Proper lymph node (LN) development requires tumor necrosis factor-related activation-induced cytokine (TRANCE) expression. Here we demonstrate that the defective LN development in TRANCE(-/)- mice correlates with a significant reduction in lymphotoxin (LT)alphabeta(+)alpha(4)beta(7)(+)CD45(+)CD4(+)CD3(-) cells and their failure to form clusters in rudimentary mesenteric LNs. Transgenic TRANCE overexpression in TRANCE(-/)- mice results in selective restoration of this cell population into clusters, and results in full LN development. Transgenic TRANCE-mediated restoration of LN development requires LTalphabeta expression on CD45(+) CD4(+)CD3(-) cells, as LNs could not be induced in LTalpha(-/)- mice. LTalpha(-/)- mice also showed defects in the fate of CD45(+)CD4(+)CD3(-) cells similar to TRANCE(-/)- mice. Thus, we propose that both TRANCE and LTalphabeta regulate the colonization and cluster formation by CD45(+) CD4(+)CD3(-) cells in developing LNs, the degree of which appears to correlate with the state of LN organogenesis.
Lymph nodes (LNs) are important sentinal organs, populated by circulating lymphocytes and antigen-bearing cells exiting the tissue beds. Although cellular and humoral immune responses are induced in LNs by antigenic challenge, it is not known if LNs are essential for acquired immunity. We examined immune responses in mice that lack LNs due to genetic deletion of lymphotoxin ligands or in utero blockade of membrane lymphotoxin. We report that LNs are absolutely required for generating contact hypersensitivity, a T cell–dependent cellular immune response induced by epicutaneous hapten. We show that the homing of epidermal Langerhans cells in response to hapten application is specifically directed to LNs, providing a cellular basis for this unique LN function. In contrast, the spleen cannot mediate contact hypersensitivity because antigen-bearing epidermal Langerhans cells do not access splenic white pulp. Finally, we formally demonstrate that LNs provide a unique environment essential for generating this acquired immune response by reversing the LN defect in lymphotoxin-α−/− mice, thereby restoring the capacity for contact hypersensitivity.
A proliferation-inducing ligand (APRIL) is a ligand of the tumor necrosis factor (TNF) family that stimulates tumor cell growth in vitro and in vivo. Expression of APRIL is highly upregulated in many tumors including colon and prostate carcinomas. Here we identify B cell maturation antigen (BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI), two predicted members of the TNF receptor family, as receptors for APRIL. APRIL binds BCMA with higher affinity than TACI. A soluble form of BCMA, which inhibits the proliferative activity of APRIL in vitro, decreases tumor cell proliferation in nude mice. Growth of HT29 colon carcinoma cells is blocked when mice are treated once per week with the soluble receptor. These results suggest an important role for APRIL in tumorigenesis and point towards a novel anticancer strategy.
Electrophoretic variation of proteins encoded by 30 structural loci was examined among chromosomally variable populations assigned to three subspecies of Peromyscus boylii: P. b. levipes, P. b. ambiguus, and P. b. beatae. Electrophoretic evidence indicates that two genetically distinct taxa are represented within the chromosomally variable populations (FN = 48–54, 56–60) in eastern and southern México. Populations of P. b. beatae and P. b. levipes with low fundamental numbers (FN = 48–54) are distinct at one locus (Idh-1) from populations of P. b. levipes, P. b. ambiguus, and P. b. beatae with intermediate FNs (FN = 56–60). No evidence of gene flow was observed between these two taxa at a locality of sympatry.
Treatment of solid tumors with cell therapeutics will require optimal T cell persistence, fitness, and trafficking. Heterogeneous solid tumors will also have to be attacked through multiple antigens simultaneously in order to prevent resistance linked to loss of antigen expression. Here we use chimeric antigen receptor (CAR) T cells that secrete bridging proteins that act as CAR-T engagers to create an optimal platform for attacking solid tumors in the CNS.
Methods
Lentiviral vectors encoding an anti-CD19 CAR and secreted bridging proteins were created. The bridging proteins contained the CD19 extracellular domain, which is the target for the CAR, and anti-tumor antigen binding domains derived from antibodies (scFv and llama VH). The resulting anti-CD19 CAR T cells secrete the bridging proteins. These candidate cell therapeutics were evaluated for antigen binding and induction of antigen-specific cytotoxicity. An anti-CD19 CAR that secretes a CD19-anti-Her2 bridging protein has moved into development. Using the CD19-anti-Her2 bridging protein as a core module, we have begun evaluating a series of multi-antigen bridging proteins.
Results
CAR-CD19 T cells that secrete bridging proteins have potent cytotoxic activity against single- and multi-antigen-positive cells. ALETA-002 is the lead candidate lentiviral vector construct encoding the anti-CD19 CAR domain and the CD19-anti-Her2 bridging protein, and has entered a GMP viral particle development campaign. This therapeutic will be systemically administered to Her2-positive breast cancer patients who are relapsing with CNS metastases. Next, multi-antigen bridging proteins encoding an anti-Her2 scFv and anti-B7H3, anti-B7H6 or anti-IL13Ra2 llama VH were assayed for potency. Lead candidates for development for the treatment of primary CNS malignancies were identified and are being manufactured at pilot-scale in 4-plasmid lentivirus production runs.
Conclusions
The use of anti-CD19 CAR T cells that can expand off of the normal CD19-positive B cell pool enables tumor-antigen independent persistence, fitness and robust trafficking into the CNS. The use of small, modular bridging proteins allows us to leverage anti-CD19 CAR T cells and use these to attack solid tumor antigens that are present on CNS resident cancers and on CNS metastatic lesions. Novel cell therapeutics for the treatment of Her2-positive CNS metastases and heterogeneous primary CNS malignancies including glioblastoma and the pediatric gliomas have been developed.
The mumps virus (MuV) small hydrophobic protein (SH) is a type I membrane protein expressed in infected cells. SH has been reported to interfere with innate immunity by inhibiting tumor necrosis factor alpha (TNF-α)-mediated apoptosis and NF-κB activation. To elucidate the underlying mechanism, we generated recombinant MuVs (rMuVs) expressing the SH protein with an N-terminal FLAG epitope or lacking SH expression due to the insertion of three stop codons into the SH gene. Using these viruses, we were able to show that SH reduces the phosphorylation of IKKβ, IκBα, and p65 as well as the translocation of p65 into the nucleus of infected A549 cells. Reporter gene assays revealed that SH interferes not only with TNF-α-mediated NF-κB activation but also with IL-1β- and poly(I·C)-mediated NF-κB activation, and that this inhibition occurs upstream of the NF-κB pathway components TRAF2, TRAF6, and TAK1. Since SH coimmunoprecipitated with tumor necrosis factor receptor 1 (TNFR1), RIP1, and IRAK1, we hypothesize that SH exerts its inhibitory function by interacting with TNFR1, interleukin-1 receptor type 1 (IL-1R1), and TLR3 complexes in the plasma membrane of infected cells.IMPORTANCE The MuV SH has been shown to impede TNF-α-mediated NF-κB activation and is therefore thought to contribute to viral immune evasion. However, the mechanisms by which SH mediates NF-κB inhibition remained largely unknown. In this study, we show that SH interacts with TNFR1, IL-1R1, and TLR3 complexes in infected cells. We thereby not only shed light on the mechanisms of SH-mediated NF-κB inhibition but also reveal that SH interferes with NF-κB activation induced by interleukin-1β (IL-1β) and double-stranded RNA.
Solid tumors display pronounced antigen heterogeneity and clinical studies have shown that antigen escape from therapy occurs rapidly, limiting the persistence and efficacy of CAR T cells. Here we present dual and triple-antigen binding proteins that bridge CAR T cells to multiple antigens, allowing a simultaneous attack on tumor antigens by a single CAR T antibody domain. We call these CAR T Engager proteins. CAR T Engager proteins can be encoded into lentiviral vectors for secretion from CAR T cells, can be encoded into oncolytic viral vectors for secretion from transduced tumor cells, or can be engineered as biologics for injection.
Methods
CAR T Engagers contain a protein target for a CAR T cell, eg. an anti-CD19 CAR T cell. We have previously presented a Her2-binding CAR T Engager protein with potent in vivo activity against solid tumors. We used this CAR T Engager as the basis for building dual and triple antigen binding proteins. Specifically, we mapped antigen expression for Her2-positive solid tumors, Her2-positive metastases, and primary CNS tumors. Our analysis identified expression patterns of two and three antigens that would essentially saturate the cellular composition of specific solid tumors, greatly reducing the chance of antigen escape from therapy. We created the corresponding CAR T Engagers and have developed single, dual and triple antigen expression cells lines to model the activity and potency of these novel proteins, administered alongside CAR T cells.
Results
CAR T cells plus dual antigen CAR T Engagers that recognize and target Her2 and B7H3 demonstrate potent cytotoxicity against either antigen alone, and synergistic potency (2 pM) if both antigens are expressed. Similarly, triple antigen CART Engagers show single antigen binding and potent cytotoxicity which is enhanced when multiple antigens are expressed on a target cell. All of the cytotoxicity is mediated through one CAR domain expressed on the primary T cells. T cells can be pre-loaded with multi-antigen CAR T Engagers and retain cytotoxic activity. Because the underlying CAR is an anti-CD19 CAR, cell persistence and fitness is further enhanced in the presence of normal B cells.
Conclusions
The CAR T Engager platform is a robust and modular solution for the multi-antigen targeting of solid tumors. Diverse antigens can be readily targeted for diverse indications. Examples of other functional modalities that can be added will be presented.
Acknowledgements
We thank Cancer Research UK for their ongoing support.
Studies in mice and humans have revealed that the T cell, immunoglobulin, mucin (TIM) genes are associated with several atopic diseases. TIM-1 is a type I membrane protein that is expressed on T cells upon stimulation and has been shown to modulate their activation. In addition to a recently described interaction with dendritic cells, TIM-1 has also been identified as a phosphatidylserine recognition molecule, and several protein ligands have been proposed. Our understanding of its activity is complicated by the possibility that TIM-1 possesses multiple and diverse binding partners. In order to delineate the function of TIM-1, we generated monoclonal antibodies directed to a cleft formed within the IgV domain of TIM-1. We have shown here that antibodies that bind to this defined cleft antagonize TIM-1 binding to specific ligands and cells. Notably, these antibodies exhibited therapeutic activity in a humanized SCID model of experimental asthma, ameliorating inflammation, and airway hyperresponsiveness. Further experiments demonstrated that the effects of the TIM-1-specific antibodies were mediated via suppression of Th2 cell proliferation and cytokine production. These results demonstrate that modulation of the TIM-1 pathway can critically influence activated T cells in a humanized disease model, suggesting that TIM-1 antagonists may provide potent therapeutic benefit in asthma and other immune-mediated disorders.
