Juan Carlos Varela

Enrichment and Expansion with Nanoscale Artificial Antigen Presenting Cells for Adoptive Immunotherapy.

Adoptive immunotherapy (AIT) can mediate durable regression of cancer, but widespread adoption of AIT is limited by the cost and complexity of generating tumor-specific T cells. Here we develop an Enrichment + Expansion strategy using paramagnetic, nanoscale artificial antigen presenting cells (aAPC) to rapidly expand tumor-specific T cells from rare naïve precursors and predicted neo-epitope responses. Nano-aAPC are capable of enriching rare tumor-specific T cells in a magnetic column and subsequently activating them to induce proliferation. Enrichment + Expansion resulted in greater than 1000-fold expansion of both mouse and human tumor-specific T cells in 1 week, with nano-aAPC based enrichment conferring a proliferation advantage during both in vitro culture and after adoptive transfer in vivo. Robust T cell responses were seen not only for shared tumor antigens, but also for computationally predicted neo-epitopes. Streamlining the rapid generation of large numbers of tumor-specific T cells in a cost-effective fashion through Enrichment + Expansion can be a powerful tool for immunotherapy.

An Innovative Microarray Strategy Identities Informative Molecular Markers for the Detection of Micrometastatic Breast Cancer

There is increasing evidence that molecular detection of micrometastatic breast cancer in the axillary lymph nodes (ALN) of breast cancer patients can improve staging. Molecular analyses of samples obtained from the Minimally Invasive Molecular Staging of Breast Cancer Trial (n = 489 patients) indicate that whereas the majority of molecular markers are informative for the detection of metastatic breast cancer (significant disease burden), only a few are sensitive for the detection of micrometastatic disease (limited disease burden). Frequency distribution and linear regression analyses reveal that relative levels of gene expression are highly correlated with apparent sensitivity for the detection of micrometastic breast cancer (P < 0.05). These data provides statistical validation of the concept that the most informative markers for detection of micrometastatic disease are those that are most highly expressed in metastatic disease. To test this hypothesis, we developed an innovative microarray strategy. RNA from a metastatic breast cancer ALN was diluted into RNA from a normal lymph node and analyzed using Affymetrix microarrays. Expression analysis indicated that only two genes [mammaglobin (mam) and trefoil factor 1 (TFF1)] were significantly overexpressed at a dilution of 1:50. Real-time reverse transcription-PCR analysis of pathology-negative ALN (n = 72) confirm that of all the markers tested, mam and TFF1 have the highest apparent sensitivity for detection of micrometastatic breast cancer. We conclude that a dilutional microarray approach is a simple and reliable method for the identification of informative molecular markers for the detection of micrometastatic cancer.

Complement blockade with a C1 esterase inhibitor in paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, clonal, hematopoietic stem cell disorder that manifests with a complement-mediated hemolytic anemia, bone marrow failure, and a propensity for thrombosis. These patients experience both intra- and extravascular hemolysis in the context of underlying complement activation. Currently eculizumab effectively blocks the intravascular hemolysis PNH. There remains an unmet clinical need for a complement inhibitor with activity early in the complement cascade to block complement at the classical and alternative pathways. C1 esterase inhibitor (C1INH) is an endogenous human plasma protein that has broad inhibitory activity in the complement pathway through inhibition of the classical pathway by binding C1r and C1s and inhibits the mannose-binding lectin-associated serine proteases in the lectin pathway. In this study, we show that commercially available plasma derived C1INH prevents lysis induced by the alternative complement pathway of PNH erythrocytes in human serum. Importantly, C1INH was able to block the accumulation of C3 degradation products on CD55 deficient erythrocytes from PNH patient on eculizumab therapy. This could suggest a role for inhibition of earlier phases of the complement cascade than that currently inhibited by eculizumab for incomplete or nonresponders to that therapy.

Antigen-specific T cell Redirectors: a nanoparticle based approach for redirecting T cells

Redirection of T cells to target and destroy tumors has become an important clinical tool and major area of research in tumor immunology. Here we present a novel, nanoparticle-based approach to selectively bind antigen-specific cytotoxic T cells (CTL) and redirect them to kill tumors, termed ATR (Antigen-specific T cell Redirectors). ATR were generated by decorating nanoparticles with both an antigen-specific T cell binding moiety, either peptide loaded MHC-Ig dimer or clonotypic anti-TCR antibody, and a model tumor cell binding moiety, anti-CD19 antibody to engage CD19+ tumor cells. ATR stably bind tumor cells and CTL in a dose dependent fashion and stimulate antigen-specific conjugate formation between those cells. ATR induced redirected lysis of tumor cells in vitro, as demonstrated by 51Cr-release killing. In vivo ATR administration led to reduced tumor growth in a SCID/beige human lymphoma treatment model. In summary, ATR represent a novel, nanoparticle based approach for redirecting antigen-specific CTL to kill tumors.

Paroxysmal nocturnal hemoglobinuria and the age of therapeutic complement inhibition.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease of hematopoietic stem cells due to a mutation in the PIG-A gene leading to a deficiency of GPI-anchored proteins. Lack of two specific GPI-anchored proteins, CD55 and CD59, leads to uncontrolled complement activation that result in both intravascular and extravascular hemolysis. Free hemoglobin leads to nitric oxide depletion that mediates the pathophysiology of some of the common clinical signs of PNH. Clinical symptoms of PNH include evidence of hemolytic anemia, bone marrow failure, smooth muscle dystonias and thromboses. Treatment options for patients with PNH include bone marrow transplantation, a therapy associated with high morbidity and mortality, or treatment with the complement inhibitor eculizumab. Eculizumab is a first-in-class anti-complement drug that in PNH has been shown to block complement-mediated hemolysis, reduce transfusion dependency, reduce thromboembolic complications and improve the quality of life (QoL) of patients.

Antigen-specific T cell redirectors (ATR) for antigen-specific redirection of T cells to tumors

Immunotherapy is the modulation of a patients immune system to treat illness. Unfortunately many T cell based attempts have failed due to the fact that existing tumor-specific T cells are mostly anergic or tolerized and ex vivo generated T cells are often already of exhausted phenotype. Therefore, investigators have developed alternative approaches including bispecific antibody technology to redirect fully functional non-tumor specific T cells to the tumor. This has been primarily accomplished through targeting CD3, which is expressed on all T cells to engage and redirect them towards a molecule that is expressed on the tumor cells. Here we present a novel nanoparticle based approach to selectively target cytotoxic T cells (CTL) and re-direct them to kill tumors, termed ATR (Antigen-specific T cell Redirectors). ATR were generated by coupling either MHC-Ig dimer or clonotypic anti-TCR antibody 1B2 to target the effector T cell population and an anti-CD19 to re-direct those to CD19+ tumor target cells onto 50-100nm nanoparticles. Flow cytometry and microscope based data confirm that the described ATR phenotype efficiently and stably stain tumor and T cells in a dose dependent manner, and ATR mediate antigen-specific conjugate formation of effector T cells and tumor target cells. We further developed two clinically relevant protocols to test and optimize our ATR in vitro. First a pre-treatment approach in which the effector T cells are pre-incubated with ATR mimicking an adoptive transfer approach and second a co-culture protocol that mimics an active immunotherapy approach of direct ATR injection. Antigen-specific ATR mediated re-direction of T cells to tumor target cells was demonstrated in 51Cr-release killing assays at low E:T ratios. Variation of ATR target-cell: effector-cell targeting molecule ratio could further increase efficacy. Finally, intra tumoral ATR injection induced T cell re-direction and reduced tumor growth in a s.c. Raji/SCIDbeige treatment model. In summary this data demonstrates that ATR target and redirect antigen-specific CTL to tumor cells that would otherwise not be recognized and mediates their lysis. ATR can be used to develop new innovative immunotherapeutic approaches for all cancers that can be targeted with antibodies or antibody-like molecules. Furthermore, ATR could also be used in conjunction with virus-specific immunization to specifically increase the targeted CTL population. Ultimately, we expect ATR and their potential for clinical applications to increase our understanding of tumor immunotherapy through T cell redirection.

Enrichment and expansion with nanoscale artificial antigen presenting cells for T cell adoptive immunotherapy

Adoptive T cell therapy can mediate durable regression of cancer [1]. While pre-existing anti-tumor responses can only be cultured from a minority of cancer patients [2], T cells specific for a wide variety of tumor antigens can be generated by stimulation of naive precursor cells with tumor antigen [3]. This culture process relies on autologous antigen presenting cells and feeder cells, which are complex biologics that must be generated for each individual patient [4], significantly increasing the cost and complexity of adoptive immunotherapy. To quickly generate large numbers of functional tumor-specific T cells from naive T cell precursors, we developed a T cell Enrichment+Expansion strategy using paramagnetic, nanoscale artificial Antigen Presenting Cells (nano-aAPC), which are capable of enriching rare tumor-specific T cells in a magnetic column and activating them. We generated up to 150,000 total Trp2-specific cells in only one week from 10 million polyclonal CD8 lymphocytes containing approximately 10 precursor cells [5]. Similar results were obtained for other tumor and model antigens, including the human tumor antigens A2-NY-ESO1 and A2-MART1. We further demonstrate that removing irrelevant bystander cells by enrichment confers a significant survival and proliferation advantage to tumor-specific T cells both during in vitro culture and after adoptive transfer in vivo. Streamlining the generation of large numbers of high-frequency tumor-specific T cells in a cost effective, reproducible fashion through Enrichment+Expansion could be a powerful addition to autologous tumor immunotherapy protocols.