Galina Denisova

T Cells Engineered With Chimeric Antigen Receptors Targeting NKG2D Ligands Display Lethal Toxicity in Mice

Ligands for the NKG2D receptor are overexpressed on tumors, making them interesting immunotherapy targets. To assess the tumoricidal properties of T cells directed to attack NKG2D ligands, we engineered murine T cells with two distinct NKG2D-based chimeric antigen receptors (CARs): (i) a fusion between the NKG2D receptor and the CD3ζ chain and (ii) a conventional second-generation CAR, where the extracellular domain of NKG2D was fused to CD28 and CD3ζ. To enhance the CAR surface expression, we also engineered T cells to coexpress DAP10. In vitro functionality and surface expression levels of all three CARs was greater in BALB/c T cells than C57BL/6 T cells, indicating strain-specific differences. Upon adoptive transfer of NKG2D-CAR-T cells into syngeneic animals, we observed significant clinical toxicity resulting in morbidity and mortality. The severity of these toxicities varied between the CAR configurations and paralleled their in vitro NKG2D surface expression. BALB/c mice were more sensitive to these toxicities than C57BL/6 mice, consistent with the higher in vitro functionality of BALB/c T cells. Treatment with cyclophosphamide prior to adoptive transfer exacerbated the toxicity. We conclude that while NKG2D ligands may be useful targets for immunotherapy, the pursuit of NKG2D-based CAR-T cell therapies should be undertaken with caution.

A novel computer algorithm improves antibody epitope prediction using affinity-selected mimotopes: a case study using monoclonal antibodies against the West Nile virus E protein.

Understanding antibody function is often enhanced by knowledge of the specific binding epitope. Here, we describe a computer algorithm that permits epitope prediction based on a collection of random peptide epitopes (mimotopes) isolated by antibody affinity purification. We applied this methodology to the prediction of epitopes for five monoclonal antibodies against the West Nile virus (WNV) E protein, two of which exhibit therapeutic activity in vivo. This strategy was validated by comparison of our results with existing F(ab)-E protein crystal structures and mutational analysis by yeast surface display. We demonstrate that by combining the results of the mimotope method with our data from mutational analysis, epitopes could be predicted with greater certainty. The two methods displayed great complementarity as the mutational analysis facilitated epitope prediction when the results with the mimotope method were equivocal and the mimotope method revealed a broader number of residues within the epitope than the mutational analysis. Our results demonstrate that the combination of these two prediction strategies provides a robust platform for epitope characterization.

Designed ankyrin repeat proteins are effective targeting elements for chimeric antigen receptors

BackgroundAdoptive cell transfer of tumor-specific T lymphocytes (T cells) is proving to be an effective strategy for treating established tumors in cancer patients. One method of generating these cells is accomplished through engineering bulk T cell populations to express chimeric antigen receptors (CARs), which are specific for tumor antigens. Traditionally, these CARs are targeted against tumor antigens using single-chain antibodies (scFv). Here we describe the use of a designed ankyrin repeat protein (DARPin) as the tumor-antigen targeting domain.MethodsWe prepared second generation anti-HER2 CARs that were targeted to the tumor antigen by either a DARPin or scFv. The CARs were engineered into human and murine T cells. We then compared the ability of CARs to trigger cytokine production, degranulation and cytotoxicity.ResultsThe DARPin CARs displayed reduced surface expression relative to scFv CARs in murine cells but both CARs were expressed equally well on human T cells, suggesting that there may be a processing issue with the murine variants. In both the murine and human systems, the DARPin CARs were found to be highly functional, triggering cytokine and cytotoxic responses that were similar to those triggered by the scFv CARs.ConclusionsThese findings demonstrate the utility of DARPins as CAR-targeting agents and open up an avenue for the generation of CARs with novel antigen binding attributes.

Applying bioinformatics for antibody epitope prediction using affinity-selected mimotopes – relevance for vaccine design

To properly characterize protective polyclonal antibody responses, it is necessary to examine epitope specificity. Most antibody epitopes are conformational in nature and, thus, cannot be identified using synthetic linear peptides. Cyclic peptides can function as mimetics of conformational epitopes (termed mimotopes), thereby providing targets, which can be selected by immunoaffinity purification. However, the management of large collections of random cyclic peptides is cumbersome. Filamentous bacteriophage provides a useful scaffold for the expression of random peptides (termed phage display) facilitating both the production and manipulation of complex peptide libraries. Immunoaffinity selection of phage displaying random cyclic peptides is an effective strategy for isolating mimotopes with specificity for a given antiserum. Further epitope prediction based on mimotope sequence is not trivial since mimotopes generally display only small homologies with the target protein. Large numbers of unique mimotopes are required to provide sufficient sequence coverage to elucidate the target epitope. We have developed a method based on pattern recognition theory to deal with the complexity of large collections of conformational mimotopes. The analysis consists of two phases: 1) The learning phase where a large collection of epitope-specific mimotopes is analyzed to identify epitope specific “signs” and 2) The identification phase where immunoaffinity-selected mimotopes are interrogated for the presence of the epitope specific “signs” and assigned to specific epitopes. We are currently using computational methods to define epitope “signs” without the need for prior knowledge of specific mimotopes. This technology provides an important tool for characterizing the breadth of antibody specificities within polyclonal antisera.

Tryptamine induces tryptophanyl-tRNA synthetase-mediated neurodegeneration with neurofibrillary tangles in human cell and mouse models

The neuropathological hallmarks of Alzheimer’s disease (AD) and other taupathies include neurofibrillary tangles and plaques. Despite the fact that only 2–10% of AD cases are associated with genetic mutations, no nontrasgenic or metabolic models have been generated to date. The findings of tryptophanyl-tRNA synthetase (TrpRS) in plaques of the AD brain were reported recently by the authors. Here it is shown that expression of cytoplasmic-TrpRS is inversely correlated with neurofibrillary degeneration, whereas a nonionic detergent-insoluble presumably aggregated TrpRS is simultaneously accumulated in human cells treated by tryptamine, a metabolic tryptophan analog that acts as a competitive inhibitor of TrpRS. TrpRS-N-terminal peptide self-assembles in double-helical fibrils in vitro. Herein, tryptamine causes neuropathy characterized by motor and behavioral deficits, hippocampal neuronal loss, neurofibrillary tangles, amyloidosis, and glucose decrease in mice. Tryptamine induced the formation of helical fibrillary tangles in both hippocampal neurons and glia. Taken together with the authors’ previous findings of tryptamine-induced nephrotoxicity and filamentous tangle formation in kidney cells, the author’s data indicates a general role of tryptamine in cell degeneration and loss. It is concluded that tryptamine as a component of a normal diet can induce neurodegeneration at the concentrations, which might be consumed along with food. Tryptophan-dependent tRNAtrp aminoacylation catalyzed by TrpRS can be inhibited by its substrate tryptophan at physiological concentrations was demonstrated. These findings indicate that the dietary supplementation with tryptophan as a tryptamine competitor may not counteract the deleterious influence of tryptamine. The pivotal role of TrpRS in protecting against neurodegeneration is suggested, providing an insight into the pathogenesis and a possible treatment of neurodegenerative diseases.

Deciphering epitope specificities within polyserum using affinity selection of random peptides and a novel algorithm based on pattern recognition theory.

While numerous strategies have been developed to map epitope specificities for monoclonal antibodies, few have been designed for elucidating epitope specificity within complex polysera. We have developed a novel algorithm based on pattern recognition theory that can be used to characterize the breadth of epitope specificities within a polyserum based on affinity selection of random peptides. To attribute these random peptides to a specific epitope, the sequences of the affinity-selected peptides were matched against a database of random peptides selected using well-described monoclonal antibodies. To test this novel algorithm, we employed polyserum from patients infected with West Nile virus and isolated 109 unique sequences which were recognized selectively by serum from West Nile virus-infected patients but not uninfected patients. Through application of our algorithm, it was possible to match 20% of the polyserum-selected peptides to the database of peptides isolated by affinity selection using monoclonal antibodies against the virus envelope protein. Statistical analysis demonstrated that the peptides selected with the polyserum could not be attributed to the peptide database by chance. This novel algorithm provides the basis for further development of methods to characterize the breadth of epitope recognition within a complex pool of antibodies.

Characterizing complex polysera produced by antigen-specific immunization through the use of affinity-selected mimotopes.

Background Antigen-based (as opposed to whole organism) vaccines are actively being pursued for numerous indications. Even though different formulations may produce similar levels of total antigen-specific antibody, the composition of the antibody response can be quite distinct resulting in different levels of therapeutic activity. Methodology/Principal Findings Using plasmid-based immunization against the proto-oncogene HER-2 as a model, we have demonstrated that affinity-selected epitope mimetics (mimotopes) can provide a defined signature of a polyclonal antibody response. Further, using novel computer algorithms that we have developed, these mimotopes can be used to predict epitope targets. Conclusions/Significance By combining our novel strategy with existing methods of epitope prediction based on physical properties of an individual protein, we believe that this method offers a robust method for characterizing the breadth of epitope-specificity within a specific polyserum. This strategy is useful as a tool for monitoring immunity following vaccination and can also be used to define relevant epitopes for the creation of novel vaccines.

Tri-functional T cell receptor antigen coupler (Tri-TAC): a novel method to direct T cells against tumors

Engineering T cells with chimeric antigen receptors (CARs) is proving to be an effective method for directing T cells to attack tumors in an MHC-independent manner. Current generation CARs aim to recapitulate T cell signaling by incorporating modular functional components of the TCR and costimulatory molecules. Development of next generation CARs has relied upon trial and error evaluation of signaling domains. We sought to develop an alternate method to re-direct the T cell receptor which does not rely upon the incorporation of signaling domains into the chimeric receptor. To this end, we developed a tri-functional molecule which is membrane-anchored and redirects the TCR in the presence of tumor antigen. We also included components of the CD4 co-receptor to provide requisite Lck signaling upon ligation of the tumor antigen. Our prototype receptor was directed against the HER-2 proto-oncogene. We have determined that engineering peripheral blood T cells with this novel receptor (termed a Tri-TAC) engenders tumor-antigen specific activation of numerous T cell functions, including cytokine production, degranulation and cytolysis - equivalent to, if not greater than, a 2nd generation CAR bearing the CD28 and CD3zeta signaling domains. Future iterations of the engineered T cells will include chimeric costimulatory receptors to enhance T cell functionality and reduce off target toxicity. This research was supported by the Canadian Institutes of Health Research and the Terry Fox Foundation.

Preclinical evaluation of a MAGE-A3 vaccination utilizing the oncolytic Maraba virus currently in first-in-human trials

ABSTRACT Multiple immunotherapeutics have been approved for cancer patients, however advanced solid tumors are frequently refractory to treatment. We evaluated the safety and immunogenicity of a vaccination approach with multimodal oncolytic potential in non-human primates (NHP) (Macaca fascicularis). Primates received a replication-deficient adenoviral prime, boosted by the oncolytic Maraba MG1 rhabdovirus. Both vectors expressed the human MAGE-A3. No severe adverse events were observed. Boosting with MG1-MAGEA3 induced an expansion of hMAGE-A3-specific CD4+ and CD8+ T-cells with the latter peaking at remarkable levels and persisting for several months. T-cells reacting against epitopes fully conserved between simian and human MAGE-A3 were identified. Humoral immunity was demonstrated by the detection of circulating MAGE-A3 antibodies. These preclinical data establish the capacity for the Ad:MG1 vaccination to engage multiple effector immune cell populations without causing significant toxicity in outbred NHPs. Clinical investigations utilizing this program for the treatment of MAGE-A3-positive solid malignancies are underway (NCT02285816, NCT02879760).

The chimeric TAC receptor co-opts the T cell receptor yielding robust anti-tumor activity without toxicity

Engineering T cells with chimeric antigen receptors (CARs) is an effective method for directing T cells to attack tumors, but may cause adverse side effects such as the potentially lethal cytokine release syndrome. Here the authors show that the T cell antigen coupler (TAC), a chimeric receptor that co-opts the endogenous TCR, induces more efficient anti-tumor responses and reduced toxicity when compared with past-generation CARs. TAC-engineered T cells induce robust and antigen-specific cytokine production and cytotoxicity in vitro, and strong anti-tumor activity in a variety of xenograft models including solid and liquid tumors. In a solid tumor model, TAC-T cells outperform CD28-based CAR-T cells with increased anti-tumor efficacy, reduced toxicity, and faster tumor infiltration. Intratumoral TAC-T cells are enriched for Ki-67+ CD8+ T cells, demonstrating local expansion. These results indicate that TAC-T cells may have a superior therapeutic index relative to CAR-T cells.Chimeric antigen receptors (CARs) are effective tools for directing T cell killing of tumors, but may cause adverse side effects. Here the authors show that coupling of antigen-recognition and CD3-binding in a modular format induces more efficient anti-tumour responses but reduced toxicity when compared with current CARs.