour growth is the consequence of a complex interplay between cancer cells and their microenvironment. Recently, a new global transcriptomic immune classification of solid tumours has identified six immune subtypes (ISs) (C1-C6). Our aim was to specifically characterise ISs in colorectal cancer (CRC) and assess their interplay with the consensus molecular subtypes (CMSs). METHODS: Clinical and molecular information, including CMSs and ISs, were obtained from The Cancer Genome Atlas (TCGA) (N = 625). Immune cell populations, differential gene expression and gene set enrichment analysis were performed to characterise ISs in the global CRC population by using CMSs. RESULTS: Only 5 ISs were identified in CRC, predominantly C1 wound healing (77%) and C2 IFN-γ dominant (17%). CMS1 showed the highest proportion of C2 (53%), whereas C1 was particularly dominant in CMS2 (91%). CMS3 had the highest representation of C3 inflammatory (7%) and C4 lymphocyte depleted ISs (4%), whereas all C6 TGF-ß dominant cases belonged to CMS4 (2.3%). Prognostic relevance of ISs in CRC substantially differed from that reported for the global TCGA, and ISs had a greater ability to stratify the prognosis of CRC patients than CMS classification. C2 had higher densities of CD8, CD4 activated, follicular helper T cells, regulatory T cells and neutrophils and the highest M1/M2 polarisation. C2 had a heightened activation of pathways related to the immune system, apoptosis and DNA repair, mTOR signalling and oxidative phosphorylation, whereas C1 was more dependent of metabolic pathways. CONCLUSIONS: The correlation of IS and CMS allows a more precise categorisation of patients with relevant clinical and biological implications, which may be valuable tools to improve tailored therapeutic interventions in CRC patients.

A novel Carcinoembryonic Antigen (CEA)-Targeted Trimeric Immunotoxin shows significantly enhanced Antitumor Activity in Human Colorectal Cancer Xenografts.

Immunotoxins are chimeric molecules, which combine antibody specificity to recognize and bind with high-affinity tumor-associated antigens (TAA) with the potency of the enzymatic activity of a toxin, in order to induce the death of target cells. Current immunotoxins present some limitations for cancer therapy, driving the need to develop new prototypes with optimized properties. Herein we describe the production, purification and characterization of two new immunotoxins based on the gene fusion of the anti-carcinoembryonic antigen (CEA) single-chain variable fragment (scFv) antibody MFE23 to α-sarcin, a potent fungal ribotoxin. One construct corresponds to a conventional monomeric single-chain immunotoxin design (IMTXCEAαS), while the other one takes advantage of the trimerbody technology and exhibits a novel trimeric format (IMTXTRICEAαS) with enhanced properties compared with their monomeric counterparts, including size, functional affinity and biodistribution, which endow them with an improved tumor targeting capacity. Our results show the highly specific cytotoxic activity of both immunotoxins in vitro, which was enhanced in the trimeric format compared to the monomeric version. Moreover, the trimeric immunotoxin also exhibited superior antitumor activity in vivo in mice bearing human colorectal cancer xenografts. Therefore, trimeric immunotoxins represent a further step in the development of next-generation therapeutic immunotoxins.

Balanced secretion of anti-CEA × anti-CD3 diabody chains using the 2A self-cleaving peptide maximizes diabody assembly and tumor-specific cytotoxicity.

Adoptive transfer of genetically engineered human cells secreting bispecific T-cell engagers has shown encouraging therapeutic effects in preclinical models of cancer. However, reducing the toxicity and improving the effectiveness of this emerging immunotherapeutic strategy will be critical to its successful application. We have demonstrated that for gene-based bispecific antibody strategies, two-chain diabodies have a better safety profile than single-chain tandem scFvs (single-chain variable fragments), because their reduced tendency to form aggregates reduces the risk of inducing antigen-independent T-cell activation. Here, we demonstrate that the incorporation of a 2A self-processing peptide derived from foot-and-mouth disease virus conveying co-translational cleavage into a two-chain anti-CD3 × anti-CEA diabody gene enables near-equimolar expression of diabody chains 1 and 2, and thus increases the final amount of assembled diabody. This was found to maximize diabody-mediated T-cell activation and cytotoxicity against carcinoembryonic antigen-positive tumor cells.

Non-hematopoietic stem cells as factories for in vivo therapeutic protein production.

As an alternative to recombinant protein administration, ex vivo gene-modified cells may provide a novel strategy for systemic delivery of therapeutic proteins. This approach has been used in preclinical and clinical studies of a plethora of pathological conditions, including anemia, hemophilia and cancer for the production of erythropoietin, coagulation factors, immunostimulatory cytokines, recombinant antibodies and angiogenesis inhibitors. Cell delivery vehicles may also be varied: autologous or allogeneic, precursor or terminally differentiated cells, with targeting properties or immobilized in immunoprotective devices. This field did not meet the expectation raised initially, mainly because of difficulties with obtaining therapeutic plasma levels and the short lifespan of producer cells that hampered clinical application. Different non-hematopoietic stem/progenitor cells have emerged as potential delivery vehicles, since they are easy to obtain, expand and transduce, and they exhibit prolonged lifespans (with mesenchymal stem cells probably being the most popular cell type, but not the only one). Special emphasis is placed on the different routes used to deliver these cellular vehicles and the controversies about their targeting abilities.

The axonal repellent Slit2 inhibits pericyte migration: potential implications in angiogenesis.

The Slit family of secreted proteins acts through the Roundabout (Robo) receptors to repel axonal migration during central nervous system development. Emerging evidence shows that Slit/Robo interactions also play a role in angiogenesis. The effect of Robo signaling on endothelial cells has been shown to be context-dependent. However, the role of Slit/Robo in pericytes has been largely unexplored. The aim of this study was to determine the effect of Slit2 on primary human pericytes and to address the underlying mechanisms, including the receptors potentially implicated. We demonstrate that both Robo1 and Robo4 are expressed by human pericytes. In the presence of their ligand Slit2, spontaneous and PDGF-induced migration of pericytes was impaired. This antimigratory activity of Slit-2 correlated with the inhibition of actin-based protrusive structures. Interestingly, human pericyte interaction with immobilized Slit2 was inhibited in the presence of anti-Robo1 and anti-Robo4 blocking antibodies, suggesting the implication of both receptors. These results add new insights into the role of Slit proteins during the angiogenic process that relies on the directional migration not only of endothelial cells but also of pericytes.

Factory neovessels: engineered human blood vessels secreting therapeutic proteins as a new drug delivery system.

Several works have shown the feasibility of engineering functional blood vessels in vivo using human endothelial cells (ECs). Going further, we explored the therapeutic potential of neovessels after gene-modifying the ECs for the secretion of a therapeutic protein. Given that these vessels are connected with the host vascular bed, we hypothesized that systemic release of the expressed protein is immediate. As a proof of principle, we used primary human ECs transduced with a lentiviral vector for the expression of a recombinant bispecific alphaCEA/alphaCD3 antibody. These ECs, along with mesenchymal stem cells as a source of mural cells, were embedded in Matrigel and subcutaneously implanted in nude mice. High antibody levels were detected in plasma for 1 month. Furthermore, the antibody exerted a therapeutic effect in mice bearing distant carcinoembryonic-antigen (CEA)-positive tumors after inoculation of human T cells. In summary, we show for the first time the therapeutic effect of a protein locally secreted by engineered human neovessels.

Inhibition of tumor growth in vivo by in situ secretion of bispecific anti-CEA x anti-CD3 diabodies from lentivirally transduced human lymphocytes.

Infiltrating T lymphocytes are found in many malignancies, but they appear to be mostly anergic and do not attack the tumor, presumably because of defective T-cell activation events. Recently, we described a strategy for the tumor-specific polyclonal activation of tumor-resident T lymphocytes based on the in situ production of recombinant bispecific antibodies (bsAbs) by transfected nonhematological cell lines. Here, we have constructed a novel HIV-1-based lentiviral vector for efficient gene transduction into various human hematopoietic cell types. Several myelomonocytic and lymphocytic cell lines secreted the anti-carcinoembryonic antigen (CEA) x anti-CD3 diabody in a functionally active form with CD3(+) T-cell lines being the most efficient secretors. Furthermore, primary human peripheral blood lymphocytes (PBLs) were also efficiently transduced and secreted high levels of functional diabody. Importantly gene-modified PBLs significantly reduced in vivo tumor growth rates in xenograft studies. These results demonstrate, for the first time, the utility of lentiviral vectors for sustained expression of recombinant bsAbs in human T lymphocytes. Such T lymphocytes, transduced ex vivo to secrete the activating diabody in autocrine fashion, may provide a promising route for a gene therapy strategy for solid human tumors.

Single-chain antibody-based gene therapy: inhibition of tumor growth by in situ production of phage-derived human antibody fragments blocking functionally active sites of cell-associated matrices.

Experimental evidence suggests that blocking the interactions between endothelial cells and extracellular matrix (ECM) components may provide a potent and general strategy to inhibit tumor neovascularization. Based on these considerations, we have focused our efforts on laminin, component of the vascular basement membrane of every tumor-associated vessel, which serves an essential role in tube formation. We screened anti-laminin single-chain antibody fragments (scFv) derived from a human phage-display library and identified one that blocks the formation of capillary-like structures in vitro. This scFv inhibits angiogenesis in vivo in the chick embryo chorioallantoic membrane assay and prevents the establishment and growth of subcutaneous tumors in mice, either when administered as bolus protein therapy or when produced locally by gene-modified tumor cells. Our work represents the first demonstration of a direct in vivo therapeutic effect of a single-chain antibody secreted by gene-modified mammalian cells. These results open the way for a new antibody-based gene therapy strategy of cancer.

Genetic approaches for antigen-selective cell therapy.

Attempts to improve the efficacy of adoptive T-cell therapies have led to the development of innovative strategies that combine the high specificity of antibody molecules with the efficient trafficking properties and effector functions of immune cells. These antigen-selective cell therapies are designed to convert therapeutically important native antigens expressed on the cell surface (tumor associated antigens, viral envelope proteins) into recruitment points of effector functions, and address the goal of major histocompatibility complex- and exogenous cytokine-independent activation of mature effector T-cells. The most promising and best characterized antigen-selective strategy is based on the genetic manipulation of the recognition specificity of T-cells by grafting the recognition specificity of a monoclonal antibody onto a lymphocyte triggering molecule (TCR-associated polypeptides, Fc epsilon RI-gamma chain). Upon encountering specific antigen, cells harboring chimeric immune receptors (CIRs) are able to undergo specific stimulation and kill antigen bearing cells in both in vitro and in vivo model systems. Initial studies have focused on terminally differentiated effector cell-based protocols. However, recent data indicate that progenitor cell-based therapies allow the permanent generation of stable populations of CIR-expressing cells of multiple lineages, leading to long-term persistent systemic immunity. Emerging gene therapy strategies are based on the use of biespecic antibody fragments. The advantages of these biespecic antibody-mediated immune recruitment (BIR) approaches (trans-recruitment and multieffector activation) could complement conventional CIR-based immunotherapies. Although further scientific progress is required regarding the selection of the ideal effector cell/s and the definition of the optimal targeting and recruitment systems, clinical trials recently initiated in patients with advanced cancer and human immunodeficiency virus infection should help us to determine the real efficacy of these approaches. The relevance of these and other emerging concepts to cell-mediated immunotherapy is discussed.

Generation and characterization of recombinant human antibodies specific for native laminin epitopes: potential application in cancer therapy.

Laminins are specific cellular regulators that directly and indirectly control activities such as cell attachment and migration, differentiation and polarity, proliferation and apoptosis, and protease expression. Considering the centrality of these issues to tumor progression, the generation of human-derived antibody fragments able to modulate laminin-regulated biological functions would allow the development of new strategies to improve treatment of cancer patients. In this report, we explore the use of phage display technology to isolate human anti-laminin antibody fragments. A library of single chain antibodies was selected using intact mouse laminin, and five different clones were identified. All the antibodies were specific for their cognate antigen, as revealed by lack of cross-reactivity with other components of the basement membranes. A more extensive characterization of the panel indicated that these antibodies recognize the native protein through conformational epitopes. All of them reduced tumor cell attachment to laminin, suggesting that domains of the laminin molecule that are recognized by these antibodies likely bind to cell-surface receptors. The antibody fragments bind to mouse, rat and human laminin. and show strong immunohistochemical reactivity with basement membranes in human and murine tissue sections. Their properties make them ideal candidates for in vivo applications.

Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.

Efficient discrimination between different densities of target antigen by tetracycline-regulatable T bodies.

Engineered T cells expressing chimeric T cell receptors (chTCRs) are of interest for cancer gene therapy but many "cancer antigens" are thought to be unsuitable targets because they are expressed at low levels on normal tissues. We therefore sought to determine whether engineered T cells expressing variable surface densities of a high-affinity chTCR could discriminate different concentrations of the targeted antigen. We plotted the relationship between chTCR density and the concentration of target antigen using Jurkat T cells expressing a hapten-binding chTCR whose expression could be modulated by tetracycline. Our analysis reveals that there is a dynamic equilibrium between cell surface density of the chTCR and the antigen density that optimally triggers T cell activation. At a fixed density of target antigen, optimal T cell activation can be achieved only within a certain range of chTCR densities, while excessive TCR signaling triggers apoptosis of the engineered T cells. Our results show that T cells can be engineered to discriminate different antigen densities and that the T cell response to a fixed concentration of antigen can be optimized by tuning the cell surface density of TCRs.

Delay in resumption of the activity of tetracycline-regulatable promoter following removal of tetracycline analogues.

The tetracycline-regulatable system (TRS) has become a widely adopted tool for modification of gene expression and analysis of gene function in mammalian cells, plants and transgenic animals. We have studied the potential application of the TRS in gene therapy, using a single vector containing both the tetracycline-controlled transactivator (tTA) and the tTA-responsive promoter (tRP) transcribing mouse GM-CSF. Stable 293 cells established using this vector were used to study the kinetics of the TRS in response to various tetracycline analogues. Dose-response studies show that doxycycline is the most potent-analogue in abolishing tTA activity. Kinetic studies indicate that, at 1,000 ng/ml, all the analogues have similar efficiencies in down-regulating the system in given time. In contrast, following the removal of the analogues, there is a temporal, dose-dependent delay in resumption of the tRP activity. The time taken for resumption of near-optimal tRP activity is approximately 48 h for tetracycline, 144 h for anhydrotetracycline, 192 h for minocycline and 216 h for doxycycline when cells were pretreated with 1000 ng/ml of these antibiotics. This property of the analogues can be employed in planning a desired course of transgene regulation.

Pharmacological control of antigen responsiveness in genetically modified T lymphocytes.

A chimeric TCR gene, comprising an anti-hapten single-chain Ab variable fragment fused to the transmembrane and cytoplasmic regions of the human TCR zeta-chain, was used to determine whether the tetracycline-regulatable system could be used to regulate gene expression in T cells. Jurkat T cells were stably transfected with a single vector encoding the tetracycline trans-activator protein, controlled by a constitutive promoter, and the chimeric TCR, under the control of a trans-activator protein-responsive promoter. In the absence of tetracyclines, the transfected T cells were shown to express the chimeric receptor on the cell surface and could be activated by its cognate Ag, leading to the secretion of IL-2. When the cells were exposed to increasing concentrations of tetracyclines, surface expression of the chimeric receptor was suppressed in a dose-dependent manner, and this suppression was sufficient to result in complete loss of responsiveness to the targeted Ag. Prolonged suppression of trans-gene expression for up to 7 days was observed after doxycycline was removed from the cultures, but eventual recovery of surface expression was complete, and the absolute time to recovery was directly proportional to the initial concentration of the drug. Pharmacologic control of trans-gene expression in gene-modified T cells will not only facilitate new approaches to the study of different aspects of T cell biology, but will also provide the basis for new gene therapy strategies.

Antigen-specific targeting of CD28-mediated T cell co-stimulation using chimeric single-chain antibody variable fragment-CD28 receptors.

T cells require two distinct signals for optimal activation. One is an antigen-specific signal and is provided by engagement of the T cell receptor (TCR). The second is an antigen-independent signal mediated by engagement of the T cell surface molecule CD28 with members of the B7 family. To endow CD28 molecules with antibody-type recognition, we have constructed chimeric single-chain antibody variable fragment (scFv)-CD28 molecules; following transfection of the genes encoding such constructs into the Jurkat human T cell line we show that they are stably expressed as functional cell surface receptors. These chimeric molecules have no apparent negative effects on the expression and signaling ability of the wild-type CD28 and TCR/CD3 molecules. When combined with signaling via the TCR/CD3 complex, these antigen-specific scFv-CD28 chimeric molecules provide signals similar to those elicited by the cross-linking of the unmodified CD28 molecules. Furthermore, the generation of double transfectants simultaneously expressing scFv-CD28 and scFv-CD3 zeta chimeras demonstrates that antigen-specific co-stimulatory signals can also synergize with signals mediated through chimeric CD3 zeta chains to secrete maximal levels of interleukin-2. Overall, our results suggest that optimal, predefined antigen-specific activation of T cells directed by the specificity of the scFv should be possible.

Isolation of tumor-derived immunoglobulin-idiotype from peripheral blood mononuclear cells in a B-cell lymphoma patient with minimal disease.

Active immunization with autologous idiotypic immunoglobulin, obtained by somatic fusion techniques, has been shown to be a useful alternative treatment in patients with B-cell lymphoma. Nevertheless, the requirement for biopsy specimens to obtain lymphoma cells could be a limitation to this therapeutic strategy. We address the question of whether peripheral blood samples containing small amounts of tumor cells can be used as appropriate fusion partners to rescue tumor-derived idiotypic proteins. In this report, we show that hybrid cells can be obtained from somatic fusions of K6H6/B5 heterohybridoma with lymphoma cells obtained from both lymph node (LN) and peripheral blood mononuclear cells (PBMC) containing only minor amounts of tumor cells. Some hybrid cells obtained from LN or PBMC fusions present an immunoglobulin (Ig) heavy-chain gene rearrangement identical with that of the original tumor and secrete identical Ig protein containing the expected H and L chains.

Delimitation of the proliferative stages in the human thymus indicates that cell expansion occurs before the expression of CD3 (T cell receptor).

Precursor of T lymphocytes undergo proliferation and maturation under the influence of the thymic microenvironment. In our study, we have attempted to determine the distribution of human postnatal thymocytes in division according to their stage of differentiation. Our data show that about 11.5% of all thymic cells are in S/G2/M phases, and that a subset of the cortical and precortical subpopulations contains most of the dividing cells. Rate of cell division is maintained at high levels from the prethymocyte precursor along the successive stages of differentiation represented by CD1+CD3-CD4-CD8- and CD1+CD3-CD4+CD8- cells. The percentage of dividing cells is maximal in an intermediate subset of CD1+CD3-CD4+CD8-CD45RO+ cells defined by the distinct expression of class I HLAdim/high molecules, which could contain cells in transit from prethymocytes to double-positive cortical cells. The CD3- fraction of the double-positive cortical cells contains most of the dividing thymocytes, although the rate of division within this subset is much less than that of the precursor CD1+CD3-CD4+CD8- cells. In a linear scheme of differentiation, cell division stops at or near the point of initiation of CD3 expression. These results suggest that in human thymus cell expansion takes place before the initiation of the positive selection process. According to this view the stringency of the selection process would require the previous generation of a large number of precursors to permit the production of sufficient numbers of mature T cells.

Differential effect of tumour necrosis factor on human thymocyte subpopulations.

We have studied the effect of tumour necrosis factor (TNF) on purified human thymocyte subpopulations. For this purpose human thymocytes were purified by negative selection with three rounds of several antibodies plus complement. TNF was able to co-stimulate in a dose-response manner the proliferation of single positive (SP) CD3+ CD4+ or CD3+ CD8+ thymocytes in the presence of optimal doses of interleukin-2 (IL-2), phytohaemagglutinin (PHA), anti-CD3 antibodies or phorbol esters. However, CD1+ CD3low CD4+ CD8+ cortical thymocytes did not proliferate significantly in response to any stimulus alone or in combination. The TNF proliferative effect on SP thymocytes was blocked by an anti-IL-2R alpha antibody. In addition, TNF enhanced the expression of the IL-2R alpha but not IL-2R beta on the cell surface of CD1- CD3+ SP thymocytes over the levels induced by the other primary stimuli, inducing as a consequence, an increase in the number of high affinity IL-2R. Furthermore, TNF was able to increase IL-2R alpha mRNA levels on SP thymocytes. On the other hand, TNF was mitogenic in the absence of any other stimulus for CD1- CD3- CD4- CD8- prethymocytes, as was IL-2, and this proliferation was not blocked by anti-IL-2R alpha antibodies. Furthermore, the proliferation of this subset in response to IL-2 and TNF was additive. TNF was able to increase directly the cell surface expression of both chains, IL-2R beta and IL-2R alpha, and the IL-2R alpha messenger RNA (mRNA) levels of CD1- CD3- CD4- CD8- prethymocytes. In summary, our results suggest that TNF may have an important role as a co-stimulatory signal in some human thymocyte subpopulations by inducing the expression of IL-2R.