Fig 4 - available from: Nature Communications
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2Q1-Fab free form structure. a Structural alignment of the free 2Q1-Fab (gray) with the 2Q1-Fab bound to the IDH2 R140Q -HLA-B*07:02 (Light Chain: light blue; Heavy Chain: deep blue). All CDR loops of the free 2Q1-Fab are colored in green. Zoomed views of the CDR loops are highlighted in boxes with CDR-H1 (yellow), -H2 (orange), and -H3 (raspberry) displaying the largest conformation changes. b Bird's-eye view of surface representation of the HLA-B*07:02 showing the positions of the 2Q1-Fab CDR loops. The CDRs of the free 2Q1-Fab structure are overlaid with the bound 2Q1-Fab to highlight the conformational change induced upon pHLA binding. The CDRs of the free 2Q1-Fab are shown in light blue (light chain) and pale green (heavy chain). The CDR loops of the bound 2Q1-Fab are colored as in (a).
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Chimeric antigen receptor (CAR) T cells have emerged as a promising class of therapeutic agents, generating remarkable responses in the clinic for a subset of human cancers. One major challenge precluding the wider implementation of CAR therapy is the paucity of tumor-specific antigens. Here, we describe the development of a CAR targeting the tumor...
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Context 1
... 2Q1-Fab fragment was purified following papain cleavage. The crystal structure of the purified 2Q1-Fab/ IDH2 R140Q -HLA-B*07:02 ternary complex ( Supplementary Fig. 4a, b, c, d) was refined to 2.9 Å resolution with one complex molecule in the asymmetric unit (Fig. 3a, Table 1). Despite the limited resolution of the data, clear electron density was observed at the binding interface for the IDH2 R140Q peptide and the CDRs of the 2Q1-Fab ( Supplementary Fig. 4e). ...
Context 2
... crystal structure of the purified 2Q1-Fab/ IDH2 R140Q -HLA-B*07:02 ternary complex ( Supplementary Fig. 4a, b, c, d) was refined to 2.9 Å resolution with one complex molecule in the asymmetric unit (Fig. 3a, Table 1). Despite the limited resolution of the data, clear electron density was observed at the binding interface for the IDH2 R140Q peptide and the CDRs of the 2Q1-Fab ( Supplementary Fig. 4e). The 2Q1-Fab docks onto IDH2 R140Q -HLA-B*07:02 in a parallel orientation with a docking angle of 20° ( Fig. 3b) and was shifted towards the C-terminus of the IDH2 R140Q peptide with an incident angle of 19°. ...
Context 3
... rmsd values of 0.25-0.32 Å. Overall, the structures of the four copies are similar, with the only striking difference being the conformation of CDR-H3 ( Supplementary Fig. 6). This is likely a result of the conformational flexibility inherent in this loop. Fig. 6). All three CDRs of the 2Q1 heavy chain exhibited significant conformational changes (Fig. 4a). Specifically, binding to the C-terminal end of HLA-B*07:02 α1 and the N-terminal end of α2 induces conformational changes in CDR-H1 and -H2, respectively, while binding to the C-terminal end of the IDH2 peptide induces a conformational shift in CDR-H3, thus optimizing binding to pHLA (Fig. 4a, ...
Context 4
... chain exhibited significant conformational changes (Fig. 4a). Specifically, binding to the C-terminal end of HLA-B*07:02 α1 and the N-terminal end of α2 induces conformational changes in CDR-H1 and -H2, respectively, while binding to the C-terminal end of the IDH2 peptide induces a conformational shift in CDR-H3, thus optimizing binding to pHLA (Fig. 4a, ...
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Avoidance of immune destruction is recognized as one of the hallmarks of cancer development. Although first predicted as a potential antitumor treatment modality more than 50 years ago, the widespread clinical use of cancer immunotherapies has only recently become a reality. Cancer immunotherapy works by reactivation of a stalled pre-existing immun...
Citations
... T-cell recognition of patient-derived AML cells has been demonstrated for two IDH2 R140Q peptides, i.e., IQNILGGTVF in HLA-B*35:43 and TIQNILGGTV in HLA-B*15:01, but not for SPNGTIQNIL in HLA-B*07:02. T-cell recognition of cell lines overexpressing the IDH2 R140Q gene has also been shown by Hwang et al. [26], who developed CAR-T-cells containing a TCR mimic antibody domain specifically targeting SPNGTIQNIL in HLA-B*07:02. These CAR-T-cells exhibited reactivity against the exogenous peptide with low avidity (EC50~10 µg/mL). ...
DNA methyltransferase 3A (DNMT3A) and isocitrate dehydrogenase 1 and 2 (IDH1/2) are genes involved in epigenetic regulation, each mutated in 7–23% of patients with acute myeloid leukemia. Here, we investigated whether hotspot mutations in these genes encode neoantigens that can be targeted by immunotherapy. Five human B-lymphoblastoid cell lines expressing common HLA class I alleles were transduced with a minigene construct containing mutations that often occur in DNMT3A or IDH1/2. From these minigene-transduced cell lines, peptides were eluted from HLA class I alleles and analyzed using tandem mass spectrometry. The resulting data are available via ProteomeXchange under the identifier PXD050560. Mass spectrometry revealed an HLA-A*01:01-binding DNMT3AR882H peptide and an HLA-B*07:02-binding IDH2R140Q peptide as potential neoantigens. For these neopeptides, peptide–HLA tetramers were produced to search for specific T-cells in healthy individuals. Various T-cell clones were isolated showing specific reactivity against cell lines transduced with full-length DNMT3AR882H or IDH2R140Q genes, while cell lines transduced with wildtype genes were not recognized. One T-cell clone for DNMT3AR882H also reacted against patient-derived acute myeloid leukemia cells with the mutation, while patient samples without the mutation were not recognized, thereby validating the surface presentation of a DNMT3AR882H neoantigen that can potentially be targeted in acute myeloid leukemia via immunotherapy.
... TCRm Ab 2Q1 specifically recognizes a neoantigen derived from isocitrate dehydrogenase 2 (IDH2 R140Q ) (SPNGTIQNIL) presented by HLA-B*07:02 (64). CAR T cells constructed from 2Q1 were cytotoxic against IDH2 R140Q -bearing target cells. ...
... CAR T cells constructed from 2Q1 were cytotoxic against IDH2 R140Q -bearing target cells. Structures of wild-type IDH2-HLA-B*02 and mutant IDH2 R140Q -HLA-B*02 complexes showed that the peptides bound in essentially identical conformations and that P7 Arg/ Gln (wild-type and mutant amino acids) is buried deep within the peptide-binding groove of the MHC molecule (64). In the structure of TCRm Ab 2Q1 bound to IDH2 R140Q -HLA-B*02, the only direct interaction between P7 Gln and 2Q1 is a hydrogen bond linking P7 Gln to V H CDR3 Arg102. ...
... In the structure of TCRm Ab 2Q1 bound to IDH2 R140Q -HLA-B*02, the only direct interaction between P7 Gln and 2Q1 is a hydrogen bond linking P7 Gln to V H CDR3 Arg102. Elimination of this hydrogen bond by mutating V H CDR3 Arg102 to alanine completely abrogated TCRm Ab binding (64). 2Q1 docks onto B A FIGURE 7 Presentation of PIK3CA H1047L neoepitope by HLA-C*03:01. ...
Adoptive cell therapy (ACT) with tumor-specific T cells has been shown to mediate durable cancer regression. Tumor-specific T cells are also the basis of other therapies, notably cancer vaccines. The main target of tumor-specific T cells are neoantigens resulting from mutations in self-antigens over the course of malignant transformation. The detection of neoantigens presents a major challenge to T cells because of their high structural similarity to self-antigens, and the need to avoid autoimmunity. How different a neoantigen must be from its wild-type parent for it to induce a T cell response is poorly understood. Here we review recent structural and biophysical studies of T cell receptor (TCR) recognition of shared cancer neoantigens derived from oncogenes, including p53R175H, KRASG12D, KRASG12V, HHATp8F, and PIK3CAH1047L. These studies have revealed that, in some cases, the oncogenic mutation improves antigen presentation by strengthening peptide–MHC binding. In other cases, the mutation is detected by direct interactions with TCR, or by energetically driven or other indirect strategies not requiring direct TCR contacts with the mutation. We also review antibodies designed to recognize peptide–MHC on cell surfaces (TCR-mimic antibodies) as an alternative to TCRs for targeting cancer neoantigens. Finally, we review recent computational advances in this area, including efforts to predict neoepitope immunogenicity and how these efforts may be advanced by structural information on peptide–MHC binding and peptide–MHC recognition by TCRs.
... The total buried surface area of the V2-Fab/ KRAS G12V -HLA-A*03:01 was 1388 Å 2 , with the variable domain of the heavy chain contributing to~25% more than the variable light chain (862 Å 2 and 526 Å 2 , respectively) (Table S1). Even though this surface area was smaller compared to other known TCRm-and TCR-pHLA structures (>2000 Å 2 ) 28 , other complexes achieve high specificity with even smaller footprints 18,35 . Importantly, the regions making up the binding interface were well-resolved in the EM map, specifically the KRAS G12V peptide, the CDRs of the V2-Fab, and the HLA-A*03:01 (Supplementary Fig. 4). ...
... Sim et al. reported conformational changes in the peptide upon TCR binding to KRAS G12D 10-19 -HLA-C*08:02, but not upon binding of a different TCR to KRAS G12D 10-18 -HLA-C*08:02 23 . In addition, groups have reported examples where TCRs or TCRms reactive to mutant neoantigens exhibit strong specificity for mutant over wild-type peptides, despite minimal structural differences in the mutant vs. wild-type pHLA 35,43,44 . ...
Specificity remains a major challenge to current therapeutic strategies for cancer. Mutation associated neoantigens (MANAs) are products of genetic alterations, making them highly specific therapeutic targets. MANAs are HLA-presented (pHLA) peptides derived from intracellular mutant proteins that are otherwise inaccessible to antibody-based therapeutics. Here, we describe the cryo-EM structure of an antibody-MANA pHLA complex. Specifically, we determine a TCR mimic (TCRm) antibody bound to its MANA target, the KRASG12V peptide presented by HLA-A*03:01. Hydrophobic residues appear to account for the specificity of the mutant G12V residue. We also determine the structure of the wild-type G12 peptide bound to HLA-A*03:01, using X-ray crystallography. Based on these structures, we perform screens to validate the key residues required for peptide specificity. These experiments led us to a model for discrimination between the mutant and the wild-type peptides presented on HLA-A*03:01 based exclusively on hydrophobic interactions.
... While these immune cells may have fewer concerns of graft-vs-host disease, which could make them more suitable as off-the-shelf products, they also have their own limitations, including short life spans, limited proliferation capabilities, and inability to form memory cells. Additionally, T cells can be engineered to target tumors through tumor-neoantigenspecific TCRs (153). TCR-T cell therapy has a significant advantage in that the target is not limited to membrane antigens, although elegantly designed CAR can also recognize intracellular neoantigens in the context of MHC complexes (154). ...
Chimeric antigen receptor (CAR) T cell therapy represents a major breakthrough in cancer care since the approval of tisagenlecleucel by the Food and Drug Administration in 2017 for the treatment of pediatric and young adult patients with relapsed or refractory acute lymphocytic leukemia. As of April 2023, six CAR T cell therapies have been approved, demonstrating unprecedented efficacy in patients with B-cell malignancies and multiple myeloma. However, adverse events such as cytokine release syndrome and immune effector cell-associated neurotoxicity pose significant challenges to CAR T cell therapy. The severity of these adverse events correlates with the pretreatment tumor burden, where a higher tumor burden results in more severe consequences. This observation is supported by the application of CD19-targeted CAR T cell therapy in autoimmune diseases including systemic lupus erythematosus and antisynthetase syndrome. These results indicate that initiating CAR T cell therapy early at low tumor burden or using debulking strategy prior to CAR T cell infusion may reduce the severity of adverse events. In addition, CAR T cell therapy is expensive and has limited effectiveness against solid tumors. In this article, we review the critical steps that led to this groundbreaking therapy and explore ongoing efforts to overcome these challenges. With the promise of more effective and safer CAR T cell therapies in development, we are optimistic that a broader range of cancer patients will benefit from this revolutionary therapy in the foreseeable future.
... 23 To access intracellular antigens, neoantigen-specific CARs have been described. 24 These CARs are generally human leucocyte antigen (HLA) restricted and thus require matching with patient HLA antigen status as well as maintained HLA antigen expression by tumour cells. ...
Immunotherapy using chimeric antigen receptor (CAR)‐engineered T‐cells has achieved remarkable impact in the treatment of selected blood cancers. However, meaningful clinical efficacy against nonhaematological malignancies has largely proven elusive. In this minireview, the main challenges to successful CAR‐based intervention against solid tumours are considered. Obstacles are considered in four categories, namely target selection, trafficking of CAR‐engineered cells to tumour deposits, the need to overcome the physical, chemical and biological hurdles to immune effector function that operate within the tumour microenvironment and selection of the best host cells for CAR engineering. A range of pre‐clinical technologies that have been developed in an effort to overcome these issues are also surveyed. Although clinical progress comes dropping slow, rapid and continued advances in cellular engineering and manufacture, coupled with the emergence of several complementary interventions bodes well for the future success of CAR T‐cell immunotherapy of solid tumours. In contrast to blood cancers, solid tumours have largely proven refractory to CAR‐based immunotherapy. Obstacles include the lack of tumour‐specific targets, difficulty in entry and infiltration of tumour deposits and the myriad of immunosuppressive factors that operate within the tumour microenvironment. We also consider various host cell types for CAR engineering that have been proposed as alternatives to conventional T‐cells.
... We have focused exclusively on cell surface targets, while excluding HLA-restricted CAR Tcell approaches, given the need, in this case, for appropriate HLA matching between patient and CAR. It should be noted that CARs may also be targeted against HLA-restricted neoantigens [4], and the interested reader is also referred to [5] for an overview of HLArestricted CAR-based immunotherapeutic approaches. ...
Simple Summary
A rapidly emerging new approach to treat cancer involves collection of patient immune white blood cells and genetic re-programming of the cells to express a new cancer-detecting receptor called a CAR. This approach has revolutionized the treatment of some blood cancers, whereas solid tumours, which account for 90% of all cancers, are proving much more difficult to treat using this approach. A major challenge in this respect relates to the identification of targets that differentiate cancer from healthy cells. In a partner review, we consider clinical data already collected when CAR technology has been applied against solid tumours that express 30 different targets. Here, we explore emerging candidates for which such clinical data are not available yet, but where other data provide information about potential suitability as future clinical targets.
Abstract
Immunotherapy with CAR T-cells has revolutionised the treatment of B-cell and plasma cell-derived cancers. However, solid tumours present a much greater challenge for treatment using CAR-engineered immune cells. In a partner review, we have surveyed data generated in clinical trials in which patients with solid tumours that expressed any of 30 discrete targets were treated with CAR-based immunotherapy. That exercise confirms that efficacy of this approach falls well behind that seen in haematological malignancies, while significant toxic events have also been reported. Here, we consider approximately 60 additional candidates for which such clinical data are not available yet, but where pre-clinical data have provided support for their advancement to clinical evaluation as CAR target antigens.
... To screen for neoantigens, comparative whole-exome or whole-genome sequencing of matched tumour and non-malignant samples is usually performed to identify tumour-specific non-synonymous somatic mutations, followed by RNA-seq and mass spectrometry to confirm expression of the mutated mRNAs and peptides 169 . Several neoantigens have already been adopted as promising CAR targets for the treatment of haematological or solid malignancies 41,43 . For example, EGFR variant III (EGFRvIII) is a neoantigen specifically expressed in certain solid tumours, including a subset of glioblastomas, leading to ongoing clinical trials of EGFRvIII-directed CAR T cells (such as NCT03726515, NCT03283631 and NCT03638206). ...
Despite the notable success of chimeric antigen receptor (CAR) T cell therapies in the treatment of certain haematological malignancies, challenges remain in optimizing CAR designs and cell products, improving response rates, extending the durability of remissions, reducing toxicity and broadening the utility of this therapeutic modality to other cancer types. Data from multidimensional omics analyses, including genomics, epigenomics, transcriptomics, T cell receptor-repertoire profiling, proteomics, metabolomics and/or microbiomics, provide unique opportunities to dissect the complex and dynamic multifactorial phenotypes, processes and responses of CAR T cells as well as to discover novel tumour targets and pathways of resistance. In this Review, we summarize the multidimensional cellular and molecular profiling technologies that have been used to advance our mechanistic understanding of CAR T cell therapies. In addition, we discuss current applications and potential strategies leveraging multi-omics data to identify optimal target antigens and other molecular features that could be exploited to enhance the antitumour activity and minimize the toxicity of CAR T cell therapy. Indeed, fully utilizing multi-omics data will provide new insights into the biology of CAR T cell therapy, further accelerate the development of products with improved efficacy and safety profiles, and enable clinicians to better predict and monitor patient responses.
... Experimental peptidome binding assays performed on three early-generation TCRms (17,19,20) suggested that they were unlikely to achieve the levels of specificity required for therapeutic applications (22,23). However, several TCRms with apparently high specificity have been reported in the past year, fostering renewed interest in this therapeutic modality and resulting in a more than doubling of the number of crystal structures of TCRm:pMHC complexes (24)(25)(26)(27)(28)(29). These include an anti-Wilms' Tumor Antigen 1 (WT1) TCRm (24) and an anti-alpha ferroprotein (AFP) TCRm (29) that have both progressed to clinical trials. ...
... These include an anti-Wilms' Tumor Antigen 1 (WT1) TCRm (24) and an anti-alpha ferroprotein (AFP) TCRm (29) that have both progressed to clinical trials. Two neoantigen peptide:MHCspecific TCRms were also identified that achieve complete selectivity over their wildtype peptide equivalents each differing by just a single residue mutation (25,26). ...
... We found twelve pMHC-binding antibodies, eleven of which have binding modes that transect the peptide binding groove (17)(18)(19)(20)(24)(25)(26)(27) and one alloantibody (30) that engages only the MHC and so was not classified as a TCRm. After filtering for interface redundancy (see Methods), we identified 10 representative TCRm:pMHC complexes (9 MHC class I, 1 MHC class II), 60 representative TCR:pMHC complexes (52 MHC class I, 8 MHC class II), and 824 representative antibody:antigen complexes. ...
T-cell receptor-mimetic antibodies (TCRms) targeting disease-associated peptides presented by Major Histocompatibility Complexes (pMHCs) are set to become a major new drug modality. However, we lack a general understanding of how TCRms engage pMHC targets, which is crucial for predicting their specificity and safety. Several new structures of TCRm:pMHC complexes have become available in the past year, providing sufficient initial data for a holistic analysis of TCRms as a class of pMHC binding agents. Here, we profile the complete set of TCRm:pMHC complexes against representative TCR:pMHC complexes to quantify the TCR-likeness of their pMHC engagement. We find that intrinsic molecular differences between antibodies and TCRs lead to fundamentally different roles for their heavy/light chains and Complementarity-Determining Region loops during antigen recognition. The idiotypic properties of antibodies may increase the likelihood of TCRms engaging pMHCs with less peptide selectivity than TCRs. However, the pMHC recognition features of some TCRms, including the two TCRms currently in clinical trials, can be remarkably TCR-like. The insights gained from this study will aid in the rational design and optimisation of next-generation TCRms.
... Commonly applied phage display-based approaches for identification of antigen-specific scFvs rely on multiple rounds of biopanning. On the basis of extensive experience with this process, we postulated that the direct assessment of scFv phage binding without the intermediate growth phases could accelerate and improve the identification of desirable scFvs [23][24][25][26] . To test this hypothesis, we first developed a quantitative NGS based phage binding assay called SLISY (Sequencing-Linked ImmunoSorbent assaY). ...
The therapeutic applications of antibodies are manifold and the emergence of SARS-CoV-2 provides a cogent example of the value of rapidly identifying biologically active antibodies. We describe an approach called SLISY (Sequencing-Linked ImmunoSorbent assaY) that in a single experiment can assess the binding specificity of millions of clones, be applied to any screen that links DNA sequence to a potential binding moiety, and requires only a single round of biopanning. We demonstrate this approach using an scFv library applied to cellular and protein targets to identify specific or broadly reacting antibodies. For a cellular target, we use paired HLA knockout cell lines to identify a panel of antibodies specific to HLA-A3. For a protein target, SLISY identifies 1279 clones that bound to the Receptor Binding Domain of the SARS-CoV-2 spike protein, with >40% of tested clones also neutralizing its interaction with ACE2 in in vitro assays. Using a multi-comparison SLISY against the Beta, Gamma, and Delta variants, we recovered clones that exhibited broad-spectrum neutralizing potential in vitro. By evaluating millions of scFvs simultaneously against multiple targets, SLISY allows the rapid identification of candidate scFvs with defined binding profiles facilitating the identification of antibodies with the desired biological activity. The covid pandemic has highlighted the need for rapid antibody development. Here, authors develop an approach called SLISY, which uses NGS with phage display to simultaneously assess millions of clones to rapidly isolate specific antibodies against SARS-CoV-2 and its evolving variants.
... We have now demonstrated the relationship between rare HLA-I alleles and PD-L1 by functional CD8+ T cell cytotoxicity tests. This interaction raises intriguing questions and provides new insights into mechanisms of current immunotherapies [40,41]. ...
Simple Summary
Immunotherapy targeting immune checkpoint pathways have recently attracted great attention in cancer treatment, but better strategies are needed to identify patients likely to benefit from it. The major histocompatibility complex (MHC) class I expression in cancer cells greatly influences the outcome of T cell-mediated immunotherapy. Here, we determined the prevalent HLA class I allelic variants in a sarcoma population. We characterized patient CD8+ T-cells and demonstrated low cytolysis to autologous tumor cells. Moreover, we used a co-culture model of autologous T-cells and PD-L1-deficient or positive biopsies of rare sarcomas to determine whether HLA-I influences tumor survival.
Abstract
The major histocompatibility complex (MHC) class I expression in cancer cells has a crucial impact on the outcome of T cell-mediated cancer immunotherapy. We now determined the HLA class I allelic variants and their expression in PD-L1-deficient and positive rare sarcoma tissues. Tumor tissues were HLA-I classified based on HLA-A and -B alleles, and for class II, the HLA-DR-B by Taqman genomic PCRs. The HLA-A24*:10-B73*:01 haplotype was the most common. A general down-regulation or deletion of HLA-B mRNA and HLA-A was observed, compared to HLA-DR-B. HLA-I was almost too low to be detectable by immunohistochemistry and 32% of grade III cases were positive to PD-L1. Functional cytotoxic assays co-culturing patient biopsies with autologous T cells were used to assess their ability to kill matched tumor cells. These results establish that deletion of HLA-I loci together with their down-regulation in individual patient restrict the autologous lymphocyte cytotoxic activity, even in the presence of the immune checkpoint blocking antibody, Nivolumab. Additionally, the proposed cytotoxic test suggests a strategy to assess the sensitivity of tumor cells to T cell-mediated attack at the level of the individual patient.
