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.

Chimeric antigen receptors enable superior control of HIV replication by rapidly killing infected cells.

Engineered T cells hold great promise to become part of an effective HIV cure strategy, but it is currently unclear how best to redirect T cells to target HIV. To gain insight, we generated engineered T cells using lentiviral vectors encoding one of three distinct HIV-specific T cell receptors (TCRs) or a previously optimized HIV-targeting chimeric antigen receptor (CAR) and compared their functional capabilities. All engineered T cells had robust, antigen-specific polyfunctional cytokine profiles when mixed with artificial antigen-presenting cells. However, only the CAR T cells could potently control HIV replication. TCR affinity enhancement did not augment HIV control but did allow TCR T cells to recognize common HIV escape variants. Interestingly, either altering Nef activity or adding additional target epitopes into the HIV genome bolstered TCR T cell anti-HIV activity, but CAR T cells remained superior in their ability to control HIV replication. To better understand why CAR T cells control HIV replication better than TCR T cells, we performed a time course to determine when HIV-specific T cells were first able to activate Caspase 3 in HIV-infected targets. We demonstrated that CAR T cells recognized and killed HIV-infected targets more rapidly than TCR T cells, which correlates with their ability to control HIV replication. These studies suggest that the speed of target recognition and killing is a key determinant of whether engineered T cell therapies will be effective against infectious diseases.

Structural insights into engineering a T-cell receptor targeting MAGE-A10 with higher affinity and specificity for cancer immunotherapy.

BACKGROUND: T-cell receptor (TCR) immunotherapy is becoming a viable modality in cancer treatment with efficacy in clinical trials. The safety of patients is paramount, so innovative cell engineering methods are being employed to exploit adaptive immunity while controlling the factors governing antigen receptor (ie, TCR) specificity and cross-reactivity. We recently reported a TCR engineering campaign and selectivity profiling assay (X-scan) targeting a melanoma antigen gene (MAGE)-A10 peptide. This helped to distinguish between two well-performing TCRs based on cross-reactivity potential during preclinical drug evaluation, allowing one to be advanced to T-cell immunotherapeutic clinical trials. Here, we present three-dimensional structural information on those TCRs, highlighting engineering improvements and molecular mechanisms likely underpinning differential selectivity. METHODS: Parental and engineered TCRs were purified and crystallized either alone or complexed to human leucocyte antigen (HLA)-A*02:01 presenting the MAGE-A10 9-mer peptide, GLYDGMEHL (pHLA/MAGE-A10-9). Using X-ray diffraction, we solved four high-resolution crystal structures and evaluated them relative to previously reported functional results. RESULTS: The unligated parental TCR displayed similar complementarity-determining region (CDR) loop conformations when bound to pHLA/MAGE-A10-9; a rigid-body movement of TCR beta chain variable domain (TRBV) relative to TCR alpha chain variable domain helped optimal pHLA engagement. This first view of an HLA-bound MAGE-A10 peptide revealed an intrachain non-covalent 'staple' between peptide Tyr3 and Glu7. A subtle Glu31-Asp mutation in ßCDR1 of the parental TCR generated a high-affinity derivative. Its pHLA-complexed structure shows that the shorter Asp leans toward the pHLA with resulting rigid-body TRBV shift, creating localized changes around the peptide's C-terminus. Structural comparison with a less selective TCR indicated that differential cross-reactivity to MAGE-A10 peptide variants is most readily explained by alterations in surface electrostatics, and the size and geometry of TCR-peptide interfacial cavities. CONCLUSIONS: Modest changes in engineered TCRs targeting MAGE-A10 produced significantly different properties. Conformational invariance of TCR and antigen peptide plus more space-filling CDR loop sequences may be desirable properties for clinically relevant TCR-pHLA systems to reduce the likelihood of structurally similar peptide mimics being tolerated by a TCR. Such properties may partially explain why the affinity-enhanced, in vitro-selected TCR has been generally well tolerated in patients.

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.

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.

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.

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.

Monoclonal TCR-redirected tumor cell killing.

T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)­mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.

Natural variations at position 93 of the invariant Vα24-Jα18 α chain of human iNKT-cell TCRs strongly impact on CD1d binding.

Human invariant natural killer T (NKT) cell TCRs bind to CD1d via an "invariant" Vα24-Jα18 chain (iNKTα) paired to semi-invariant Vß11 chains (iNKTß). Single-amino acid variations at position 93 (p93) of iNKTα, immediately upstream of the "invariant" CDR3α region, have been reported in a substantial proportion of human iNKT-cell clones (4-30%). Although p93, a serine in most human iNKT-cell TCRs, makes no contact with CD1d, it could affect CD1d binding by altering the conformation of the crucial CDR3α loop. By generating recombinant refolded iNKT-cell TCRs, we show that natural single-nucleotide variations in iNKTα, translating to serine, threonine, asparagine or isoleucine at p93, exert a powerful effect on CD1d binding, with up to 28-fold differences in affinity between these variants. This effect was observed with CD1d loaded with either the artificial α-galactosylceramide antigens KRN7000 or OCH, or the endogenous glycolipid ß-galactosylceramide, and its importance for autoreactive recognition of endogenous lipids was demonstrated by the binding of variant iNKT-cell TCR tetramers to cell surface expressed CD1d. The serine-containing variant showed the strongest CD1d binding, offering an explanation for its predominance in vivo. Complementary molecular dynamics modeling studies were consistent with an impact of p93 on the conformation of the CDR3α loop.

High affinity soluble ILT2 receptor: a potent inhibitor of CD8(+) T cell activation.

Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin super-family receptor ILT2 (synonyms: LIR1, MIR7, CD85j), we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex (MHC) class I molecules. Produced in a dimeric form, either by chemical cross-linking with bivalent polyethylene glycol (PEG) derivatives or as a genetic fusion with human IgG Fc-fragment, the mutants exhibited a further increase in ligand-binding strength due to the avidity effect, with resident half-times (t(1/2)) on the surface of MHC I-positive cells of many hours. The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors (TCRs). In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8(+) cytotoxic T lymphocytes (CTLs) in the presence of their target cells, with subnanomolar potency and in a dose-dependent manner. As a selective inhibitor of CD8(+) CTL responses, the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.

Human TCR-binding affinity is governed by MHC class restriction.

T cell recognition is initiated by the binding of TCRs to peptide-MHCs (pMHCs), the interaction being characterized by weak affinity and fast kinetics. Previously, only 16 natural TCR/pMHC interactions have been measured by surface plasmon resonance (SPR). Of these, 5 are murine class I, 5 are murine class II, and 6 are human class I-restricted responses. Therefore, a significant gap exists in our understanding of human TCR/pMHC binding due to the limited SPR data currently available for human class I responses and the absence of SPR data for human class II-restricted responses. We have produced a panel of soluble TCR molecules originating from human T cells that respond to naturally occurring disease epitopes and their cognate pMHCs. In this study, we compare the binding affinity and kinetics of eight class-I-specific TCRs (TCR-Is) to pMHC-I with six class-II-specific TCRs (TCR-IIs) to pMHC-II using SPR. Overall, there is a substantial difference in the TCR-binding equilibrium constants for pMHC-I and pMHC-II, which arises from significantly faster on-rates for TCRs binding to pMHC-I. In contrast, the off-rates for all human TCR/pMHC interactions fall within a narrow window regardless of class restriction, thereby providing experimental support for the notion that binding half-life is the principal kinetic feature controlling T cell activation.