Artificial Antigen Presenting Cells for Detection and Desensitization of Autoreactive T cells Associated with Type 1 Diabetes.

Autoimmune diseases and in particular type 1 diabetes rely heavily on treatments that target the symptoms rather than prevent the underlying disease. One of the barriers to better therapeutic strategies is the inability to detect and efficiently target rare autoreactive T-cell populations that are major drivers of these conditions. Here, we develop a unique artificial antigen-presenting cell (aAPC) system from biocompatible polymer particles that allows specific encapsulation of bioactive ingredients. Using our aAPC, we demonstrate that we are able to detect rare autoreactive CD4 populations in human patients, and using mouse models, we demonstrate that our particles are able to induce desensitization in the autoreactive population. This system provides a promising tool that can be used in the prevention of autoimmunity before disease onset.

The Intracellular Proteome as a Source for Novel Targets in CAR-T and T-Cell Engagers-Based Immunotherapy.

The impressive clinical success of cancer immunotherapy has motivated the continued search for new targets that may serve to guide potent effector functions in an attempt to efficiently kill malignant cells. The intracellular proteome is an interesting source for such new targets, such as neo-antigens and others, with growing interest in their application for cell-based immunotherapies. These intracellular-derived targets are peptides presented by MHC class I molecules on the cell surface of malignant cells. These disease-specific class I HLA-peptide complexes can be targeted by specific TCRs or by antibodies that mimic TCR-specificity, termed TCR-like (TCRL) antibodies. Adoptive cell transfer of TCR engineered T cells and T-cell-receptor-like based CAR-T cells, targeted against a peptide-MHC of interest, are currently tested as cancer therapeutic agents in pre-clinical and clinical trials, along with soluble TCR- and TCRL-based agents, such as immunotoxins and bi-specific T cell engagers. Targeting the intracellular proteome using TCRL- and TCR-based molecules shows promising results in cancer immunotherapy, as exemplified by the success of the anti-gp100/HLA-A2 TCR-based T cell engager, recently approved by the FDA for the treatment of unresectable or metastatic uveal melanoma. This review is focused on the selection and isolation processes of TCR- and TCRL-based targeting moieties, with a spotlight on pre-clinical and clinical studies, examining peptide-MHC targeting agents in cancer immunotherapy.

Phenotypic Models of CAR T-Cell Activation Elucidate the Pivotal Regulatory Role of CAR Downmodulation.

Adoptive cell immunotherapy with chimeric antigen receptor (CAR) showed limited potency in solid tumors, despite durable remissions for hematopoietic malignancies. Therefore, an investigation of ways to enhance the efficacy of CARs' antitumor response has been engaged upon. We previously examined the interplay between the biophysical parameters of CAR binding (i.e., affinity, avidity, and antigen density), as regulators of CAR T-cell activity and detected nonmonotonic behaviors of affinity and antigen density and an interrelation between avidity and antigen density. Here, we built an evolving phenotypic model of CAR T-cell regulation, which suggested that receptor downmodulation is a key determinant of CAR T-cell function. We verified this assumption by measuring and manipulating receptor downmodulation and intracellular signaling processes. CAR downmodulation inhibition, via actin polymerization inhibition, but not inhibition of regulatory inhibitory phosphatases, was able to increase CAR T-cell responses. In addition, we documented trogocytosis in CAR T cells that depends on actin polymerization. In summary, our study modeled the parameters that govern CAR T-cell engagement and revealed an underappreciated mechanism of T-cell regulation. These results have a potential to predict and therefore advance the rational design of CAR T cells for adoptive cell treatments.See related article on p. 872.

Shaping Functional Avidity of CAR T Cells: Affinity, Avidity, and Antigen Density That Regulate Response.

Chimeric antigen receptors (CARs) are immunoreceptors that redirect T cells to selectively kill tumor cells. Given their clinical successes in hematologic malignancies, there is a strong aspiration to advance this immunotherapy for solid cancers; hence, molecular CAR design and careful target choice are crucial for their function. To evaluate the functional significance of the biophysical properties of CAR binding (i.e., affinity, avidity, and antigen density), we generated an experimental system in which these properties are controllable. We constructed and characterized a series of CARs, which target the melanoma tumor-associated antigen Tyr/HLA-A2, and in which the affinity of the single-chain Fv binding domains ranged in KD from 4 to 400 nmol/L. These CARs were transduced into T cells, and each CAR T-cell population was sorted by the level of receptor expression. Finally, the various CAR T cells were encountered with target cells that present different levels of the target antigen. We detected nonmonotonic behaviors of affinity and antigen density, and an interrelation between avidity and antigen density. Antitumor activity measurements in vitro and in vivo corroborated these observations. Our study contributes to the understanding of CAR T-cell function and regulation, having the potential to improve therapies by the rational design of CAR T cells.See related article on p. 946.

Recruitment of Oligoclonal Viral-Specific T cells to Kill Human Tumor Cells Using Single-Chain Antibody-Peptide-HLA Fusion Molecules.

Tumor progression is often associated with the development of diverse immune escape mechanisms. One of the main tumor escape mechanism is HLA loss, in which human solid tumors exhibit alterations in HLA expression. Moreover, tumors that present immunogenic peptides via class I MHC molecules are not susceptible to CTL-mediated lysis, because of the relatively low potency of the tumor-specific CLTs. Here, we present a novel cancer immunotherapy approach that overcomes these problems by using the high affinity and specificity of antitumor antibodies to recruit potent antiviral memory CTLs to attack tumor cells. We constructed a recombinant molecule by genetic fusion of a cytomegalovirus (CMV)-derived peptide pp65 (NLVPMVATV) to scHLA-A2 molecules that were genetically fused to a single-chain Fv Ab fragment specific for the tumor cell surface antigen mesothelin. This fully covalent fusion molecule was expressed in E. coli as inclusion bodies and refolded in vitro. The fusion molecules could specifically bind mesothelin-expressing cells and mediate their lysis by NLVPMVATV-specific HLA-A2-restricted human CTLs. More importantly, these molecules exhibited very potent antitumor activity in vivo in a nude mouse model bearing preestablished human tumor xenografts that were adoptively transferred along with human memory CTLs. These results represent a novel and powerful approach to immunotherapy for solid tumors, as demonstrated by the ability of the CMV-scHLA-A2-SS1(scFv) fusion molecule to mediate specific and efficient recruitment of CMV-specific CTLs to kill tumor cells.

Functional comparison of engineered T cells carrying a native TCR versus TCR-like antibody-based chimeric antigen receptors indicates affinity/avidity thresholds.

Adoptive transfer of Ag-specific T lymphocytes is an attractive form of immunotherapy for cancers. However, acquiring sufficient numbers of host-derived tumor-specific T lymphocytes by selection and expansion is challenging, as these cells may be rare or anergic. Using engineered T cells can overcome this difficulty. Such engineered cells can be generated using a chimeric Ag receptor based on common formats composed from Ag-recognition elements such as αß-TCR genes with the desired specificity, or Ab variable domain fragments fused with T cell-signaling moieties. Combining these recognition elements are Abs that recognize peptide-MHC. Such TCR-like Abs mimic the fine specificity of TCRs and exhibit both the binding properties and kinetics of high-affinity Abs. In this study, we compared the functional properties of engineered T cells expressing a native low affinity αß-TCR chains or high affinity TCR-like Ab-based CAR targeting the same specificity. We isolated high-affinity TCR-like Abs recognizing HLA-A2-WT1Db126 complexes and constructed CAR that was transduced into T cells. Comparative analysis revealed major differences in function and specificity of such CAR-T cells or native TCR toward the same antigenic complex. Whereas the native low-affinity αß-TCR maintained potent cytotoxic activity and specificity, the high-affinity TCR-like Ab CAR exhibited reduced activity and loss of specificity. These results suggest an upper affinity threshold for TCR-based recognition to mediate effective functional outcomes of engineered T cells. The rational design of TCRs and TCR-based constructs may need to be optimized up to a given affinity threshold to achieve optimal T cell function.

Melanoma cells present high levels of HLA-A2-tyrosinase in association with instability and aberrant intracellular processing of tyrosinase.

Short-lived protein translation products are proposed to be a major source of substrates for major histocompatibility complex (MHC) class I antigen processing and presentation; however, a direct link between protein stability and the presentation level of MHC class I-peptide complexes has not been made. We have recently discovered that the peptide Tyr((369-377)) , derived from the tyrosinase protein is highly presented by HLA-A2 on the surface of melanoma cells. To examine the molecular mechanisms responsible for this presentation, we compared characteristics of tyrosinase in melanoma cells lines that present high or low levels of HLA-A2-Tyr((369-377)) complexes. We found no correlation between mRNA levels and the levels of HLA-A2-Tyr((369-377)) presentation. Co-localization experiments revealed that, in cell lines presenting low levels of HLA-A2-Tyr((369-377)) complexes, tyrosinase co-localizes with LAMP-1, a melanosome marker, whereas in cell lines presenting high HLA-A2-Tyr((369-377)) levels, tyrosinase localizes to the endoplasmic reticulum. We also observed differences in tyrosinase molecular weight and glycosylation composition as well as major differences in protein stability (t(1/2) ). By stabilizing the tyrosinase protein, we observed a dramatic decrease in HLA-A2-tyrosinase presentation. Our findings suggest that aberrant processing and instability of tyrosinase are responsible for the high presentation of HLA-A2-Tyr((369-377)) complexes and thus shed new light on the relationship between intracellular processing, stability of proteins, and MHC-restricted peptide presentation.

T-cell receptor-like antibodies: novel reagents for clinical cancer immunology and immunotherapy.

Major histocompatibility complex class I molecules play a central role in the immune response against a variety of cells that have undergone malignant transformation by shaping the T-cell repertoire and presenting peptide antigens from endogeneous antigens to CD8+ cytotoxic T-cells. Diseased tumor or virus-infected cells are present on class I major histocompatibility complex molecule peptides that are derived from tumor-associated antigens or viral-derived proteins. Due to their unique specificity, such major histocompatibility complex-peptide complexes are a desirable target for novel approaches in immunotherapy. Targeted delivery of toxins or other cytotoxic drugs to cells which express specific major histocompatibility complex-peptide complexes that are involved in the immune response against cancer or viral infections would allow for a specific immunotherapeutic treatment of these diseases. It has recently been demonstrated that antibodies with the antigen-specific, major histocompatibility complex-restricted specificity of T-cells can be generated by taking advantage of the selection power of phage display technology. In addition to their tumor targeting capabilities, antibodies that mimic the fine specificity of T-cell receptors can serve as valuable research reagents that enable study of human class I peptide-major histocompatibility complex ligand presentation, as well as T-cell receptor peptide-major histocompatibility complex interactions. T-cell receptor-like antibody molecules may prove to be useful tools for studying major histocompatibility complex class I antigen presentation in health and disease as well as for therapeutic purposes in cancer, infectious diseases and autoimmune disorders.

Targeting an extracellular epitope of the human multidrug resistance protein 1 (MRP1) in malignant cells with a novel recombinant single chain Fv antibody.

Inherent and acquired multidrug resistance (MDR) is characterized by a simultaneous resistance to diverse anticancer drugs and is a major impediment towards curative chemotherapy of cancer. Hence one important goal is to develop strategies aimed at specific targeting of major anticancer drug efflux transporters of the ATP-binding cassette (ABC) superfamily including multidrug resistance protein 1 -MRP1 (ABCC1). To date, no monoclonal antibody has been isolated that can target an extracellular MRP1 epitope. Using a phage display approach, we have isolated a recombinant single-chain Fv (scFv) antibody that specifically reacts with the extracellular N-terminus of the human MRP1. Flow cytometric analysis revealed that this scFv fragment binds specifically to various viable human tumor cells that display variable MRP1 expression levels but not to MRP1 null cells. Furthermore, this scFv antibody failed to react with tumor cells that overexpress other members of the MRP family that have an extracellular N-terminus (MRP2 and MRP3) as well as with MRP4, MRP5, and breast cancer resistance protein. Flow cytometric analysis also showed a good correlation between the fluorescence intensity of the anti-MRP1 scFv antibody and MRP1 levels in viable tumor cells. These findings constitute the first successful isolation of a small recombinant scFv antibody directed to an extracellular epitope of the MRP1 in viable malignant cells. These novel small Fv-based recombinant antibodies that possess superior tumor penetration capabilities may possibly be used to selectively target drugs or tumor cells that express MRP-1.

Disruption of P-glycoprotein anticancer drug efflux activity by a small recombinant single-chain Fv antibody fragment targeted to an extracellular epitope.

Inherent and acquired MDR is characterized by simultaneous resistance to diverse anticancer drugs and continues to be a major impediment in the curative chemotherapy of cancer. The MDR1 gene product, Pgp, is an ATP-driven efflux pump, which extrudes a variety of dissimilar hydrophobic cytotoxic compounds from MDR cells. Pgp overexpression results in MDR of tumor cell lines in vitro as well as of a variety of human malignancies. Thus, one major goal is to develop strategies aimed at specifically disrupting Pgp drug-efflux activity. To this end, we have developed a small recombinant antibody capable of potent reversal of MDR, by disrupting Pgp drug-efflux activity. Using a phage display approach, we isolated a small scFv recombinant antibody fragment that specifically reacts with the first extracellular loop of human Pgp. This scFv fragment binds specifically to various Pgp-overexpressing human MDR carcinoma cell lines, consequently disrupts Pgp drug-efflux function and thereby reverses the MDR phenotype. We have successfully disrupted anticancer drug-extrusion pump activity in MDR cells using a small recombinant scFv fragment. We propose that these novel small Fv-based recombinant antibody molecules may lead to the development of a new class of antibody fragment-based agents that specifically inhibit Pgp drug extrusion. Hence, these small recombinant antibody fragments may be applied in combination chemotherapy to overcome MDR in various human cancers.