Pappanaicken R. Kumaresan

Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells

BACKGROUND T cells expressing antigen-specific chimeric antigen receptors (CARs) improve outcomes for CD19-expressing B cell malignancies. We evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR. METHODS T cells were genetically modified using DNA plasmids from the SB platform to stably express a second-generation CD19-specific CAR and selectively propagated ex vivo with activating and propagating cells (AaPCs) and cytokines. Twenty-six patients with advanced non-Hodgkin lymphoma and acute lymphoblastic leukemia safely underwent hematopoietic stem cell transplantation (HSCT) and infusion of CAR T cells as adjuvant therapy in the autologous (n = 7) or allogeneic settings (n = 19). RESULTS SB-mediated genetic transposition and stimulation resulted in 2,200- to 2,500-fold ex vivo expansion of genetically modified T cells, with 84% CAR expression, and without integration hotspots. Following autologous HSCT, the 30-month progression-free and overall survivals were 83% and 100%, respectively. After allogeneic HSCT, the respective 12-month rates were 53% and 63%. No acute or late toxicities and no exacerbation of graft-versus-host disease were observed. Despite a low antigen burden and unsupportive recipient cytokine environment, CAR T cells persisted for an average of 201 days for autologous recipients and 51 days for allogeneic recipients. CONCLUSIONS CD19-specific CAR T cells generated with SB and AaPC platforms were safe, and may provide additional cancer control as planned infusions after HSCT. These results support further clinical development of this nonviral gene therapy approach. TRIAL REGISTRATION Autologous, NCT00968760; allogeneic, NCT01497184; long-term follow-up, NCT01492036. FUNDING National Cancer Institute, private foundations, and institutional funds. Please see Acknowledgments for details.

Bioengineering T cells to target carbohydrate to treat opportunistic fungal infection

Significance Patients with compromised T-cell function are at risk for opportunistic fungal infections. We have developed a novel approach to restore immunity by using a fungal pattern-recognition receptor Dectin-1 to redirect T-cell specificity to carbohydrate antigen in the fungal cell wall. We did so by genetically modifying T cells using the nonviral Sleeping Beauty gene-transfer system to enforce expression of a chimeric antigen receptor (CAR) that recapitulates the specificity of Dectin-1 (D-CAR). The D-CAR+ T cells can be electroporated and propagated on artificial activating and propagating cells in a manner suitable for human application, enabling this immunology to be translated into immunotherapy. This approach has implications for genetically modifying T cells to express CARs with specificity for carbohydrate and thus broadening their application in the investigational treatment of pathogens and malignancies. Clinical-grade T cells are genetically modified ex vivo to express chimeric antigen receptors (CARs) to redirect their specificity to target tumor-associated antigens in vivo. We now have developed this molecular strategy to render cytotoxic T cells specific for fungi. We adapted the pattern-recognition receptor Dectin-1 to activate T cells via chimeric CD28 and CD3-ζ (designated “D-CAR”) upon binding with carbohydrate in the cell wall of Aspergillus germlings. T cells genetically modified with the Sleeping Beauty system to express D-CAR stably were propagated selectively on artificial activating and propagating cells using an approach similar to that approved by the Food and Drug Administration for manufacturing CD19-specific CAR+ T cells for clinical trials. The D-CAR+ T cells exhibited specificity for β-glucan which led to damage and inhibition of hyphal growth of Aspergillus in vitro and in vivo. Treatment of D-CAR+ T cells with steroids did not compromise antifungal activity significantly. These data support the targeting of carbohydrate antigens by CAR+ T cells and provide a clinically appealing strategy to enhance immunity for opportunistic fungal infections using T-cell gene therapy.

CS1 (CRACC, CD319) Induces Proliferation and Autocrine Cytokine Expression on Human B Lymphocytes

CS1 (CRACC, CD319), a member of the CD2 family of cell surface receptors, is implicated in the activation of NK cell-mediated cytotoxicity. Previous studies showed that CS1 is also expressed on activated B cells. However, the functional role of CS1 in human B-lymphocytes is not known. Two isoforms of CS1, CS1-L and CS1-S, are expressed in human NK cells that differentially regulate NK cell function. CS1-L contains immunoreceptor tyrosine-based switch motifs in its cytoplasmic domain whereas CS1-S lacks immunoreceptor tyrosine-based switch motifs. In this study, we show that human B lymphocytes express only the CS1-L isoform, and its expression is up-regulated upon B cell activation with various stimulators. Moreover, anti-CS1 mAb strongly enhanced proliferation of both freshly isolated as well as activated B cells. The enhanced proliferation effects of CS1 were most prominent on B cells activated by anti-CD40 mAbs and/or hrIL-4. The effects of CS1 on B cell proliferation were shown on both naive and memory B cells. Human cytokine microarray and quantitative real-time PCR results indicated that CS1 activation enhanced mRNA transcripts of flt3 ligand, lymphotoxin A, TNF, and IL-14. Neutralizing Abs against lymphotoxin A, TNF-α, and/or flt3 ligand abolished the ability of CS1 on the B cell proliferation. These results suggest that activation of B lymphocytes, through surface CS1, may be mediated through secretion of autocrine cytokines and CS1 may play a role in the regulation of B lymphocyte proliferation during immune responses.

C-reactive protein induces release of both endothelial microparticles and circulating endothelial cells in vitro and in vivo: further evidence of endothelial dysfunction.

BACKGROUND Inflammation is pivotal in atherosclerosis. A key early event in atherosclerosis is endothelial dysfunction. C-reactive protein (CRP), the prototypic marker of inflammation in humans, is a risk marker for cardiovascular disease, and there is mounting evidence to support its role in atherothrombosis. CRP has been shown to promote endothelial dysfunction both in vitro and in vivo. Emerging biomarkers of endothelial dysfunction include circulating endothelial cells (CECs) and endothelial microparticles (EMPs). However, there is a paucity of data examining the effect of CRP on CEC and EMP production in vitro and in vivo. METHODS In this report, we treated human aortic endothelial cells (HAECs) with increasing concentrations of CRP (0-50 μg/mL) or boiled CRP. We counted CECs and EMPs by flow cytometry. RESULTS Although CRP treatment resulted in a significant increase in release of both CECs and EMPs, boiled CRP failed to have an effect. Pretreatment of HAECs with sepiapterin or diethylenetriamine NONOate, both of which preserve nitric oxide (NO), resulted in attenuation of CRPs effects on CECs and EMPs. CD32 and CD64 blocking antibodies but not CD16 antibody or lectin-like oxidized LDL receptor 1 small interfering RNA (LOX-1 siRNA) prevented CRP-induced production of CECs and EMPs. Furthermore, delivery of human CRP to Wistar rats compared with human serum albumin resulted in significantly increased CECs and EMPs, corroborating the in vitro findings. CONCLUSIONS We provide novel data that CRP, via NO deficiency, promotes endothelial dysfunction by inducing release of CECs and EMPs, which are biomarkers of endothelial dysfunction.

The Use of One-Bead One-Compound Combinatorial Library Technology to Discover High-Affinity αvβ3 Integrin and Cancer Targeting Arginine-Glycine-Aspartic Acid Ligands with a Built-in Handle

The αvβ3 integrin, expressed on the surface of various normal and cancer cells, is involved in numerous physiologic processes such as angiogenesis, apoptosis, and bone resorption. Because this integrin plays a key role in angiogenesis and metastasis of human tumors, αvβ3 integrin ligands are of great interest to advances in targeted therapy and cancer imaging. In this report, one-bead one-compound (OBOC) combinatorial libraries containing the arginine-glycine-aspartic acid (RGD) motif were designed and screened against K562 myeloid leukemia cells that had been transfected with the human αvβ3 integrin gene. Cyclic peptide LXW7 was identified as a leading ligand with a built-in handle that binds specifically to αvβ3 and showed comparable binding affinity (IC50 = 0.68 ± 0.08 μmol/L) to some of the well-known RGD “head-to-tail” cyclic pentapeptide ligands reported in the literature. The biotinylated form of LXW7 ligand showed similar binding strength as LXW7 against αvβ3 integrin, whereas biotinylated RGD cyclopentapeptide ligands revealed a 2- to 8-fold weaker binding affinity than their free forms. LXW7 was able to bind to both U-87MG glioblastoma and A375M melanoma cell lines, both of which express high levels of αvβ3 integrin. In vivo and ex vivo optical imaging studies with the biotinylated ligand/streptavidin-Cy5.5 complex in nude mice bearing U-87MG or A375M xenografts revealed preferential uptake of biotinylated LXW7 in tumor. When compared with biotinylated RGD cyclopentapeptide ligands, biotinylated LXW7 showed higher tumor uptake but lower liver uptake. Mol Cancer Ther; 9(10); 2714–23. ©2010 AACR.

Genetic Engineering of T Cells to Target HERV-K, an Ancient Retrovirus on Melanoma.

Purpose: The human endogenous retrovirus (HERV-K) envelope (env) protein is a tumor-associated antigen (TAA) expressed on melanoma but not normal cells. This study was designed to engineer a chimeric antigen receptor (CAR) on T-cell surface, such that they target tumors in advanced stages of melanoma. Experimental Design: Expression of HERV-K protein was analyzed in 220 melanoma samples (with various stages of disease) and 139 normal organ donor tissues using immunohistochemical (IHC) analysis. HERV-K env–specific CAR derived from mouse monoclonal antibody was introduced into T cells using the transposon-based Sleeping Beauty (SB) system. HERV-K env–specific CAR+ T cells were expanded ex vivo on activating and propagating cells (AaPC) and characterized for CAR expression and specificity. This includes evaluating the HERV-K–specific CAR+ T cells for their ability to kill A375-SM metastasized tumors in a mouse xenograft model. Results: We detected HERV-K env protein on melanoma but not in normal tissues. After electroporation of T cells and selection on HERV-K+ AaPC, more than 95% of genetically modified T cells expressed the CAR with an effector memory phenotype and lysed HERV-K env+ tumor targets in an antigen-specific manner. Even though there is apparent shedding of this TAA from tumor cells that can be recognized by HERV-K env–specific CAR+ T cells, we observed a significant antitumor effect. Conclusions: Adoptive cellular immunotherapy with HERV-K env–specific CAR+ T cells represents a clinically appealing treatment strategy for advanced-stage melanoma and provides an approach for targeting this TAA on other solid tumors. Clin Cancer Res; 21(14); 3241–51. ©2015 AACR.

Rapid discovery of death ligands with one-bead-two-compound combinatorial library methods

The one-bead-one-compound (OBOC) technology enables one to generate thousands to millions of chemical molecules on resin beads (90 μm diameter) such that each bead displays 10(13) copies of the same chemical entity. Whole-cell binding assays have been developed to screen OBOC combinatorial libraries for ligands that bind to specific cell surface receptors. While very powerful, this screening method does not address the downstream cell signaling properties of the binding ligand. We have modified the OBOC technology by introducing a fixed known cell adhesion ligand to the outer layer of each bead. This one-bead-two-compound (OB2C) library configuration allows the bound cells to interact with the random immobilized chemical molecules on each bead. The bound cells can then be probed for specific cellular responses such as apoptosis and activation or inhibition of a specific cell signaling pathway. To validate this concept, an OB2C combinatorial library was created such that a random hexapeptide plus a high affinity lymphoma targeting ligand LLP2A were displayed on each bead. This LLP2A-X(6) OB2C library was then screened with human T-cell leukemia cells (Molt-4) for cell death responses. After 5 days of incubation, propidium iodide was added to the bead library to stain dead cells. Beads coated by red fluorescent cells were isolated for sequence analysis. Two ligands identified by this method, when added to the lymphoid cancer cells, were able to induce cell death.

Rainbow beads: A color coding method to facilitate high-throughput screening and optimization of one-bead one-compound combinatorial libraries

We have developed a new color-encoding method that facilitates high-throughput screening of one-bead one-compound (OBOC) combinatorial libraries. Polymer beads displaying chemical compounds or families of compounds are stained with oil-based organic dyes that are used as coding tags. The color dyes do not affect cell binding to the compounds displayed on the surface of the beads. We have applied such rainbow beads in a multiplex manner to discover and profile ligands against cell surface receptors. In the first application, a series of OBOC libraries with different scaffolds or motifs are each color-coded; small samples of each library are then combined and screened concurrently against live cells for cell attachment. Preferred libraries can be rapidly identified and selected for subsequent large-scale screenings for cell surface binding ligands. In a second application, beads with a series of peptide analogues (e.g., alanine scan) are color-coded, combined, and tested for binding against a specific cell line in a single-tissue culture well; the critical residues required for binding can be easily determined. In a third application, ligands reacting against a series of integrins are color-coded and used as a readily applied research tool to determine the integrin profile of any cell type. One major advantage of this straightforward and yet powerful method is that only an ordinary inverted microscope is needed for the analysis, instead of sophisticated (and expensive) fluorescent microscopes or flow cytometers.

Targeting Btk/Etk of prostate cancer cells by a novel dual inhibitor

Btk and Etk/BMX are Tec-family non-receptor tyrosine kinases. Btk has previously been reported to be expressed primarily in B cells and has an important role in immune responses and B-cell malignancies. Etk has been shown previously to provide a strong survival and metastasis signal in human prostate cancer cells, and to confer androgen independence and drug resistance. While the role of Etk in prostate carcinogenesis is well established, the functions of Btk in prostate cancer have never been investigated, likely due to the perception that Btk is a hematopoietic, but not epithelial, kinase. Herein, we found that Btk is overexpressed in prostate cancer tissues and prostate cancer cells. The level of Btk in prostate cancer tissues correlates with cancer grades. Knockdown of Btk expression selectively inhibits the growth of prostate cancer cells, but not that of the normal prostate epithelial cells, which express very little Btk. Dual inhibition of Btk and Etk has an additive inhibitory effect on prostate cancer cell growth. To explore Btk and Etk as targets for prostate cancer, we developed a small molecule dual inhibitor of Btk and Etk, CTN06. Treatment of PC3 and other prostate cancer cells, but not immortalized prostate epithelial cells with CTN06 resulted in effective cell killing, accompanied by the attenuation of Btk/Etk signals. The killing effect of CTN06 is more potent than that of commonly used inhibitors against Src, Raf/VEGFR and EGFR. CTN06 induces apoptosis as well as autophagy in human prostate cancer cells, and is a chemo-sensitizer for docetaxel (DTX), a standard of care for metastatic prostate cancer patients. CTN06 also impeded the migration of human prostate cancer cells based on a ‘wound healing’ assay. The anti-cancer effect of CTN06 was further validated in vivo in a PC3 xenograft mouse model.

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-γ). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.