Efficacy and toxicity management of CAR-T-cell immunotherapy: a matter of responsiveness control or tumour-specificity?

Chimaeric antigen receptor (CAR)-expressing T-cells have demonstrated potent clinical efficacy in patients with haematological malignancies. However, the use of CAR-T-cells targeting solid tumour-associated antigens (TAAs) has been limited by organ toxicities related to activation of T-cell effector functions through the CAR. Most existing CARs recognize TAAs, which are also found in normal tissues. CAR-T-cell-mediated destruction of normal tissues constitutes a major roadblock to CAR-T-cell therapy, and must be avoided or mitigated. There is a broad range of strategies for modulating antigen responsiveness of CAR-T-cells, with varying degrees of complexity. Some of them might ameliorate the acute and chronic toxicities associated with current CAR constructs. However, further embellishments to CAR therapy may complicate clinical implementation and possibly create new immunogenicity issues. In contrast, the development of CARs targeting truly tumour-specific antigens might circumvent on-target/off-tumour toxicities without adding additional complexity to CAR-T-cell therapies, but these antigens have been elusive and may require novel selection strategies for their discovery.

Proteasome activator complex PA28 identified as an accessible target in prostate cancer by in vivo selection of human antibodies.

Antibody cancer therapies rely on systemically accessible targets and suitable antibodies that exert a functional activity or deliver a payload to the tumor site. Here, we present proof-of-principle of in vivo selection of human antibodies in tumor-bearing mice that identified a tumor-specific antibody able to deliver a payload and unveils the target antigen. By using an ex vivo enrichment process against freshly disaggregated tumors to purge the repertoire, in combination with in vivo biopanning at optimized phage circulation time, we have identified a human domain antibody capable of mediating selective localization of phage to human prostate cancer xenografts. Affinity chromatography followed by mass spectrometry analysis showed that the antibody recognizes the proteasome activator complex PA28. The specificity of soluble antibody was confirmed by demonstrating its binding to the active human PA28αß complex. Whereas systemically administered control phage was confined in the lumen of blood vessels of both normal tissues and tumors, the selected phage spread from tumor vessels into the perivascular tumor parenchyma. In these areas, the selected phage partially colocalized with PA28 complex. Furthermore, we found that the expression of the α subunit of PA28 [proteasome activator complex subunit 1 (PSME1)] is elevated in primary and metastatic human prostate cancer and used anti-PSME1 antibodies to show that PSME1 is an accessible marker in mouse xenograft tumors. These results support the use of PA28 as a tumor marker and a potential target for therapeutic intervention in prostate cancer.

The profile of tumor antigens which can be targeted by immunotherapy depends upon the tumor's anatomical site.

Previously, we showed that vesicular stomatitis virus (VSV) engineered to express a cDNA library from human melanoma cells (ASMEL, Altered Self Melanoma Epitope Library) was an effective systemic therapy to treat subcutaneous (s.c.) murine B16 melanomas. Here, we show that intravenous treatment with the same ASMEL VSV-cDNA library was an effective treatment for established intra-cranial (i.c.) melanoma brain tumors. The optimal combination of antigens identified from the ASMEL which treated s.c. B16 tumors (VSV-N-RAS+VSV-CYTC-C+VSV-TYRP-1) was ineffective against i.c. B16 brain tumors. In contrast, combination of VSV-expressed antigens-VSV-HIF-2α+VSV-SOX-10+VSV-C-MYC+VSV-TYRP1-from ASMEL which was highly effective against i.c. B16 brain tumors, had no efficacy against the same tumors growing subcutaneously. Correspondingly, i.c. B16 tumors expressed a HIF-2α(Hi), SOX-10(Hi), c-myc(Hi), TYRP1, N-RAS(lo)Cytc(lo) antigen profile, which differed significantly from the HIF-2α(lo), SOX-10(lo), c-myc(lo), TYRP1, N-RAS(Hi)Cytc(Hi) phenotype of s.c. B16 tumors, and was imposed upon the tumor cells by CD11b(+) cells within the local brain tumor microenvironment. Combining T-cell costimulation with systemic VSV-cDNA treatment, long-term cures of mice with established i.c. tumors were achieved in about 75% of mice. Our data show that the anatomical location of a tumor profoundly affects the profile of antigens that it expresses.

Engineered human tumor xenografts with functional human vascular networks.

Animal models of human tumor angiogenesis would have multiple applications. However, they are extremely difficult to standardize. In this work, we tried to generate human tumor xenografts containing a human vascular bed in immunodeficient mice by subcutaneous co-implantation of matrigel-embedded human endothelial cells (EC), human mesenchymal stem cells (MSC) as a source of mural cells and HT1080 human fibrosarcoma cells. Unfortunately, in this context human EC were rapidly substituted by their murine counterparts, and by day 16 post-implantation human CD34 positive cells were hardly detectable in intratumoral vessels. In an attempt to inhibit host EC colonization of human xenografts and promote human EC grafting, we investigated the effect of radiation prior to implantation on the vascularization and growth of tumor xenografts. Nude mice underwent either localized (implantation area) or sublethal whole-body exposure to radiation. Localized radiation inhibited both human and murine neovascularization, and even the tumor growth rate was remarkably decreased when compared to control unirradiated mice and sublethally whole-body irradiated mice. Interestingly, numerous human vessels were detectable in sublethally irradiated mice at day 30, with murine EC only over passing human EC when spontaneous hematopoietic reconstitution has taken place. This observation strongly suggests the implication of bone marrow-derived murine endothelial precursors in tumor neovascularization. In summary, we have established a model of human tumor neovascularization that is amenable to both the study of molecular aspects in the angiogenic process and the evaluation of potential new antiangiogenic drugs.

Tumor immunotherapy using gene-modified human mesenchymal stem cells loaded into synthetic extracellular matrix scaffolds.

Mesenchymal stem cells (MSCs) are appealing as gene therapy cell vehicles given their ease of expansion and transduction. However, MSCs exhibit immunomodulatory and proangiogenic properties that may pose a risk in their use in anticancer therapy. For this reason, we looked for a strategy to confine MSCs to a determined location, compatible with a clinical application. Human MSCs genetically modified to express luciferase (MSC(luc)), seeded in a synthetic extracellular matrix (sECM) scaffold (sentinel scaffold) and injected subcutaneously in immunodeficient mice, persisted for more than 40 days, as assessed by bioluminescence imaging in vivo. MSCs modified to express a bispecific alpha-carcinoembryonic antigen (alphaCEA)/alphaCD3 diabody (MSC(dAb)) and seeded in an sECM scaffold (therapeutic scaffolds) supported the release of functional diabody into the bloodstream at detectable levels for at least 6 weeks after implantation. Furthermore, when therapeutic scaffolds were implanted into CEA-positive human colon cancer xenograft-bearing mice and human T lymphocytes were subsequently transferred, circulating alphaCEA/alphaCD3 diabody activated T cells and promoted tumor cell lysis. Reduction of tumor growth in MSC(dAb)-treated mice was statistically significant compared with animals that only received MSC(luc). In summary, we report here for the first time that human MSCs genetically engineered to secrete a bispecific diabody, seeded in an sECM scaffold and implanted in a location distant from the primary tumor, induce an effective antitumor response and tumor regression.

Immunogenicity of self tumor associated proteins is enhanced through protein truncation.

We showed previously that therapy with Vesicular Stomatitis Virus (VSV) expressing tumor-associated proteins eradicates established tumors. We show here that when cellular cDNA were cloned into VSV which retained their own poly-A signal, viral species emerged in culture which had deleted the cellular poly-A signal and also contained a truncated form of the protein coding sequence. Typically, the truncation occurred such that a Tyrosine-encoding codon was converted into a STOP codon. We believe that the truncation of tumor-associated proteins expressed from VSV in this way occurred to preserve the ability of the virus to replicate efficiently. Truncated cDNA expressed from VSV were significantly more effective than full length cDNA in treating established tumors. Moreover, tumor therapy with truncated cDNA was completely abolished by depletion of CD4+ T cells, whereas therapy with full length cDNA was CD8+ T cell dependent. These data show that the type/potency of antitumor immune responses against self-tumor-associated proteins can be manipulated in vivo through the nature of the self protein (full length or truncated). Therefore, in addition to generation of neoantigens through sequence mutation, immunological tolerance against self-tumor-associated proteins can be broken through manipulation of protein integrity, allowing for rational design of better self-immunogens for cancer immunotherapy.

Anti-metastatic action of FAK inhibitor OXA-11 in combination with VEGFR-2 signaling blockade in pancreatic neuroendocrine tumors.

The present study sought to determine the anti-tumor effects of OXA-11, a potent, novel small-molecule amino pyrimidine inhibitor (1.2 pM biochemical IC(50)) of focal adhesion kinase (FAK). In studies of cancer cell lines, OXA-11 inhibited FAK phosphorylation at phospho-tyrosine 397 with a mechanistic IC(50) of 1 nM in TOV21G tumor cells, which translated into functional suppression of proliferation in 3-dimensional culture with an EC(50) of 9 nM. Studies of OXA-11 activity in TOV21G tumor-cell xenografts in mice revealed a pharmacodynamic EC(50) of 1.8 nM, indicative of mechanistic inhibition of pFAK [Y397] in these tumors. OXA-11 inhibited TOV21G tumor growth in a dose-dependent manner and also potentiated effects of cisplatin on tumor cell proliferation and apoptosis in vitro and on tumor growth in mice. Studies of pancreatic neuroendocrine tumors in RIP-Tag2 transgenic mice revealed OXA-11 suppression of pFAK [Y397] and pFAK [Y861] in tumors and liver. OXA-11 given daily from age 14 to 17 weeks reduced tumor vascularity, invasion, and when given together with the anti-VEGFR-2 antibody DC101 reduced the incidence, abundance, and size of liver metastases. Liver micrometastases were found in 100 % of mice treated with vehicle, 84 % of mice treated with OXA-11, and 79 % of mice treated with DC101 (19-24 mice per group). In contrast, liver micrometastases were found in only 52 % of 21 mice treated with OXA-11 plus DC101, and those present were significantly smaller and less numerous. Together, these findings indicate that OXA-11 is a potent and selective inhibitor of FAK phosphorylation in vitro and in vivo. OXA-11 slows tumor growth, potentiates the anti-tumor actions of cisplatin and--when combined with VEGFR-2 blockade--reduces metastasis of pancreatic neuroendocrine tumors in RIP-Tag2 mice.

CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors.

A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CAR(v2)) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CAR(v2) fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells.Molecular Therapy-Nucleic Acids (2013) 2, e93; doi:10.1038/mtna.2013.19; published online 21 May 2013.

Basement membrane-rich organoids with functional human blood vessels are permissive niches for human breast cancer metastasis.

Metastatic breast cancer is the leading cause of death by malignancy in women worldwide. Tumor metastasis is a multistep process encompassing local invasion of cancer cells at primary tumor site, intravasation into the blood vessel, survival in systemic circulation, and extravasation across the endothelium to metastasize at a secondary site. However, only a small percentage of circulating cancer cells initiate metastatic colonies. This fact, together with the inaccessibility and structural complexity of target tissues has hampered the study of the later steps in cancer metastasis. In addition, most data are derived from in vivo models where critical steps such as intravasation/extravasation of human cancer cells are mediated by murine endothelial cells. Here, we developed a new mouse model to study the molecular and cellular mechanisms underlying late steps of the metastatic cascade. We have shown that a network of functional human blood vessels can be formed by co-implantation of human endothelial cells and mesenchymal cells, embedded within a reconstituted basement membrane-like matrix and inoculated subcutaneously into immunodeficient mice. The ability of circulating cancer cells to colonize these human vascularized organoids was next assessed in an orthotopic model of human breast cancer by bioluminescent imaging, molecular techniques and immunohistological analysis. We demonstrate that disseminated human breast cancer cells efficiently colonize organoids containing a functional microvessel network composed of human endothelial cells, connected to the mouse circulatory system. Human breast cancer cells could be clearly detected at different stages of the metastatic process: initial arrest in the human microvasculature, extravasation, and growth into avascular micrometastases. This new mouse model may help us to map the extravasation process with unprecedented detail, opening the way for the identification of relevant targets for therapeutic intervention.

The heterotrimeric laminin coiled-coil domain exerts anti-adhesive effects and induces a pro-invasive phenotype.

Laminins are large heterotrimeric cross-shaped extracellular matrix glycoproteins with terminal globular domains and a coiled-coil region through which the three chains are assembled and covalently linked. Laminins are key components of basement membranes, and they serve as attachment sites for cell adhesion, migration and proliferation. In this work, we produced a recombinant fragment comprising the entire laminin coiled-coil of the α1-, ß1-, and γ1-chains that assemble into a stable heterotrimeric coiled-coil structure independently of the rest of the molecule. This domain was biologically active and not only failed to serve as a substrate for cell attachment, spreading and focal adhesion formation but also inhibited cell adhesion to laminin when added to cells in a soluble form at the time of seeding. Furthermore, gene array expression profiling in cells cultured in the presence of the laminin coiled-coil domain revealed up-regulation of genes involved in cell motility and invasion. These findings were confirmed by real-time quantitative PCR and zymography assays. In conclusion, this study shows for the first time that the laminin coiled-coil domain displays anti-adhesive functions and has potential implications for cell migration during matrix remodeling.

Lymphocyte display: a novel antibody selection platform based on T cell activation.

Since their onset, display technologies have proven useful for the selection of antibodies against a variety of targets; however, most of the antibodies selected with the currently available platforms need to be further modified for their use in humans, and are restricted to accessible antigens. Furthermore, these platforms are not well suited for in vivo selections. We present here a novel cell based antibody display platform, which takes advantage of the functional capabilities of T lymphocytes. The display of antibodies on the surface of T lymphocytes, as a part of a chimeric-immune receptor (CIR) mediating signaling, may ideally link the antigen-antibody interaction to a demonstrable change in T cell phenotype, due to subsequent expression of the early T cell activation marker CD69. In this proof-of-concept, an in vitro selection was carried out using a human T cell line lentiviral-transduced to express a tumor-specific CIR on the surface, against a human tumor cell line expressing the carcinoembryonic antigen. Based on an effective interaction between the CIR and the tumor antigen, we demonstrated that combining CIR-mediated activation with FACS sorting of CD69(+) T cells, it is possible to isolate binders to tumor specific cell surface antigen, with an enrichment factor of at least 10(3)-fold after two rounds, resulting in a homogeneous population of T cells expressing tumor-specific CIRs.

Long-term in vivo imaging of human angiogenesis: critical role of bone marrow-derived mesenchymal stem cells for the generation of durable blood vessels.

Angiogenesis is a multistep process that encompasses complex molecular and cellular interactions that can not be recapitulated in vitro. Here, we demonstrate that vasculature generated from lentivirally transduced human primary endothelial cells expressing firefly luciferase and co-implanted with human bone marrow mesenchymal stem cells in immunodeficient mice can be assessed quantitatively by in vivo whole body bioluminescence imaging for more than 120 days. Luciferase activity correlated with the formation of a network of functional, mature blood vessels of human nature inside the implant that critically depend on the presence of mesenchymal stem cells. In summary, our study offers an unprecedented opportunity to perform long-term serial analysis of the molecular events involved in the angiogenic process and monitoring responses to anti-angiogenic agents.