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Adoptive T cell therapy with T cells expressing affinity-enhanced TCRs has shown promising results in phase 1/2 clinical trials for solid and hematological tumors. However, depth and durability of responses to adoptive T cell therapy can suffer from an inhibitory tumor microenvironment. A common immune-suppressive agent is TGF-β, which is secreted by tumor cells and cells recruited to the tumor. We investigated whether human T cells could be engineered to be resistant to inhibition by TGF-β. Truncating the intracellular signaling domain from TGF-β receptor (TGFβR) II produces a dominant-negative receptor (dnTGFβRII) that dimerizes with endogenous TGFβRI to form a receptor that can bind TGF-β but cannot signal. We previously generated specific peptide enhanced affinity receptor TCRs recognizing the HLA-A*02–restricted peptides New York esophageal squamous cell carcinoma 1 (NY-ESO-1)157–165/l-Ag family member-1A (TCR: GSK3377794, formerly NY-ESO-1c259) and melanoma Ag gene A10254–262 (TCR: ADP-A2M10, formerly melanoma Ag gene A10c796). In this article, we show that exogenous TGF-β inhibited in vitro proliferation and effector functions of human T cells expressing these first-generation high-affinity TCRs, whereas inhibition was reduced or abolished in the case of second-generation TCRs coexpressed with dnTGFβRII (e.g., GSK3845097). TGF-β isoforms and a panel of TGF-β–associated genes are overexpressed in a range of cancer indications in which NY-ESO-1 is commonly expressed, particularly in synovial sarcoma. As an example, immunohistochemistry/RNAscope identified TGF-β–positive cells close to T cells in tumor nests and stroma, which had low frequencies of cells expressing IFN-γ in a non–small cell lung cancer setting. Coexpression of dnTGFβRII may therefore improve the efficacy of TCR-transduced T cells.

Patients with hepatocellular carcinoma (HCC) have a poor prognosis and limited therapeutic options. Alpha-fetoprotein (AFP) is often expressed at high levels in HCC and is an established clinical biomarker of the disease. Expression of AFP in nonmalignant liver can occur, particularly in a subset of progenitor cells and during chronic inflammation, at levels typically lower than in HCC. This cancer-specific overexpression indicates that AFP may be a promising target for immunotherapy. We verified expression of AFP in normal and diseased tissue and generated an affinity-optimized T-cell receptor (TCR) with specificity to AFP/HLA-A*02+ tumors. Expression of AFP was investigated using database searches, by qPCR, and by immunohistochemistry (IHC) analysis of a panel of human tissue samples, including normal, diseased, and malignant liver. Using in vitro mutagenesis and screening, we generated a TCR that recognizes the HLA-A*02-restricted AFP158-166 peptide, FMNKFIYEI, with an optimum balance of potency and specificity. These properties were confirmed by an extension of the alanine scan (X-scan) and testing TCR-transduced T cells against normal and tumor cells covering a variety of tissues, cell types, and human leukocyte antigen (HLA) alleles. Conclusion: We have used a combination of physicochemical, in silico, and cell biology methods for optimizing a TCR for improved affinity and function, with properties that are expected to allow TCR-transduced T cells to differentiate between antigen levels on nonmalignant and cancer cells. T cells transduced with this TCR constitute the basis for a trial of HCC adoptive T-cell immunotherapy.

The antiviral effects of hepatitis C virus (HCV)-specific CD8 T cells have been shown in an HCV replicon system but not in an authentic infectious HCV cell culture (HCVcc) system. Here, we developed tools to examine the antigenicity of HCV-infected HLA-A2-positive Huh7.5 hepatoma cells (Huh7.5A2 cells) in activating HCV-specific CD8 T cells and the downstream antiviral effects. Infectious HCV epitope mutants encoding the well-defined genotype 1a-derived HLA-A2-restricted HCV NS3-1073 or NS5-2594 epitope were generated from a genotype 2a-derived HCV clone (Jc1Gluc2A) by site-directed mutagenesis. CD8 T-cell lines specific for NS3-1073 and NS5-2594 were expanded from HCV-seropositive persons by peptide stimulation in vitro or engineered from HCV-seronegative donor T cells by transduction of a lentiviral vector expressing HCV-specific T-cell receptors. HCV-specific CD8 T cells were cocultured with Huh7.5 cells that were pulsed with titrating doses of HCV epitope peptides or infected with HCV epitope mutants. HCV-specific CD8 T-cell activation (CD107a, gamma interferon, macrophage inflammatory protein 1β, tumor necrosis factor alpha) was dependent on the peptide concentrations and the relative percentages of HCV-infected Huh7.5A2 cells. HCV-infected Huh7.5A2 cells activated HCV-specific CD8 T cells at levels comparable to those achieved with 0.1 to 2 μM pulsed peptides, providing a novel estimate of the level at which endogenously processed HCV epitopes are presented on HCV-infected cells. While HCV-specific CD8 T-cell activation with cytolytic and antiviral effects was blunted by PD-L1 expression on HCV-infected Huh7.5A2 cells, resulting in the improved viability of Huh7.5A2 cells, PD-1 blockade reversed this effect, producing enhanced cytolytic elimination of HCV-infected Huh7.5A2 cells. Our findings, obtained using an infectious HCVcc system, show that the HCV-specific CD8 T-cell function is modulated by antigen expression levels, the percentage of HCV-infected cells, and the PD-1/PD-L1 pathways and has antiviral and cytotoxic effects. IMPORTANCE We developed several novel molecular and immunological tools to study the interactions among HCV, HCV-infected hepatocytes, and HCV-specific CD8 T cells. Using these tools, we show the level at which HCV-infected hepatoma cells present endogenously processed HCV epitopes to HCV-specific CD8 T cells with antiviral and cytotoxic effects. We also show the marked protective effect of PD-L1 expression on HCV-infected hepatoma cells against HCV-specific CD8 T cells.

Single and dual amino acid substitution variants were generated in the TCR CDRs of three TCRs that recognize tumor-associated Ags. Substitutions that enhance the reactivity of TCR gene-modified T cells to the cognate Ag complex were identified using a rapid RNA-based transfection system. The screening of a panel of variants of the 1G4 TCR, that recognizes a peptide corresponding to amino acid residues 157–165 of the human cancer testis Ag NY-ESO-1 (SLLMWITQC) in the context of the HLA-A*02 class I allele, resulted in the identification of single and dual CDR3α and CDR2β amino acid substitutions that dramatically enhanced the specific recognition of NY-ESO-1+/HLA-A*02+ tumor cell lines by TCR gene-modified CD4+ T cells. Within this group of improved TCRs, a dual substitution in the 1G4 TCR CDR3α chain was identified that enhanced Ag-specific reactivity in gene-modified CD4+ and CD8+ T cells. Separate experiments on two distinct TCRs that recognize the MART-1 27–35 (AAGIGILTV) peptide/HLA-A*02 Ag complex characterized single amino acid substitutions in both TCRs that enhanced CD4+ T cell Ag-specific reactivity. These results indicate that simple TCR substitution variants that enhance T cell function can be identified by rapid transfection and assay techniques, providing the means for generating potent Ag complex-specific TCR genes for use in the study of T cell interactions and in T cell adoptive immunotherapy.

Tumor-associated human telomerase reverse transcriptase (hTERT) is expressed in >85% of human tumors but not in most normal cells. As a result, this antigen has received considerable attention from those interested in cancer immunotherapy. Specifically, there has been strong interest in MHC class I–associated peptides derived from hTERT because these are expressed on the cell surface and thus may enable the targeting of tumor cells. Much of this interest has focused on peptide 540–548, ILAKFLHWL, which was predicted to exhibit the strongest binding to the common HLA A*0201 presenting molecule. The hTERT540–548 peptide is currently being assessed in therapeutic vaccination trials; however, there is controversy surrounding whether it is naturally processed and presented on the surface of neoplastic cells. Here, we generate two highly sensitive reagents to assess the presentation of hTERT540–548 on tumor cells: (a) a CD8+ CTL clone, and (b) a recombinant T-cell receptor (TCR) that binds with picomolar affinity and a half-life exceeding 14 h. This TCR enables the identification of individual HLA A2-hTERT540–548 complexes on the cell surface. The use of both this TCR and the highly antigen-sensitive CTL clone shows that the hTERT540–548 peptide cannot be detected on the surface of tumor cells, indicating that this peptide is not a naturally presented epitope. We propose that, in future, rigorous methods must be applied for the validation of peptide epitopes used for clinical applications. [Mol Cancer Ther 2007;6(7):2081–91]

Despite recent therapeutic advances, multiple myeloma (MM) remains largely incurable. Herein we report results of a phase I/II trial to evaluate the safety and activity of autologous T-cells engineered to express an affinity-enhanced T-cell receptor (TCR) recognizing a naturally processed peptide shared by the cancer-testis antigens NY-ESO-1 and LAGE-1. Twenty patients with antigen-positive MM received an average 2.4×109 engineered T cells two days after autologous stem cell transplant (ASCT). Infusions were well-tolerated without clinically apparent cytokine release syndrome, despite high IL-6 levels. Engineered T-cells expanded, persisted, trafficked to marrow and exhibited a cytotoxic phenotype. Persistence of engineered T cells in blood was inversely associated with NY-ESO-1 levels in the marrow. Disease progression was associated with loss of T cell persistence or antigen escape, consistent with the expected mechanism of action of the transferred T cells. Encouraging clinical responses were observed in 16 of 20 patients (80%) with advanced disease, with a median progression free survival of 19.1 months. NY-ESO-1/LAGE-1 TCR-engineered T-cells were safe, trafficked to marrow and showed extended persistence that correlated with clinical activity against antigen-positive myeloma.

Certain polysulphated polyanions have been shown to have prophylactic effects on the progression of transmissible spongiform encephalopathy disease, presumably because they bind to prion protein (PrP). Until now, the difficulty of obtaining large quantities of native PrP has precluded detailed studies of these interactions. We have over-expressed murine recombinant PrP (recPrP), lacking its glycophosphoinositol membrane anchor, in modified mammalian cells. Milligram quantities of secreted, soluble and partially glycosylated protein were purified under non-denaturing conditions and the identities of mature-length aglycosyl recPrP and two cleavage fragments were determined by electrospray MS. Binding was assessed by surface plasmon resonance techniques using both direct and competitive ligand-binding approaches. recPrP binding to immobilized polyanions was enhanced by divalent metal ions. Polyanion binding was strong and showed complex association and dissociation kinetics that were consistent with ligand-directed recPrP aggregation. The differences in the binding strengths of recPrP to pentosan polysulphate and to other sulphated polyanions were found to parallel their in vivo anti-scrapie and in vitro anti-scrapie-specific PrP formation potencies. When recPrP was immobilized by capture on metal-ion chelates it was found, contrary to expectation, that the addition of polyanions promoted the dissociation of the protein.

An obstacle to cancer immunotherapy has been that the affinity of T-cell receptors (TCRs) for antigens expressed in tumors is generally low. We initiated clinical testing of engineered T cells expressing an affinity-enhanced TCR against HLA-A*01–restricted MAGE-A3. Open-label protocols to test the TCRs for patients with myeloma and melanoma were initiated. The first two treated patients developed cardiogenic shock and died within a few days of T-cell infusion, events not predicted by preclinical studies of the high-affinity TCRs. Gross findings at autopsy revealed severe myocardial damage, and histopathological analysis revealed T-cell infiltration. No MAGE-A3 expression was detected in heart autopsy tissues. Robust proliferation of the engineered T cells in vivo was documented in both patients. A beating cardiomyocyte culture generated from induced pluripotent stem cells triggered T-cell killing, which was due to recognition of an unrelated peptide derived from the striated muscle-specific protein titin. These patients demonstrate that TCR-engineered T cells can have serious and not readily predictable off-target and organ-specific toxicities and highlight the need for improved methods to define the specificity of engineered TCRs.

MAGE A3, which belongs to the family of cancer-testis antigens, is an attractive target for adoptive therapy given its reactivation in various tumors and limited expression in normal tissues. We developed an affinity-enhanced T cell receptor (TCR) directed to a human leukocyte antigen (HLA)-A*01-restricted MAGE A3 antigen (EVDPIGHLY) for use in adoptive therapy. Extensive preclinical investigations revealed no off-target antigen recognition concerns; nonetheless, administration to patients of T cells expressing the affinity-enhanced MAGE A3 TCR resulted in a serious adverse event (SAE) and fatal toxicity against cardiac tissue. We present a description of the preclinical in vitro functional analysis of the MAGE A3 TCR, which failed to reveal any evidence of off-target activity, and a full analysis of the post-SAE in vitro investigations, which reveal cross-recognition of an off-target peptide. Using an amino acid scanning approach, a peptide from the muscle protein Titin (ESDPIVAQY) was identified as an alternative target for the MAGE A3 TCR and the most likely cause of in vivo toxicity. These results demonstrate that affinity-enhanced TCRs have considerable effector functions in vivo and highlight the potential safety concerns for TCR-engineered T cells. Strategies such as peptide scanning and the use of more complex cell cultures are recommended in preclinical studies to mitigate the risk of off-target toxicity in future clinical investigations.

We investigated whether TCRs restricted to the more ubiquitously expressed MHC class I molecules could be used to redirect human regulatory T cells (Tregs). Using a series of HLA-A2–restricted TCRs that recognize the same peptide-MHC class I complex (pMHC) with affinities varying up to 3500 fold, we observed that TCR affinity had no effect on the ability of the introduced TCRs to confer potent Ag-specific suppressive activity. Surprisingly, we found a naturally occurring, low-affinity MHC class I–restricted TCR specific for an NY-ESO-1 epitope that was unable to redirect a functional CD4 T-effector cell response could confer potent antigen-specific suppressive activity when expressed in Tregs and severely impair the expansion of highly functional HIV-1GAG–specific CD8 T cells expressing a high-affinity TCR. This suppressive activity was only observed when both Ags were presented by the same cell, and no suppression was observed when the target Ags were put in distinct cells. These studies underscore the clinical utility of using MHC class I–restricted TCRs to endow Tregs with specificity to control autoimmune disease and highlight the conditions in which this approach would have most therapeutic benefit.

Meeting abstracts Preclinical safety testing for adoptive immunotherapies utilising T cell receptor-(TCR) engineered T cells relies on stringent target validation and a robust pre-clinical specificity testing strategy. Specific safety concerns include on-target off-tumour toxicity and off-target

Previously, we have described the use of phage display to generate high affinity disulfide bond-linked T cell receptors (TCRs). The affinities of the mutant TCRs were analysed after refolding of separately expressed alpha and beta chains from Escherichia coli inclusion bodies. This approach is only suitable for the analysis of small numbers of TCR variants. An attractive alternative would be soluble expression within the bacterial periplasm, but the generic production of TCRs within the E. coli periplasm has so far not proved successful. Here we show that functional, soluble TCR can be produced within the cytoplasm of trxB gor mutant E. coli strains, with maximum yields of 3.4 mg/l. We also investigated the effect of coexpressing the folding modulators Skp and DsbC finding that the TCR expression levels were largely unaffected by these chaperones. Importantly, we demonstrated that the amount of protein purified from 50 ml starter cultures was sufficient to show functionality of the TCR by specific antigen binding in both ELISA and surface plasmon resonance (SPR) assays. This TCR production method has the potential to allow rapid and medium throughput analysis of affinity-matured TCRs selected from TCR phage display libraries.

Congenic mouse strains VM/Dk and VM-Sinc s7/Dk differ at the Sinc gene, which controls the incubation period of scrapie in mice; VM/Dk mice are Sinc p7p7 and VM-Sinc s7/Dk mice are Sinc s7s7. Restriction fragment length polymorphism and DNA sequencing analysis demonstrated that the PrP genes also differ in these strains, confirming the close genetic linkage of Sinc and PrP. Using the restriction enzyme HhaI, we have shown that at least 100 kb of DNA flanking the PrP gene differs between the two strains, and therefore the congruence of the Sinc and PrP genes is still not certain.

We examined the activity of human T cells engineered to express variants of a single TCR (1G4) specific for the cancer/testis Ag NY-ESO-1, generated by bacteriophage display with a wide range of affinities (from 4 μM to 26 pM). CD8+ T cells expressing intermediate- and high-affinity 1G4 TCR variants bound NY-ESO-1/HLA-A2 tetramers with high avidity and Ag specificity, but increased affinity was associated with a loss of target cell specificity of the TCR gene-modified cells. T cells expressing the highest affinity TCR (KD value of 26 pM) completely lost Ag specificity. The TCRs with affinities in the midrange, KD 5 and 85 nM, showed specificity only when CD8 was absent or blocked, while the variant TCRs with affinities in the intermediate range—with KD values of 450 nM and 4 μM—demonstrated Ag-specific recognition. Although the biological activity of these two relatively low-affinity TCRs was comparable to wild-type reactivity in CD8+ T cells, introduction of these TCR dramatically increased the reactivity of CD4+ T cells to tumor cell lines.

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Adoptive immunotherapy for cancer has been limited by a lack of antigen specificity, low levels of target expression, and failure to break self-tolerance. We hypothesized that infusion of genetically modified tumor-specific T cells following autologous stem cell transplant (ASCT) may overcome these barriers for multiple myeloma (MM). To test this, we conducted a phase I/II clinical trial ([NCT01352286][1]) in which T cells engineered with an HLA-A*0201 restricted, affinity-enhanced TCR recognizing NY-ESO-1 / LAGE-1 peptides (NY-ESOc259-T), were infused in the setting of profound lymphodepletion that accompanies high-dose chemotherapy given with ASCT. HLA-A*0201 MM patients eligible for ASCT, with antigen positive tumor were enrolled. NY-ESOc259-T was manufactured in a 10 day process using anti-CD3/CD28 microbeads and lentiviral vector, and was administered two days following ASCT. IMWG criteria were used to assess response at day 100 with the addition of a near complete response category (nCR) due to the common occurrence of oligoclonal banding observed following rapid post-ASCT immune reconstitution. Blood and marrow samples were taken at multiple timepoints for serum cytokine analysis, NY-ESOc259-T persistence and trafficking, multiparameter flow analysis to examine the phenotype and function of NY-ESOc259-T, and tumor biomarker analysis. 25 of 29 enrolled patients were infused. A mean of 2.8 × 109 engineered cells were administered (range 8.3 × 108-4.2 × 109), and the average transduction efficiency was 33% (range 30%-45%). Patients tended to have advanced disease (64% chromosomal abnormalities, and 24% prior ASCT). At 3 months, 67% (16/24) and 58% (14/24) of patients were in VGPR and nCR or better, respectively. Infusions were well-tolerated and no cytokine release syndrome was reported. NY-ESOc259-T persisted at 6 months in all but one patient, and in a subset of patients at 2 years; marrow infiltration was consistently observed from day 7 through day 180. NY-ESOc259-T initially displayed a dominant activated effector phenotype which converted towards a dominant effector memory phenotype by 1 year post infusion, in a pattern that mirrored clinical responses. Persisting cells demonstrated a polyfunctional response (IFN-γ and TNF-α) with a cytotoxic (CD107a and granzyme B) signature without overexpression of exhaustion markers (PD-1, LAG-3, and TIM-3). Tumor biomarker analysis is ongoing. MM relapse occurred in 13/25 patients. This data show that NY-ESOc259-T cells exhibit robust trafficking and expansion, durable persistence without exhaustion, and follow a natural immune expansion and contraction pattern consistent with an antigen-driven mechanism of action. Relapse correlated with a loss of persistence or tumor antigen escape, suggesting that targeting multiple antigens and maintenance infusions may increase durable remissions. Citation Format: Aaron Rapoport, Edward Stadtmauer, Luca Melchiori, Ryan Wong, Eduardo Davila, Gwendolyn Binder-Scholl, Tom Holdich, Dan Vogl, Brendan Weiss, Jeffrey Finkelstein, Simon Lacey, Sarah Bond, Marylene Fortin, Yoav Peretz, Joanna Brewer, Alan Bennett, Andrew Gerry, Nick Pumphrey, Helen Tayton-Martin, Lilliam Ribeiro, Ashraf Badros, Saul Yanovich, Nancy Hardy, Jean Yared, Naseem Kerr, Sunita Philip, Sandra Wesphal, Bruce L. Levine, Carl June, Michael Kalos, Bent Jakobsen. NY-ESO T cells administered post ASCT for MM exhibit extended functionality without exhaustion in a natural pattern of effector and memory programming. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4701. doi:10.1158/1538-7445.AM2015-4701 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01352286&atom=%2Fcanres%2F75%2F15_Supplement%2F4701.atom

Engineering T cells with enhanced affinity T cell receptors (TCRs) is a promising therapeutic strategy for treating cancer. Engineered TCRs overcome the limitations of naturally occurring cancer-antigen specific TCRs which are typically of extremely low affinity and fail to efficiently recognize tumor cells. However, the use TCRs with enhanced affinity for adoptive therapy carries with it the risk that they might recognize normal tissue, either as on-target or off-target reactivity. Two fatalities in a clinical trial of an affinity-enhanced MAGE-A3-specific TCR have highlighted the need for more biologically relevant cellular testing systems to minimize cross-reactivity-related toxicity in future adoptive immunotherapy trials. Cellular Dynamics International Inc. supply a range of iCell products that are highly pure induced pluripotent stem (iPS) cell-derived cells that reproduce normal human biology in an in vitro setting. The advantage of iCell cultures over standard cultures of primary cells is that they better represent human organs with respect to their antigen presentation by HLA. In this study, a panel of iCell sub-types, representing major organs of the human body, were used to test the reactivity of T cells transduced with an enhanced affinity TCR for HLA-A2-restricted peptide antigen NY-ESO-1/LAGE-1157-165. IncuCyte FLR technology, which enables direct visualization of caspase-3/7-dependent apoptosis at 37°C in real time, was used to assess the killing of the iCells by NY-ESO TCR-transduced T cells over a 4 day time course. While potent killing by the NY-ESO TCR-transduced T cells was observed against an HLA-A2+, NY-ESO-1+/LAGE-1+ tumor cell line, A375, no reactivity was observed against any of the tested iCells sub-types unless the iCells were pulsed with 1 µM NY-ESO peptide. The screening of TCR-transduced T cells against biologically appropriate cell cultures, such as iCells, further advances preclinical safety testing of new TCRs for use in adoptive immunotherapy. Citation Format: Katherine J. Adams, Andrew B. Gerry, Joanna E. Brewer, Nicholas J. Pumphrey, Alan D. Bennett, Bent K. Jakobsen. The use of induced pluripotent stem (iPS) cells for the safety testing of enhanced affinity TCR-transduced T cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2801. doi:10.1158/1538-7445.AM2014-2801

Recombinant aglycosyl murine prion protein (recPrP) has been over-expressed in a CHO cell line using the glutamine synthetase (GS) expression system. The recPrP lacks the glycophosphoinositol (GPI) anchor by which it is normally attached to the cell surface, and thus, the product is secreted into the medium. The aim of this work is to provide large quantities of mature length recPrP for structural and physicochemical studies. Currently, recPrP production is carried out in 500 mL spinner flask cultures, in which the cells are attached to Cytodex™ microcarriers. As serum interferes with the detection and purification of recPrP, our present production process includes a cell growth phase in medium containing 10% (v/v) dialysed foetal calf serum (dFCS), followed by a production phase in serum-free medium. These cultures yield up to 3 mg L-1 recPrP prior to purification. The concentration of mature length recPrP (23 kDa) usually increases over the first three days of the serum-free production phase, but then declines shortly after peak titres are attained. This loss of product in CHO cell cultures has been associated with the specific cleavage of the mature length prion protein into two fragments of 14 kDa and 9 kDa, not seen with bacterial (E. coli) host expression systems. Site specific cleavage not only compromises the productivity of the CHO cell system, but presents an obstacle in the preparation of homogeneous, mature length protein for x-ray crystallography studies. To date, attempts to control this problem with protease inhibitors have met with limited success. In this paper we provide evidence that site-specific cleavage of recPrP is a cell-associated event and describe a semi-continuous harvesting and medium replenishment approach to prevent accumulation of cleavage products in production cultures.

Background Despite recent therapeutic advances, multiple myeloma (MM) remains primarily an incurable cancer. Patients experiencing rapid recovery of T cells post autologous stem cell transplant (auto-SCT) may have improved outcomes, and spontaneous cellular responses to tumor can occur, suggesting immune mediated control of tumor is possible. We and others have investigated therapeutic cancer vaccines that have shown promise in pilot studies, in particular following post-transplant infusion of activated autologous T cells. However, efficacy of these approaches may be limited by thymic selection which restricts the repertoire of T cell receptors (TCRs) to low affinity TCRs that cannot recognize the low level of antigen present on most tumor cells. We hypothesized that incorporation of affinity-enhanced tumor antigen-specific TCRs into autologous T cells infused post-transplant would overcome this limitation and improve response rates in the post auto-SCT setting. Methods We report interim results of a Phase II clinical trial (NCT01352286) to evaluate the safety and activity of autologous T cells genetically engineered to express an affinity-enhanced TCR that recognizes the NY-ESO-1/LAGE-1 peptide complex HLA‐A*0201‐SLLMWITQC; these cells are infused in the setting of profound lymphodepletion that accompanies high dose chemotherapy administered during auto-SCT. Patients with high risk or relapsed MM, who are HLA‐A*0201 positive, and whose tumor is positive for NY-ESO-1 and/or LAGE-1 by RT-PCR are eligible. CD25 depleted CD4 and CD8 T cells are activated and expanded using anti-CD3/CD28 antibody conjugated microbeads, and genetically modified with a lentiviral vector containing the TCR construct at a multiplicity of infection of 1. Engineered T cells are administered four days after high dose melphalan and two days following auto-SCT, at a dose range of 1-10 billion total cells with a minimum gene modification requirement of 10%. Patients are evaluated for MM responses in accordance with the IMWG criteria at 6 weeks, and 3 and 6 months post infusion. At 3 months, patients start lenalidomide maintenance. The initial 6 patient phase is complete and a 20 patient extension phase is ongoing. Results Prior to enrollment on study, patients had received a median of 3 prior therapies including 6 with prior transplant. 50% of tumors contained high risk chromosomal abnormalities, and NY-ESO-1 expression is correlated with adverse prognosis. 20 patients (average age of 57) have been infused with an average of 2.3 X 109 engineered T cells (range 4.5 X 108-3.9 X 109); this reflects an average clinical scale transduction efficiency of 34% (range 18% – 49%). Infusions have been well tolerated, and the majority of adverse events were related to the high dose melphalan. Possibly related SAEs were neutropenia and thrombocytopenia, and GI and metabolism disorders including diarrhea, colitis, hyponatremia and hypomagnesemia. 10, 4, 2, and 2 patients have reached the 1 year, 9 month, 6 month and 3 month assessment timepoints, respectively, and 17/20 patients are alive. Best response by day 100 is sCR/CR in 2/15 (13%), nCR in 10/15 (67%), and PR in 3/15% (20%), which compares favorably to historic responses in patients undergoing first or second transplant. Engineered T cells expanded and persisted in blood and marrow at 180 days by Q-PCR and flow-cytometry in all but one case (Figure). 7 patients progressed after day 100, which was accompanied either by loss of engineered T cells or loss of tumor antigen. Detailed phenotyping and functional analysis of engineered T cells, and correlates with clinical responses, is underway. Summary This is the first clinical evaluation of engineered T cells in the MM setting. Infusions are safe, well tolerated, and are associated with encouraging responses in a high risk myeloma population. A study evaluating the engineered T cells in a non-transplant study is underway. Disclosures: Stadtmauer: Celgene: Consultancy. Binder-Scholl:Adaptimmune: Employment. Smethurst:Adaptimmune: Employment. Brewer:Adaptimmune: Employment. Bennett:Adaptimmune: Employment. Gerry:Adaptimmune: Employment. Pumphrey:Adaptimmune: Employment. Tayton-Martin:Adaptimmune: Employment. Ribeiro:Adaptimmune: Employment. Levine:Novartis: cell and gene therapy IP, cell and gene therapy IP Patents & Royalties. Jakobsen:Adaptimmune: Employment. Kalos:Novartis corporation: CART19 technology, CART19 technology Patents & Royalties; Adaptive biotechnologies: Member scientific advisory board , Member scientific advisory board Other. June:Novartis: Patents & Royalties, Research Funding.

The transmissible spongiform encephalopathies are characterized by conversion of a host protein, PrPC (cellular prion protein), to a protease-resistant isoform, PrPSc (prion protein scrapie isoform). The importance of the highly-flexible, N-terminal region of PrP has recently become more widely appreciated, particularly the biological activities associated with its metal ion binding domain and its potential to form a poly(L-proline) II (PPII) helix. Circular dichroism spectroscopy of an N-terminal peptide, PrP37-53, showed that the PPII helix is formed in aqueous buffer; as it also contains a Xaa-Pro-Gly consensus sequence, it may act as a substrate for the collagen modifying enzyme prolyl 4-hydroxylase. Direct evidence for this modifi-cation was obtained by mass spectrometry and Edman sequencing in recombinant mouse PrP secreted from stably transfected Chinese hamster ovary cells. Almost complete conversion of proline to 4-hydroxyproline occurs specifically at residue Pro44 of this murine protein; the same hydroxylated residue was detected, at lower levels, in PrPSc from the brains of scrapie infected mice. Cation binding and/or post-translational hydroxylation of this region of PrP may regulate its role in the physiology and pathobiology of the cell.

Advanced atherosclerotic lesions are characterized by lipid accumulation, chronic inflammation, and defective efferocytosis; an ideal therapy should address all aspects of this multi-factorial disease. Ixmyelocel-T, an expanded autologous multicellular therapy showing clinical promise in the treatment of severe diseases associated with atherosclerosis, includes a novel population of M2-like macrophages. We examined the properties of these macrophages to determine their potential mechanisms of action in atherosclerotic states. Flow cytometry revealed that ixmyelocel-T macrophages express surface receptors of alternative activation (CD206 and CD163) and scavenger receptors involved in lipid and apoptotic cell uptake (CD36, LDLR, CD91, MSR1, and MerTK). ELISA analysis revealed ixmyelocel-T macrophages secrete substantial levels of IL-10 (1060 278 vs. 1978 313 pg/ml, p < 0.05) and IL-1ra (12673 1242 vs. 37576 3650 pg/ml, p < 0.001) before and after overnight LPS stimulation. Baseline secretion of the pro-inflammatory cytokines IL-1b, TNFa, and IL-12 is minimal and remains low after LPS stimulation. Fluorescent microscopy and flow cytometry revealed that ixmyelocel-T readily phagocytosed apoptotic cells. Apoptosis analyzed by a caspase 3/7 assay demonstrated that ixmyelocel-T isn’t sensitive to the lipotoxic effects of modified cholesterol (87 10.78 vs. 100 14.58% of control). Real time PCR revealed that the cholesterol transporter genes ABAC1 (1.49 0.32 vs. 0.42 0.11 fold change, p < 0.01) and ABCG1 (0.41 0.13 vs. 0.07 0.03 fold change, p < 0.05) are both significantly upregulated when ixmyelocel-T macrophages are loaded with cholesterol. Ixmyelocel-T also exhibits enhanced ApoAI mediated cholesterol efflux (13.91 1.90 vs. 3.42 0.31%, p < 0.001 compared to THP-1). In addition, ixmyelocel-T effluxed cholesterol in vivo in using a mouse model for reverse cholesterol transport. These biological properties of ixmyelocel-T macrophages suggest that this cellular therapy may exert beneficial effects on atherosclerotic disease by providing macrophages that potentially limit atheroma development through secretion of anti-inflammatory cytokines, removal of apoptotic cells, and promotion of cholesterol efflux.

Background: Adoptive immunotherapy for cancer has been limited by lack of antigen specificity, low levels of target expression, and failure to break self-tolerance. We are conducting an early phase clinical trial (NCT01352286) attempting to overcome these barriers using T cells engineered with an HLA-A0201 restricted, affinity-enhanced TCR that recognizes an epitope expressed by the NY-ESO-1 and LAGE-1 cancer testis antigens; these cells are infused in the setting of profound lymphodepletion that accompanies high-dose chemotherapy with autologous stem cell transplant (aSCT) for patients with high risk or relapsed multiple myeloma (MM). Methods: Inclusion criteria include: 1) eligibility for aSCT, 2) PS of 0-2, 3) high risk MM or relapse after prior therapy, 4) HLA-A0201 positive, and 5) NY-ESO-1 and/or LAGE-1 positive tumor by PCR. CD25 depleted T cells are activated and expanded using anti-CD3/28 antibody conjugated microbeads, and genetically modified with a lentiviral vector. T cells are administered four days after high dose melphalan and two days following auto-SCT. Patients are evaluated for MM responses in accordance with the IMWG criteria at 6 weeks, and 3 and 6 months. At 3 months, patients with adequate marrow function start lenalidomide maintenance. Blood and marrow are monitored for persistence of engineered cells by qPCR and by surface expression of the NY-ESO-1 / LAGE-1 TCR using dextramerTM reagents. NY-ESO-1 and LAGE-1 antigen expression in marrow was assessed by qRT-PCR at baseline and post infusion. Results: As of November 2012, 21 patients have been enrolled, 15 have been infused; 4 were taken off study prior to infusion due to disease progression. An average of 2.7 x 109 engineered T cells were administered per patient (range 8.3 x 108-4.2 x 109), and the average transduction efficiency was 33% (range 30%-45%). More than 50% (8/15) of patients have high risk chromosomal abnormalities, and 3 (20%) have received prior aSCT. At 3 months post aSCT, 73% of patients were in a very good partial response (VGPR) or better. Gastrointestinal toxicity resulting from autologous GVHD (aGVHD) occurred in a subset of patients at a higher rate than reported following aSCT alone or aSCT and T cell infusion, and was resolved in all cases. Infused T cells typically showed peak expansion in blood at day 14, followed by durable persistence in blood and marrow at 6-12 months in all but one patient. Disease progression is typically accompanied by very low levels or loss of engineered T cell persistence or loss of target antigen on tumor. Conclusions: We report for the first time that possible correlates of clinical response in this study include persistence of engineered T cells and loss of antigen, suggesting specific activity of the infused cells. Infusions are well tolerated with a possible risk of manageable aGVHD. Citation Format: Aaron P. Rapoport, Edward A. Stadtmauer, Dan T. Vogl, Brendan Weiss, Gwendolyn K. Binder-Scholl, Dominic P. Smethurst, Jeffrey Finkelstein, Irina Kulikovskaya, Minnal Gupta, Erica Suppa, Tatiana Mikheeva, Joanna E. Brewer, Alan D. Bennett, Andrew B. Gerry, Nick J. Pumphrey, Helen K. Tayton-Martin, Lilliam Ribeiro, Elizabeth Veloso, Zhaohui Zheng, Ashraf Z. Bados, Saul Yanovich, Gorgun Akpek, Karen Dengel, Naseem Kerr, Sunita Philip, Kelly-Marie Betts, Sandra Westphal, Bruce L. Levine, Bent K. Jakobsen, Carl H. June, Michael Kalos. Correlates of clinical response following autologous stem cell transplant and adoptive immunotherapy with engineered T cells expressing an affinity-enhanced T cell receptor in patients with mutliple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4575. doi:10.1158/1538-7445.AM2013-4575

Abstract 755 Background: Adoptive transfer of T cells genetically engineered to express affinity-enhanced T cell receptors (TcRs) specific for self-antigens is one approach to generate potent anti-tumor immunity, and this approach is being evaluated in a number of clinical trials. Although persistence of engineered T cells is positively correlated with response rates, a recurring observation to-date has been poor T cell persistence beyond the initial treatment window. We have recently initiated a Phase II clinical trial (NCT01352286) in patients with recurrent multiple myeloma (MM), to evaluate the safety and activity of autologous T cells genetically engineered to express an HLA-A201 restricted, affinity-enhanced TCR that targets an epitope shared by the NY-ESO-1 and LAGE-1 cancer testis antigens commonly expressed by MM. Clinical study details and outcomes are reported elsewhere in this meeting (Rapoport, A, Stadtmauer, E. et al.). Here we report the in vivo expansion, bioactivity, trafficking, durable persistence, and anti-tumor activity of TcR modified cells in this trial. Methods: Subjects received high-dose melphalan, infusion of autologous hematopoietic stem cells on day 0, and infusion of gene engineered cells (GEC) on day +2. Persistence of GEC was assessed by quantitative ABI-Taqman based PCR and a qualified assay that detects the lentivirus backbone sequence. Surface expression of the introduced affinity-enhanced TcR α/β receptor was detected by multiparametric flow cytometry and TcR-specific dextramer™reagents. NY-ESO-1 and LAGE-1 antigen expression in marrow samples was assessed by quantitative ABI-taqman based PCR and inventoried primer/probe sets. Results: As of August 2012, we have screened marrow from 46 patients for NY-ESO- and LAGE-1 expression. Of these, 29 (63%) and 17 (37%) expressed LAGE-1 and NY-ESO-1, respectively by PCR. LAGE-1 co-expression was observed in all cases with NY-ESO-1. To date 12 patients have been infused with GEC. Robust engraftment was observed, with peak levels of gene-marked cells in peripheral blood and marrow ranging from 1 × 102-1 × 103 cells/μL and peak levels detected within the first 21 days post infusion. By day +100 the absolute number of GEC showed patient-specific and variable contraction, with a subset of patients maintaining GEC at 1 × 102-1 × 103 cells/μL, and a second subset where GEC contracted to 1 × 101-1 × 102cells/μL. Trafficking to and persistence of GEC in marrow was observed. At both early (up to day +30) and, in a subset of patients, late timepoints (post day+42), circulating CD3+ T cells could be detected with surface expression of the affinity enhanced TcR. Engineered cells are detected in all patients at their latest timepoint by Q-PCR. To date we see no evidence for TCR mispairing with endogenous TCR. Tumor-antigen and tumor-specific targeting by GEC was evaluated using Q-PCR and quantifying transcript levels of NY-ESO-1, LAGE-1, and CD138 (as a plasma cell marker) in marrow biopsies at enrollment, and days +42, +100, and +180. In the initial cohort of 5 patients, selective reappearance of CD138 transcript is observed in 4 cases (2 sCR, 1 VGPR, 1 PR), with non-detectable or very weak expression of LAGE-1/NY-ESO-1 transcripts, suggesting specific targeting of tumor cells that express LAGE-1/NY-ESO-1. In the fifth patient, who has progressive disease and low engineered cell persistence, CD138 and LAGE-1/NY-ESO-1 transcripts are both present above pre-treatment levels. A second infusion was recently given to this patient. Summary: Our studies show for the first time that adoptive transfer of affinity-enhanced TCR engineered T cells in MM patients results in prolonged T cell persistence, homing to marrow, and anti-tumor activity. Disclosures: Binder-Scholl: Adaptimmune: Employment. Smethurst:Adaptimmune Ltd: Employment. Brewer:Adaptimmune Ltd: Employment. Bennett:Adaptimmune Ltd: Employment. Gerry:Adaptimmune Ltd: Employment. Pumphrey:Adaptimmune Ltd: Employment. Tayton-Martin:Adaptimmune Ltd: Employment. Levine:TxCell: Consultancy, Membership on an entity's Board of Directors or advisory committees; University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight, financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties. Jakobsen:Adaptimmune Ltc: Employment. June:Novartis: Research Funding, entitled to receive royalties from patents licensed to Novartis, entitled to receive royalties from patents licensed to Novartis Patents & Royalties.

Abstract 472 Background: Despite recent therapeutic advances, multiple myeloma (MM) remains primarily an incurable cancer. Patients experiencing rapid recovery of T cells post auto-SCT may have improved outcomes, and spontaneous cellular responses to tumor can occur, suggesting immune mediated control of tumor is possible. We and others have investigated therapeutic cancer vaccines that have shown promise in pilot studies. However, vaccine efficacy may be limited by thymic selection which restricts the repertoire of T cell receptors (TCRs) to low affinity TCRs that cannot recognize the reduced level of antigen present on most tumor cells. We hypothesized that incorporation of affinity-enhanced tumor antigen specific TCRs into autologous T cells would overcome this limitation. Methods: We report interim results of a Phase II clinical trial (NCT01352286) to evaluate the safety and activity of autologous T cells genetically engineered to express an HLA-A201 restricted, affinity-enhanced TCR targeting an epitope shared by the NY-ESO-1 and LAGE-1 tumor antigens (gene engineered cells, or GEC). Inclusion criteria include: 1) eligibility for auto-SCT, 2) PS of 0–2, 3) high risk MM or relapse after prior therapy (prior allo-SCT excluded), 4) HLA-A201 positive, and 5) NY-ESO-1 and/or LAGE-1 positive tumor. GEC are manufactured from monocyte and T regulatory cell depleted apheresis, followed by lentivector gene transfer and T cell expansion by bead-based artificial antigen presenting cells. GEC are administered four days after high dose melphalan and two days following auto-SCT, at a dose range of 1–10 billion cells. Patients are evaluated for MM responses in accordance with the IMWG criteria at 6 weeks, and 3 and 6 months post infusion. At 3 months, patients start lenalidomide maintenance. The initial 6 patient phase is complete and a 20 patient extension phase is ongoing. Results: 19 patients have been enrolled, 12 have been infused, 3 are pending infusion, and 4 were taken off study prior to infusion due to disease progression. The average TCR transfer efficiency was 31.9% (range 18.2%-45%). 6, 4, and 1 patients have reached the 6 month, 3 month, and 6 week post infusion MM assessment time points, respectively. All treated patients are alive. 3/11 (27%) and 7/11 (64%) patients have a stringent CR or best response of VGPR or better, respectively; 3/11 (27%) have stable or partial responses, and 1/11 (9%) had progressive disease. In patients with VGPR, oligoclonal banding was common, marrow showed no expansion of clonal plasma cells, and all had normalized light chain ratios, suggesting at most minimal residual disease. These results are encouraging, especially considering that 3/11 patients had prior ASCT and 8/11 had adverse cytogenetics. GEC infusions have been well tolerated. The majority of adverse events are related to the high dose melphalan. Several patients had a transient skin rash with lymphocytosis and others had a diarrheal syndrome that occurred later than expected for melphalan-induced mucositis. In all cases GI toxicity has been self-limited or treatable with a short dose of corticosteroids. Possibly related SAEs were cytopenias such as neutropenia and thrombocytopenia, and GI and metabolism disorders such as diarrhea, colitis, hyponatremia and hypomagnesemia. GEC have been detected for up to 1 year at 1% in the circulation and bone marrow. Detailed correlative analysis is reported elsewhere in this meeting (Kalos, M. et al). Summary: This is the first test of GEC in the MM setting. Infusion of GEC administered post auto-SCT is safe, well tolerated, and leads to high response rate in a high risk myeloma population. Phase II accrual continues and a study evaluating GEC outside of auto-SCT is planned. Disclosures: Stadtmauer: celgene: Consultancy; millenium: Consultancy. Binder-Scholl:Adaptimmune : Employment. Brewer:Adaptimmune Ltd: Employment. Bennett:Adaptimmune Ltd: Employment. Gerry:Adaptimmune Ltd: Employment. Pumphrey:Adaptimmune Ltd: Employment. Smethurst:Adaptimmune Ltd: Employment. Tayton-Martin:Adaptimmune Ltd: Employment. Lilliam:Adaptimmune: Employment. Levine:TxCell: Consultancy, Membership on an entity's Board of Directors or advisory committees; University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight, financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties. Jakobsen:Adaptimmune Ltc: Employment. June:Novartis: Research Funding, entitled to receive royalties from patents licensed to Novartis, entitled to receive royalties from patents licensed to Novartis Patents & Royalties.

Futurists “wave a magic wand,” insinuating thereby that tabula rasa is a psychic and sociological option. “If there were no Bureau, what would the Jewish community do?” “If there were no Jewish community, what would the world's Jews do?” “If there were no world… ?” On the surface, it's an innocuous strategy: a way to probe assumptions, a way to hone a definition of needs. Examined closely, however, it is a flawed way to explore reality. At worst, it is a device to encourage a preconceived outcome.

Although prophecy belongs to fools and children, who can but respond when an editor calls? Yet, in responding, the wisdom of the warning should serve to remind us that events whose outcomes we cannot foresee will significantly affect what supplementary Jewish education will be like in the 90's; for example

1Read at Midwest Regional Meeting, The Religious Research Association, Minneapolis, Minn., March 2, 1962

We have previously demonstrated that an improved affinity TCR generated against NYESO-1 has shown remarkable efficacy in clinical studies in multiple myeloma (NCT01352286). The same TCR is also being tested in synovial sarcoma (NCT01343043), melanoma (NCT01350401) and ovarian (NCT01567891) clinical trials. We will extend these clinical studies to include hepatocellular carcinoma shortly. In this present study, we have generated a panel of improved-affinity TCRs for a widely expressed hepatic carcinoma-specific HLA-A2-restricted peptide antigen AFP158-166. This TCR panel demonstrates stepwise increases in affinity from wildtype to approximately 500-fold greater binding to cognate antigen. These affinity-enhanced TCRs were screened for efficacy and specificity by a variety of in vitro assays, such as interferon gamma ELISpot, Incucyte™ cytotoxicity assays for visualizing caspase 3/7 activity, Luminex™ multiple cytokine secretion assays and 3D microtissue models against a variety of hepatocellular carcinoma cell lines and normal primary cell types. This TCR panel demonstrated a window of activity whereby moderately increased affinity TCRs showed maximal response to antigen positive hepatocellular carcinoma cell lines HepG2, HuH6 and JHH5-A2, whilst showing no response to primary normal hepatocytes or primary cells from other organ systems. Greater increases in TCR affinity led to off-target activity, demonstrated as responses to primary normal target cells as well as antigen positive cell lines. This data has led to selection of a lead candidate for full pre-clinical assessment that demonstrates vastly improved efficacy against hepatocellular carcinoma cell lines, yet retains specificity by absence of response to primary normal tissue. This TCR may be suitable for further clinical studies in hepatocellular carcinoma.

Meeting abstracts We report on a 26 patient phase I/II clinical trial ([NCT01352286][1]) to investigate the safety, feasibility and anti-tumor activity of T cells engineered to express an affinity-enhanced TCR that recognizes the NY-ESO-1/LAGE-1 peptide complex HLA-A*0201-SLLMWITQC. Patients with