TRBC1-targeting antibody-drug conjugates for the treatment of T cell cancers.

Antibody and chimeric antigen receptor (CAR) T cell-mediated targeted therapies have improved survival in patients with solid and haematologic malignancies1-9. Adults with T cell leukaemias and lymphomas, collectively called T cell cancers, have short survival10,11 and lack such targeted therapies. Thus, T cell cancers particularly warrant the development of CAR T cells and antibodies to improve patient outcomes. Preclinical studies showed that targeting T cell receptor ß-chain constant region 1 (TRBC1) can kill cancerous T cells while preserving sufficient healthy T cells to maintain immunity12, making TRBC1 an attractive target to treat T cell cancers. However, the first-in-human clinical trial of anti-TRBC1 CAR T cells reported a low response rate and unexplained loss of anti-TRBC1 CAR T cells13,14. Here we demonstrate that CAR T cells are lost due to killing by the patient's normal T cells, reducing their efficacy. To circumvent this issue, we developed an antibody-drug conjugate that could kill TRBC1+ cancer cells in vitro and cure human T cell cancers in mouse models. The anti-TRBC1 antibody-drug conjugate may provide an optimal format for TRBC1 targeting and produce superior responses in patients with T cell cancers.

Hydrophobic interactions dominate the recognition of a KRAS G12V neoantigen.

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.

Targeting public neoantigens for cancer immunotherapy.

Several current immunotherapy approaches target private neoantigens derived from mutations that are unique to individual patients' tumors. However, immunotherapeutic agents can also be developed against public neoantigens derived from recurrent mutations in cancer driver genes. The latter approaches target proteins that are indispensable for tumor growth, and each therapeutic agent can be applied to numerous patients. Here we review the opportunities and challenges involved in the identification of suitable public neoantigen targets and the development of therapeutic agents targeting them.

Structural engineering of chimeric antigen receptors targeting HLA-restricted neoantigens.

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-specific isocitrate dehydrogenase 2 (IDH2) with R140Q mutation presented on the cell surface in complex with a common human leukocyte antigen allele, HLA-B*07:02. Engineering of the hinge domain of the CAR, as well as crystal structure-guided optimization of the IDH2R140Q-HLA-B*07:02-targeting moiety, enhances the sensitivity and specificity of CARs to enable targeting of this HLA-restricted neoantigen. This approach thus holds promise for the development and optimization of immunotherapies specific to other cancer driver mutations that are difficult to target by conventional means.

Targeting loss of heterozygosity for cancer-specific immunotherapy.

Developing therapeutic agents with potent antitumor activity that spare normal tissues remains a significant challenge. Clonal loss of heterozygosity (LOH) is a widespread and irreversible genetic alteration that is exquisitely specific to cancer cells. We hypothesized that LOH events can be therapeutically targeted by "inverting" the loss of an allele in cancer cells into an activating signal. Here we describe a proof-of-concept approach utilizing engineered T cells approximating NOT-gate Boolean logic to target counterexpressed antigens resulting from LOH events in cancer. The NOT gate comprises a chimeric antigen receptor (CAR) targeting the allele of human leukocyte antigen (HLA) that is retained in the cancer cells and an inhibitory CAR (iCAR) targeting the HLA allele that is lost in the cancer cells. We demonstrate that engineered T cells incorporating such NOT-gate logic can be activated in a genetically predictable manner in vitro and in mice to kill relevant cancer cells. This therapeutic approach, termed NASCAR (Neoplasm-targeting Allele-Sensing CAR), could, in theory, be extended to LOH of other polymorphic genes that result in altered cell surface antigens in cancers.

Bispecific antibodies targeting mutant RAS neoantigens.

Mutations in the RAS oncogenes occur in multiple cancers, and ways to target these mutations has been the subject of intense research for decades. Most of these efforts are focused on conventional small-molecule drugs rather than antibody-based therapies because the RAS proteins are intracellular. Peptides derived from recurrent RAS mutations, G12V and Q61H/L/R, are presented on cancer cells in the context of two common human leukocyte antigen (HLA) alleles, HLA-A3 and HLA-A1, respectively. Using phage display, we isolated single-chain variable fragments (scFvs) specific for each of these mutant peptide-HLA complexes. The scFvs did not recognize the peptides derived from the wild-type form of RAS proteins or other related peptides. We then sought to develop an immunotherapeutic agent that was capable of killing cells presenting very low levels of these RAS-derived peptide-HLA complexes. Among many variations of bispecific antibodies tested, one particular format, the single-chain diabody (scDb), exhibited superior reactivity to cells expressing low levels of neoantigens. We converted the scFvs to this scDb format and demonstrated that they were capable of inducing T cell activation and killing of target cancer cells expressing endogenous levels of the mutant RAS proteins and cognate HLA alleles. CRISPR-mediated alterations of the HLA and RAS genes provided strong genetic evidence for the specificity of the scDbs. Thus, this approach could be applied to other common oncogenic mutations that are difficult to target by conventional means, allowing for more specific anticancer therapeutics.

Targeting a neoantigen derived from a common TP53 mutation.

TP53 (tumor protein p53) is the most commonly mutated cancer driver gene, but drugs that target mutant tumor suppressor genes, such as TP53, are not yet available. Here, we describe the identification of an antibody highly specific to the most common TP53 mutation (R175H, in which arginine at position 175 is replaced with histidine) in complex with a common human leukocyte antigen-A (HLA-A) allele on the cell surface. We describe the structural basis of this specificity and its conversion into an immunotherapeutic agent: a bispecific single-chain diabody. Despite the extremely low p53 peptide-HLA complex density on the cancer cell surface, the bispecific antibody effectively activated T cells to lyse cancer cells that presented the neoantigen in vitro and in mice. This approach could in theory be used to target cancers containing mutations that are difficult to target in conventional ways.

TCR ß chain-directed bispecific antibodies for the treatment of T cell cancers.

Immunotherapies such as chimeric antigen receptor (CAR) T cells and bispecific antibodies redirect healthy T cells to kill cancer cells expressing the target antigen. The pan-B cell antigen-targeting immunotherapies have been remarkably successful in treating B cell malignancies. Such therapies also result in the near-complete loss of healthy B cells, but this depletion is well tolerated by patients. Although analogous targeting of pan-T cell markers could, in theory, help control T cell cancers, the concomitant healthy T cell depletion would result in severe and unacceptable immunosuppression. Thus, therapies directed against T cell cancers require more selective targeting. Here, we describe an approach to target T cell cancers through T cell receptor (TCR) antigens. Each T cell, normal or malignant, expresses a unique TCR ß chain generated from 1 of 30 TCR ß chain variable gene families (TRBV1 to TRBV30). We hypothesized that bispecific antibodies targeting a single TRBV family member expressed in malignant T cells could promote killing of these cancer cells, while preserving healthy T cells that express any of the other 29 possible TRBV family members. We addressed this hypothesis by demonstrating that bispecific antibodies targeting TRBV5-5 (α-V5) or TRBV12 (α-V12) specifically lyse relevant malignant T cell lines and patient-derived T cell leukemias in vitro. Treatment with these antibodies also resulted in major tumor regressions in mouse models of human T cell cancers. This approach provides an off-the-shelf, T cell cancer selective targeting approach that preserves enough healthy T cells to maintain cellular immunity.

An engineered antibody fragment targeting mutant ß-catenin via major histocompatibility complex I neoantigen presentation.

Mutations in CTNNB1, the gene encoding ß-catenin, are common in colon and liver cancers, the most frequent mutation affecting Ser-45 in ß-catenin. Peptides derived from WT ß-catenin have previously been shown to be presented on the cell surface as part of major histocompatibility complex (MHC) class I, suggesting an opportunity for targeting this common driver gene mutation with antibody-based therapies. Here, crystal structures of both the WT and S45F mutant peptide bound to HLA-A*03:01 at 2.20 and 2.45 Å resolutions, respectively, confirmed the accessibility of the phenylalanine residue for antibody recognition. Phage display was then used to identify single-chain variable fragment clones that selectively bind the S45F mutant peptide presented in HLA-A*03:01 and have minimal WT or other off-target binding. Following the initial characterization of five clones, we selected a single clone, E10, for further investigation. We developed a computational model of the binding of E10 to the mutant peptide-bound HLA-A3, incorporating data from affinity maturation as initial validation. In the future, our model may be used to design clones with maintained specificity and higher affinity. Such derivatives could be adapted into either cell-based (CAR-T) or protein-based (bispecific T-cell engagers) therapies to target cancer cells harboring the S45F mutation in CTNNB1.

Direct Detection and Quantification of Neoantigens.

Many immunotherapeutic approaches under development rely on T-cell recognition of cancer-derived peptides bound to human leukocyte antigen molecules on the cell surface. Direct experimental demonstration that such peptides are processed and bound is currently challenging. Here, we describe a method that meets this challenge. The method entailed an optimized immunoprecipitation protocol coupled with two-dimensional chromatography and mass spectrometry. The ability to detect and quantify minute amounts of predefined antigens should be useful both for basic research in tumor immunology and for the development of rationally designed cancer vaccines.

The neuroprotective role of ferrostatin-1 under rotenone-induced oxidative stress in dopaminergic neuroblastoma cells.

Endoplasmic reticulum (ER) proteins including protein disulfide isomerase (PDI) are playing crucial roles in maintaining appropriate protein folding. Under nitrosative stress, an excess of nitric oxide (NO) radical species induced the S-nitrosylation of PDI cysteines which eliminate its isomerase and oxidoreductase capabilities. In addition, the S-nitrosylation-PDI complex is the cause of aggregation especially of the α-synuclein (α-syn) protein (accumulation of Lewy-body aggregates). We recently identified a potent antioxidant small molecule, Ferrostatin-1 (Fer-1), that was able to inhibit a non-apoptotic cell death named ferroptosis. Ferroptosis cell death involved the generation of oxidative stress particularly lipid peroxide. In this work, we reported the neuroprotective role of ferrostatin-1 under rotenone-induced oxidative stress in dopaminergic neuroblastoma cells (SH-SY5Y). We first synthesized the Fer-1 and confirmed that it is not toxic toward the SH-SY5Y cells at concentrations up to 12.5 µM. Second, we showed that Fer-1 compound quenched the commercially available stable radical, the 2,2-diphenyl-1-picrylhydrazyl (DPPH), in non-cellular assay at 82 %. Third, Fer-1 inhibited the ROS/RNS generated under rotenone insult in SH-SY5Y cells. Fourth, we revealed the effective role of Fer-1 in ER stress mediated activation of apoptotic pathway. Finally, we reported that Fer-1 mitigated rotenone-induced α-syn aggregation.

Generation of MANAbodies specific to HLA-restricted epitopes encoded by somatically mutated genes.

Mutant epitopes encoded by cancer genes are virtually always located in the interior of cells, making them invisible to conventional antibodies. We here describe an approach to identify single-chain variable fragments (scFvs) specific for mutant peptides presented on the cell surface by HLA molecules. We demonstrate that these scFvs can be successfully converted to full-length antibodies, termed MANAbodies, targeting "Mutation-Associated Neo-Antigens" bound to HLA. A phage display library representing a highly diverse array of single-chain variable fragment sequences was first designed and constructed. A competitive selection protocol was then used to identify clones specific for mutant peptides bound to predefined HLA types. In this way, we obtained two scFvs, one specific for a peptide encoded by a common KRAS mutant and the other by a common epidermal growth factor receptor (EGFR) mutant. The scFvs bound to these peptides only when the peptides were complexed with HLA-A2 (KRAS peptide) or HLA-A3 (EGFR peptide). We converted one scFv to a full-length antibody (MANAbody) and demonstrate that the MANAbody specifically reacts with mutant peptide-HLA complex even when the peptide differs by only one amino acid from the normal, WT form.

miR-221/222 targets adiponectin receptor 1 to promote the epithelial-to-mesenchymal transition in breast cancer.

The epithelial-to-mesenchymal transition (EMT) is a highly conserved physiological program involved in development and tissue repair; however, its aberrant activation has been implicated in accelerating the progression of a variety of cancers. In breast cancer, the microRNAs (miRNAs) miR-221 and miR-222 (miR-221/222) are differentially expressed in the clinically more aggressive basal-like subtype compared to luminal subtype of breast cancer and upregulation of miR-221/222 induces the EMT by targeting the 3' untranslated region (3'UTR) of the GATA family transcriptional repressor TRPS1 (tricho-rhino-phalangeal syndrome type 1). The complete mechanism through which miR-221/222 promotes the EMT, however, is not fully understood. We identified adiponectin receptor 1 (ADIPOR1), a receptor for the adipocytokine adiponectin, as a direct target of miR-221/222. ADIPOR1 is expressed at higher levels in the luminal compared to the basal-like subtype of breast cancer cell lines, which can be reduced by miR-221/222 targeting of its 3'UTR. In addition, miR-221/222 were negatively correlated with ADIPOR1 expression across breast cancer cell lines and tumors. ADIPOR1 depletion by siRNA in MCF10A cells induced the EMT and increased cell invasion. Depletion of ADIPOR1 by siRNA induced activation of the canonical nuclear factor-kappaB (NF-κB) and subsequent phosphorylation of signal transducer and activator of transcription 3 (STAT3) in an interleukin 6 (IL6)-dependent manner. Finally, overexpression of ADIPOR1 in the basal-like cell line, MDA-MB-231, attenuated cell invasion and promoted the mesenchymal-to-epithelial transition (MET). We conclude that ADIPOR1 negatively regulates EMT in breast cancer and provides an additional node by which miR-221/222 induces the EMT. These results suggest that ADIPOR1 may play an important role in breast cancer progression and metastasis, and could potentially offer an alternative therapeutic strategy for basal-like breast cancer patients.

Metastatic progression of prostate cancer and e-cadherin regulation by zeb1 and SRC family kinases.

Expression of E-cadherin is used to monitor the epithelial phenotype, and its loss is suggestive of epithelial-mesenchymal transition (EMT). EMT triggers tumor metastasis. Exit from EMT is marked by increased E-cadherin expression and is considered necessary for tumor growth at sites of metastasis; however, the mechanisms associated with exit from EMT are poorly understood. Herein are analyzed 185 prostate cancer metastases, with significantly higher E-cadherin expression in bone than in lymph node and soft tissue metastases. To determine the molecular mechanisms of regulation of E-cadherin expression, three stable isogenic cell lines from DU145 were derived that differ in structure, migration, and colony formation on soft agar and Matrigel. When injected into mouse tibia, the epithelial subline grows most aggressively, whereas the mesenchymal subline does not grow. In cultured cells, ZEB1 and Src family kinases decrease E-cadherin expression. In contrast, in tibial xenografts, E-cadherin RNA levels increase eight- to 10-fold despite persistent ZEB1 expression, and in all ZEB1-positive metastases (10 of 120), ZEB1 and E-cadherin proteins were co-expressed. These data suggest that transcriptional regulation of E-cadherin differs in cultured cells versus xenografts, which more faithfully reflect E-cadherin regulation in cancers in human beings. Furthermore, the aggressive nature of xenografts positive for E-cadherin and the frequency of metastases positive for E-cadherin suggest that high E-cadherin expression in metastatic prostate cancer is associated with aggressive tumor growth.

Drosophila melanogaster Cad99C, the orthologue of human Usher cadherin PCDH15, regulates the length of microvilli.

Actin-based protrusions can form prominent structures on the apical surface of epithelial cells, such as microvilli. Several cytoplasmic factors have been identified that control the dynamics of actin filaments in microvilli. However, it remains unclear whether the plasma membrane participates actively in microvillus formation. In this paper, we analyze the function of Drosophila melanogaster cadherin Cad99C in the microvilli of ovarian follicle cells. Cad99C contributes to eggshell formation and female fertility and is expressed in follicle cells, which produce the eggshells. Cad99C specifically localizes to apical microvilli. Loss of Cad99C function results in shortened and disorganized microvilli, whereas overexpression of Cad99C leads to a dramatic increase of microvillus length. Cad99C that lacks most of the cytoplasmic domain, including potential PDZ domain-binding sites, still promotes excessive microvillus outgrowth, suggesting that the amount of the extracellular domain determines microvillus length. This study reveals Cad99C as a critical regulator of microvillus length, the first example of a transmembrane protein that is involved in this process.

Phylogenetic characterization of virulence and resistance phenotypes of Pseudomonas syringae.

Individual strains of the plant pathogenic bacterium Pseudomonas syringae vary in their ability to produce toxins, nucleate ice, and resist antimicrobial compounds. These phenotypes enhance virulence, but it is not clear whether they play a dominant role in specific pathogen-host interactions. To investigate the evolution of these virulence-associated phenotypes, we used functional assays to survey for the distribution of these phenotypes among a collection of 95 P. syringae strains. All of these strains were phylogenetically characterized via multilocus sequence typing (MLST). We surveyed for the production of coronatine, phaseolotoxin, syringomycin, and tabtoxin; for resistance to ampicillin, chloramphenicol, rifampin, streptomycin, tetracycline, kanamycin, and copper; and for the ability to nucleate ice at high temperatures via the ice-nucleating protein INA. We found that fewer than 50% of the strains produced toxins and significantly fewer strains than expected produced multiple toxins, leading to the speculation that there is a cost associated with the production of multiple toxins. None of these toxins was associated with host of isolation, and their distribution, relative to core genome phylogeny, indicated extensive horizontal genetic exchange. Most strains were resistant to ampicillin and copper and had the ability to nucleate ice, and yet very few strains were resistant to the other antibiotics. The distribution of the rare resistance phenotypes was also inconsistent with the clonal history of the species and did not associate with host of isolation. The present study provides a robust phylogenetic foundation for the study of these important virulence-associated phenotypes in P. syringae host colonization and pathogenesis.