Engineering Cancer Antigen-Specific T Cells to Overcome the Immunosuppressive Effects of TGF-ß.

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-ß, 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-ß. Truncating the intracellular signaling domain from TGF-ß receptor (TGFßR) II produces a dominant-negative receptor (dnTGFßRII) that dimerizes with endogenous TGFßRI to form a receptor that can bind TGF-ß 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-ß 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 dnTGFßRII (e.g., GSK3845097). TGF-ß isoforms and a panel of TGF-ß-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-ß-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 dnTGFßRII may therefore improve the efficacy of TCR-transduced T cells.

Tuning T-Cell Receptor Affinity to Optimize Clinical Risk-Benefit When Targeting Alpha-Fetoprotein-Positive Liver Cancer.

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.

Histone Deacetylase Inhibitors Enhance CD4 T Cell Susceptibility to NK Cell Killing but Reduce NK Cell Function.

In the search for a cure for HIV-1 infection, histone deacetylase inhibitors (HDACi) are being investigated as activators of latently infected CD4 T cells to promote their targeting by cytotoxic T-lymphocytes (CTL). However, HDACi may also inhibit CTL function, suggesting different immunotherapy approaches may need to be explored. Here, we study the impact of different HDACi on both Natural Killer (NK) and CTL targeting of HIV-1 infected cells. We found HDACi down-regulated HLA class I expression independently of HIV-1 Nef which, without significantly compromising CTL function, led to enhanced targeting by NK cells. HDACi-treated HIV-1-infected CD4 T cells were also more effectively cleared than untreated controls during NK co-culture. However, HDACi impaired NK function, reducing degranulation and killing capacity. Depending on the HDACi and dose, this impairment could counteract the benefit gained by treating infected target cells. These data suggest that following HDACi-induced HLA class I down-regulation NK cells kill HIV-1-infected cells, although HDACi-mediated NK cell inhibition may negate this effect. Our data emphasize the importance of studying the effects of potential interventions on both targets and effectors.

Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma.

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.

Selection, engineering, and in vivo testing of a human leukocyte antigen-independent T-cell receptor recognizing human mesothelin.

BACKGROUND: Canonical α/ß T-cell receptors (TCRs) bind to human leukocyte antigen (HLA) displaying antigenic peptides to elicit T cell-mediated cytotoxicity. TCR-engineered T-cell immunotherapies targeting cancer-specific peptide-HLA complexes (pHLA) are generating exciting clinical responses, but owing to HLA restriction they are only able to target a subset of antigen-positive patients. More recently, evidence has been published indicating that naturally occurring α/ß TCRs can target cell surface proteins other than pHLA, which would address the challenges of HLA restriction. In this proof-of-concept study, we sought to identify and engineer so-called HLA-independent TCRs (HiTs) against the tumor-associated antigen mesothelin. METHODS: Using phage display, we identified a HiT that bound well to mesothelin, which when expressed in primary T cells, caused activation and cytotoxicity. We subsequently engineered this HiT to modulate the T-cell response to varying levels of mesothelin on the cell surface. RESULTS: The isolated HiT shows cytotoxic activity and demonstrates killing of both mesothelin-expressing cell lines and patient-derived xenograft models. Additionally, we demonstrated that HiT-transduced T cells do not require CD4 or CD8 co-receptors and, unlike a TCR fusion construct, are not inhibited by soluble mesothelin. Finally, we showed that HiT-transduced T cells are highly efficacious in vivo, completely eradicating xenografted human solid tumors. CONCLUSION: HiTs can be isolated from fully human TCR-displaying phage libraries against cell surface-expressed antigens. HiTs are able to fully activate primary T cells both in vivo and in vitro. HiTs may enable the efficacy seen with pHLA-targeting TCRs in solid tumors to be translated to cell surface antigens.

Enhancing Efficacy of TCR-engineered CD4 + T Cells Via Coexpression of CD8α.

Adoptive cell therapy with T cells expressing affinity-enhanced T-cell receptors (TCRs) is a promising treatment for solid tumors. Efforts are ongoing to further engineer these T cells to increase the depth and durability of clinical responses and broaden efficacy toward additional indications. In the present study, we investigated one such approach: T cells were transduced with a lentiviral vector to coexpress an affinity-enhanced HLA class I-restricted TCR directed against MAGE-A4 alongside a CD8α coreceptor. We hypothesized that this approach would enhance CD4 + T-cell helper and effector functions, possibly leading to a more potent antitumor response. Activation of transduced CD4 + T cells was measured by detecting CD40 ligand expression on the surface and cytokine and chemokine secretion from CD4 + T cells and dendritic cells cultured with melanoma-associated antigen A4 + tumor cells. In addition, T-cell cytotoxic activity against 3-dimensional tumor spheroids was measured. Our data demonstrated that CD4 + T cells coexpressing the TCR and CD8α coreceptor displayed enhanced responses, including CD40 ligand expression, interferon-gamma secretion, and cytotoxic activity, along with improved dendritic cell activation. Therefore, our study supports the addition of the CD8α coreceptor to HLA class I-restricted TCR-engineered T cells to enhance CD4 + T-cell functions, which may potentially improve the depth and durability of antitumor responses in patients.

Phase 1 Clinical Trial Evaluating the Safety and Anti-Tumor Activity of ADP-A2M10 SPEAR T-Cells in Patients With MAGE-A10+ Head and Neck, Melanoma, or Urothelial Tumors.

Background: ADP-A2M10 specific peptide enhanced affinity receptor (SPEAR) T-cells are genetically engineered autologous T-cells that express a high-affinity melanoma-associated antigen (MAGE)-A10-specific T-cell receptor (TCR) targeting MAGE-A10-positive tumors in the context of human leukocyte antigen (HLA)-A*02. ADP-0022-004 is a phase 1, dose-escalation trial to evaluate the safety and anti-tumor activity of ADP-A2M10 in three malignancies (https://clinicaltrials.gov: NCT02989064). Methods: Eligible patients were HLA-A*02 positive with advanced head and neck squamous cell carcinoma (HNSCC), melanoma, or urothelial carcinoma (UC) expressing MAGE-A10. Patients underwent apheresis; T-cells were isolated, transduced with a lentiviral vector containing the MAGE-A10 TCR, and expanded. Patients underwent lymphodepletion with fludarabine and cyclophosphamide prior to receiving ADP-A2M10. ADP-A2M10 was administered in two dose groups receiving 0.1×109 and >1.2 to 6×109 transduced cells, respectively, and an expansion group receiving 1.2 to 15×109 transduced cells. Results: Ten patients (eight male and two female) with HNSCC (four), melanoma (three), and UC (three) were treated. Three patients were treated in each of the two dose groups, and four patients were treated in the expansion group. The most frequently reported adverse events grade ≥3 were leukopenia (10), lymphopenia (10), neutropenia (10), anemia (nine), and thrombocytopenia (five). Two patients reported cytokine release syndrome (one each with grade 1 and grade 3), with resolution. Best response included stable disease in four patients, progressive disease in five patients, and not evaluable in one patient. ADP-A2M10 cells were detectable in peripheral blood from patients in each dose group and the expansion group and in tumor tissues from patients in the higher dose group and the expansion group. Peak persistence was greater in patients from the higher dose group and the expansion group compared with the lower dose group. Conclusions: ADP-A2M10 has shown an acceptable safety profile with no evidence of toxicity related to off-target binding or alloreactivity in these malignancies. Persistence of ADP-A2M10 in the peripheral blood and trafficking of ADP-A2M10 into the tumor was demonstrated. Because MAGE-A10 expression frequently overlaps with MAGE-A4 expression in tumors and responses were observed in the MAGE-A4 trial (NCT03132922), this clinical program closed, and trials with SPEAR T-cells targeting the MAGE-A4 antigen are ongoing.

Preclinical evaluation of an affinity-enhanced MAGE-A4-specific T-cell receptor for adoptive T-cell therapy.

A substantial obstacle to the success of adoptive T cell-based cancer immunotherapy is the sub-optimal affinity of T-cell receptors (TCRs) for most tumor antigens. Genetically engineered TCRs that have enhanced affinity for specific tumor peptide-MHC complexes may overcome this barrier. However, this enhancement risks increasing weak TCR cross-reactivity to other antigens expressed by normal tissues, potentially leading to clinical toxicities. To reduce the risk of such adverse clinical outcomes, we have developed an extensive preclinical testing strategy, involving potency testing using 2D and 3D human cell cultures and primary tumor material, and safety testing using human primary cell and cell-line cross-reactivity screening and molecular analysis to predict peptides recognized by the affinity-enhanced TCR. Here, we describe this strategy using a developmental T-cell therapy, ADP-A2M4, which recognizes the HLA-A2-restricted MAGE-A4 peptide GVYDGREHTV. ADP-A2M4 demonstrated potent anti-tumor activity in the absence of major off-target cross-reactivity against a range of human primary cells and cell lines. Identification and characterization of peptides recognized by the affinity-enhanced TCR also revealed no cross-reactivity. These studies demonstrated that this TCR is highly potent and without major safety concerns, and as a result, this TCR is now being investigated in two clinical trials (NCT03132922, NCT04044768).

Affinity-enhanced T-cell receptors for adoptive T-cell therapy targeting MAGE-A10: strategy for selection of an optimal candidate.

Circulating T-cells that have passed thymic selection generally bear T-cell receptors (TCRs) with sub-optimal affinity for cancer-associated antigens, resulting in a limited ability to detect and eliminate tumor cells. Engineering TCRs to increase their affinity for cancer targets is a promising strategy for generating T-cells with enhanced potency for adoptive immunotherapy in cancer patients. However, this manipulation also risks generating cross-reactivity to antigens expressed by normal tissue, with potentially serious consequences. Testing in animal models might not detect such cross-reactivity due to species differences in the antigenic repertoire. To mitigate the risk of off-target toxicities in future clinical trials, we therefore developed an extensive in vitro testing strategy. This approach involved systematic substitution at each position of the antigenic peptide sequence using all natural amino acids to generate a profile of peptide specificity ("X-scan"). The likelihood of off-target reactivity was investigated by searching the human proteome for sequences matching this profile, and testing against a panel of primary cell lines. Starting from a diverse panel of parental TCRs, we engineered several affinity-enhanced TCRs specific for the cancer-testis antigen MAGE-A10. Two of these TCRs had affinities and specificities which appeared to be equally optimal when tested in conventional biochemical and cellular assays. The X-scan method, however, permitted us to select the most specific and potent candidate for further pre-clinical and clinical testing.

Aqueous peroxyl radical exposure to THP-1 cells causes glutathione loss followed by protein oxidation and cell death without increased caspase-3 activity.

Protein oxidation within cells exposed to oxidative free radicals has been reported to occur in an uninhibited manner with both hydroxyl and peroxyl radicals. In contrast, THP-1 cells exposed to peroxyl radicals (ROO(*)) generated by thermo decomposition of the azo compound AAPH showed a distinct lag phase of at least 6 h, during which time no protein oxidation or cell death was observed. Glutathione appears to be the source of the lag phase as cellular levels were observed to rapidly decrease during this period. Removal of glutathione with buthionine sulfoxamine eliminated the lag phase. At the end of the lag phase there was a rapid loss of cellular MTT reducing activity and the appearance of large numbers of propidium iodide/annexin-V staining necrotic cells with only 10% of the cells appearing apoptotic (annexin-V staining only). Cytochrome c was released into the cytoplasm after 12 h of incubation but no increase in caspase-3 activity was found at any time points. We propose that the rapid loss of glutathione caused by the AAPH peroxyl radicals resulted in the loss of caspase activity and the initiation of protein oxidation. The lack of caspase-3 activity appears to have caused the cells to undergo necrosis in response to protein oxidation and other cellular damage.

Common variants of apolipoprotein A-IV differ in their ability to inhibit low density lipoprotein oxidation.

Apolipoprotein A-IV (apoA-IV) inhibits lipid peroxidation, thus demonstrating potential anti-atherogenic properties. The aim of this study was to investigate how the inhibition of low density lipoprotein (LDL) oxidation was influenced by common apoA-IV isoforms. Recombinant wild type apoA-IV (100 microg/ml) significantly inhibited the oxidation of LDL (50 microg protein/ml) by 5 microM CuSO(4) (P<0.005), but not by 100 microM CuSO(4), suggesting that it may act by binding copper ions. ApoA-IV also inhibited the oxidation of LDL by the water-soluble free-radical generator 2,2'-azobis(amidinopropane) dihydrochloride (AAPH; 1 mM), as shown by the two-fold increase in the time for half maximal conjugated diene formation (T(1/2); P<0.05) suggesting it can also scavenge free radicals in the aqueous phase. Compared to wild type apoA-IV, apoA-IV-S347 decreased T(1/2) by 15% (P=0.036) and apoA-IV-H360 increased T(1/2) by 18% (P=0.046). All apoA-IV isoforms increased the relative electrophoretic mobility of native LDL, suggesting apoA-IV can bind to LDL and acts as a site-specific antioxidant. The reduced inhibition of LDL oxidation by apoA-IV-S347 compared to wild type apoA-IV may account for the previous association of the APOA4 S347 variant with increased CHD risk and oxidative stress.

Effects of dairy products naturally enriched with cis-9,trans-11 conjugated linoleic acid on the blood lipid profile in healthy middle-aged men.

BACKGROUND: Interest in the development of dairy products naturally enriched in conjugated linoleic acid (CLA) exists. However, feeding regimens that enhance the CLA content of milk also increase concentrations of trans-18:1 fatty acids. The implications for human health are not yet known. OBJECTIVE: This study investigated the effects of consuming dairy products naturally enriched in cis-9,trans-11 CLA (and trans-11 18:1) on the blood lipid profile, the atherogenicity of LDL, and markers of inflammation and insulin resistance in healthy middle-aged men. DESIGN: Healthy middle-aged men (n = 32) consumed ultra-heat-treated milk, butter, and cheese that provided 0.151 g/d (control) or 1.421 g/d (modified) cis-9,trans-11 CLA for 6 wk. This was followed by a 7-wk washout and a crossover to the other treatment. RESULTS: Consumption of dairy products enriched with cis-9,trans-11 CLA and trans-11 18:1 did not significantly affect body weight, inflammatory markers, insulin, glucose, triacylglycerols, or total, LDL, and HDL cholesterol but resulted in a small increase in the ratio of LDL to HDL cholesterol. The modified dairy products changed LDL fatty acid composition but had no significant effect on LDL particle size or the susceptibility of LDL to oxidation. Overall, increased consumption of full-fat dairy products and naturally derived trans fatty acids did not cause significant changes in cardiovascular disease risk variables, as may be expected on the basis of current health recommendations. CONCLUSION: Dairy products naturally enriched with cis-9,trans-11 CLA and trans-11 18:1 do not appear to have a significant effect on the blood lipid profile.

NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma.

Despite recent therapeutic advances, multiple myeloma (MM) remains largely incurable. Here 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 × 10(9) engineered T cells 2 d after autologous stem cell transplant. 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, in accordance 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.

Examining the presentation of tumor-associated antigens on peptide-pulsed T2 cells.

Peptide-pulsed T2 cells are routinely used to study T-cell activation by MHC-restricted peptides derived from tumor-associated antigens (TAAs). Nevertheless, the capacity of T2 cells to present antigenic epitopes remains to be precisely quantified, primarily due to the detection limits imposed by available methods. Since naturally-processed TAA-derived epitopes have been shown to be displayed at levels as low as 10-150 copies per cell, highly sensitive detection and quantification techniques are essential to assess the natural degree of T-cell sensitivity. Here, we report the use of soluble, high-affinity T-cell receptors (TCRs) coupled with single-molecule fluorescence microscopy to quantify three reported TAA-derived epitopes on peptide-pulsed T2 cells, dissecting the relationship between concentration of exogenous peptide, number of epitopes presented, and activation of epitope-specific T cells. Our findings indicate that peptide concentrations in the low nanomolar range are required for T2 cells to present TAAs in extents that are comparable to those of malignant cells.

Identification of a Titin-derived HLA-A1-presented peptide as a cross-reactive target for engineered MAGE A3-directed T cells.

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.

A moderate reduction in extracellular pH protects macrophages against apoptosis induced by oxidized low density lipoprotein.

We investigated the effect of pH on macrophage apoptosis induced by oxidized low density lipoprotein (OxLDL), as human atherosclerotic lesions have regions of low pH. Hydroperoxide-rich and oxysterol-rich LDL caused 38% and 74% apoptosis of J774 macrophages, respectively, at 24 h, as measured by the externalization of phosphatidylserine. Native LDL, however, did not cause apoptosis. Reducing the pH of the culture medium from 7.4 to 7.0 inhibited apoptosis induced by hydroperoxide-rich or oxysterol-rich OxLDL by 61% and 46%, respectively (P < 0.001). These data were confirmed by semiquantitative analysis of cytochrome c release from mitochondria. Decreasing the extracellular pH to 7.0 reduced the uptake of hydroperoxide-rich and oxysterol-rich (125)I-labeled LDL by 82% and 42%, respectively, and reduced cell surface binding of oxysterol-rich LDL by 31%. This may explain the reduced apoptosis. Additionally, low pH did not affect OxLDL-induced apoptosis of human monocytes, which do not possess scavenger receptors for OxLDL, but reduced apoptosis of human monocyte-derived macrophages, which do possess them. Our investigations suggest that the presence of areas of low pH within atherosclerotic lesions may reduce the uptake of OxLDL and reduce macrophage apoptosis, thus affecting lesion progression.

A novel method for production of lipid hydroperoxide- or oxysterol-rich low-density lipoprotein.

LDL oxidation may be important in atherosclerosis. Extensive oxidation of LDL by copper induces increased uptake by macrophages, but results in decomposition of hydroperoxides, making it more difficult to investigate the effects of hydroperoxides in oxidised LDL on cell function. We describe here a simple method of oxidising LDL by dialysis against copper ions at 4 degrees C, which inhibits the decomposition of hydroperoxides, and allows the production of LDL rich in hydroperoxides (626+/-98 nmol/mg LDL protein) but low in oxysterols (3+/-1 nmol 7-ketocholesterol/mg LDL protein), whilst allowing sufficient modification (2.6+/-0.5 relative electrophoretic mobility) for rapid uptake by macrophages (5.49+/-0.75 microg (125)I-labelled hydroperoxide-rich LDL vs. 0.46+/-0.04 microg protein/mg cell protein in 18 h for native LDL). By dialysing under the same conditions, but at 37 degrees C, the hydroperoxides are decomposed extensively and the LDL becomes rich in oxysterols. This novel method of oxidising LDL with high yield to either a hydroperoxide- or oxysterol-rich form by simply altering the temperature of dialysis may provide a useful tool for determining the effects of these different oxidation products on cell function.