Kenneth W. Kinzler

Spatial Regulation of CAR Signaling Enables Logic-Gated Activity

Chimeric antigen receptors (CARs) can induce T cells to kill cancer cells but also to kill normal cells that express the same antigens 1 . Designing CARs to recognize combinations of antigens, via Boolean logic, can simultaneously expand the scope of targetable antigens and make CAR T cells more specific to cancer 2 . For example, one antigen may be expressed on cancer cells and normal bone marrow cells, while a second antigen may be present on the same cancer cells but only in normal lungs. If recognition of both antigens is required for T cell activation, only the cancer cells will be killed. Creating such AND-gated CAR T cells has been challenging given the need to engineer non-natural signaling mechanisms that integrate two ligand binding events into a single T cell activation stimulus 3–6 . Here, we design a fundamentally new AND-gated receptor called M ulti- AN tigen T riggered I mmune S ynapse (MANTIS), which leverages differences in extracellular receptor dimensions to regulate CAR signaling. MANTIS initially prevents CAR activity by steric blocking with a bulky extracellular domain. Upon engagement of the first antigen, MANTIS sheds this blocking domain, releasing a free CAR that can bind a second antigen and activate the T cell in an AND-gated manner. This work demonstrates how differences in extracellular receptor size can be leveraged to spatially regulate intracellular signaling pathways in response to antigen patterns, paving the way for new applications in synthetic biology and cell engineering. One sentence summary Differences in extracellular size can be leveraged to regulate CAR T cell activity for precise recognition of antigen patterns.

Bispecific antibodies and CAR T cells targeting a TP53 mutation–associated neoantigen show discordant affinity requirements

Mutation-associated neoantigens (MANAs) are highly cancer-specific targets for immunotherapy where peptides derived from intracellular mutant proteins are presented on the cell surface via HLA molecules. T cell-engaging bispecific antibodies and CAR T cells can target MANAs to eliminate cancer cells via T cell activation. However, the low antigen density of MANAs on the cell surface can limit therapeutic efficacy. Here, we investigated whether increasing the affinity of the H2 single-chain variable fragment (scFv) targeting the p53 R175H MANA (HMTEVVRHC presented on HLA-A*02:01) improves its therapeutic effect. We identified higher-affinity H2 variants via phage biopanning and a thiocyanate elution method. Increasing bispecific antibody affinity to the low nanomolar range increased cancer cell killing and tumor control in mouse xenograft models without sacrificing antigen specificity. We next asked how increasing scFv affinity impacts CAR T cell function - a matter of debate. We appended each variant scFv to a CD28z CAR, CD3γ, or the T cell receptor. In striking contrast to the bispecific antibody results, increasing CAR affinity decreased function in each CAR format due to lower T cell activation upon interaction with target cancer cells. These results have important implications for the design of future immunotherapeutic approaches targeting low-density antigens.

A plasma-based test for the measurable residual disease informs the response to treatment in AML patients

Precision targeting of autoreactive B cells in systemic lupus erythematosus using anti-9G4 idiotope synthetic immune receptor T cells

Data from Detection of Cancers Three Years prior to Diagnosis Using Plasma Cell-Free DNA

Multi-Omics Evaluation of Peritoneal Fluid in Gastroesophageal Cancer: Results From Interim Analysis

Detection of human brain cancers using genomic and immune cell characterization of cerebrospinal fluid through CSF-BAM

Minimizing and quantifying uncertainty in AI-informed decisions: Applications in medicine

NXPE1 alters the sialoglycome by acetylating sialic acids in the human colon

Mild periodic acid Schiff staining (mPAS) of human colonic tissue has been used to answer a variety of fundamental questions in germline and somatic genetics. mPAS stains sialic acids except when these glycans are modified by O-acetylation, but a full accounting of the genes contributing to sialoglycan acetylation is incomplete. Using haplotypes derived from whole genome sequencing, we identify a region on chromosome 11 that is associated with inherited differences in mPAS staining. Of the genes in this region, only haplotypes containing NXPE1 correlate perfectly with mPAS staining in the original cohort used for whole genome sequencing, as well as in a validation cohort. Transcriptomic analysis indicates that linked haplotypes are associated with altered expression of NXPE1 suggesting a possible genetic mechanism. Genetic manipulation of a common single nucleotide polymorphism observed in the haplotype region and located in NXPE1's promoter alters expression and causes changes to modified sialic acid levels supporting this mechanism. Finally, high-performance liquid chromatography (HPLC) confirms that enzymatically active NXPE1 is capable of transferring an acetyl group from acetyl coenzyme A to sialic acid in vitro. These findings suggest that NXPE1 is the long-sought gene responsible for differences in colon mPAS staining and may be the prototype of a new family of sialic acid O-acetylation-modifying genes.

Detection of Cancers Three Years prior to Diagnosis Using Plasma Cell-Free DNA

To explore how early cancers can be detected prior to clinical signs or symptoms, we assessed prospectively collected serial plasma samples from the Atherosclerosis Risk in Communities study, including 26 participants diagnosed with cancer and 26 matched controls. At the index time point, 8 of these 52 participants scored positively with a multicancer early detection test. All eight participants were diagnosed with cancer within 4 months after blood collection. In six of these eight participants, we were able to assess an earlier plasma sample collected 3.1 to 3.5 years prior to clinical diagnosis. In four of these six participants, the same mutations detected by the multicancer early detection test could be identified but at 8.6- to 79-fold lower mutant allele fractions. These results demonstrate that it is possible to detect ctDNA more than 3 years prior to clinical diagnosis and provide benchmark sensitivities required for this purpose. SIGNIFICANCE: Earlier detection is a promising strategy to reduce cancer mortality. For cancers of all stages, therapies are more effective with a lower disease burden. In this study, we demonstrate that ctDNA is detectable 3 years or more before cancer diagnosis and provide estimates for the sensitivity required to achieve such very early detection. See related commentary by Crisafulli et al., p. 1774.

ME3BP-7 is a targeted cytotoxic agent that rapidly kills pancreatic cancer cells expressing high levels of monocarboxylate transporter MCT1

Nearly 30% of pancreatic ductal adenocarcinomas (PDACs) exhibit a marked overexpression of monocarboxylate transporter 1 (MCT1) offering a unique opportunity for therapy. However, biochemical inhibitors of MCT1 have proven unsuccessful in clinical trials. In this study, we present an alternative approach using 3-bromopyruvate (3BP) to target MCT1 overexpressing PDACs. 3BP is a cytotoxic agent that is known to be transported into cells via MCT1, but its clinical usefulness has been hampered by difficulties in delivering the drug systemically. We describe here a novel microencapsulated formulation of 3BP (ME3BP-7), which is effective against a variety of PDAC cells in vitro and remains stable in serum. Furthermore, systemically administered ME3BP-7 significantly reduces pancreatic cancer growth and metastatic spread in multiple orthotopic models of pancreatic cancer with manageable toxicity. ME3BP-7 is, therefore, a prototype of a promising new drug, in which the targeting moiety and the cytotoxic moiety are both contained within the same single small molecule.

A Blood-Based Assay for Detection of Patients with Advanced Adenomas

Screening for colorectal cancer with blood-based testing should detect advanced adenomas (AA), facilitating more effective cancer prevention. We evaluated four different methods to detect AAs in plasma: (i) a machine learning algorithm, Signatures of fragment Length (SignaL), based on cell-free DNA (cfDNA) fragmentation; (ii) a “Protein-17” assay measuring 17 cancer-associated proteins; (iii) a global aneuploidy score (GAS); and (iv) cfDNA mutation analysis querying 15 genes commonly mutated in colorectal cancer. Existing data from study populations with and without cancer were utilized to determine 99.5% specificity thresholds. We studied 40 AA cases and 32 colonoscopy-negative controls. SignaL detected 9/40 AAs [22.5%, 95% confidence interval (CI), 12.3%–37.5%] at 100% specificity (95% CI, 89.3%–100%). Protein-17 detected 5/40 AAs (12.5%, 95% CI, 5.5%–26.1%) including three cases not identified by SignaL, at 100% specificity (95% CI, 89.3%–100%). GAS detected 11/40 AAs (27.5%, 95% CI, 16.1%–42.8%) but resulted in 2/32 positive controls (93.8% specificity, 95% CI, 79.9%–98.3%). Combining SignaL, the Protein-17 assay, and GAS at the 99.5% specificity thresholds resulted in detection of 16/40 AAs (40%, 95% CI, 26.3%–55.4%) at 93.8% specificity (95% CI, 79.9%–98.3%). Of 32/40 evaluable AA plasma samples, only one was cfDNA mutation positive at level of significance of 0.01. A blood-based assay based on the analysis of repeated sequence elements plus proteins seems to be able to detect a considerable fraction of patients with AA at relatively high specificity. Large, prospective studies are required to determine whether this approach can add to the options currently available for screening patients for premalignant lesions of the colon. SIGNIFICANCE: Blood-based screening for colorectal cancer could improve testing uptake and outcomes. We propose novel methods to detect AAs in plasma using cfDNA fragmentation patterns, cancer-associated proteins, and aneuploidy with high specificity. Larger studies are needed to validate clinical utility.

Identification of nonsense-mediated decay inhibitors that alter the tumor immune landscape

Despite exciting developments in cancer immunotherapy, its broad application is limited by the paucity of targetable antigens on the tumor cell surface. As an intrinsic cellular pathway, nonsense-mediated decay (NMD) conceals neoantigens through the destruction of the RNA products from genes harboring truncating mutations. We developed and conducted a high-throughput screen, based on the ratiometric analysis of transcripts, to identify critical mediators of NMD in human cells. This screen implicated disruption of kinase SMG1’s phosphorylation of UPF1 as a potential disruptor of NMD. This led us to design a novel SMG1 inhibitor, KVS0001, that elevates the expression of transcripts and proteins resulting from human and murine truncating mutations in vitro and murine cells in vivo. Most importantly, KVS0001 concomitantly increased the presentation of immune-targetable human leukocyte antigens (HLA) class I-associated peptides from NMD-downregulated proteins on the surface of human cancer cells. KVS0001 provides new opportunities for studying NMD and the diseases in which NMD plays a role, including cancer and inherited diseases.

Detection of human brain cancers using genomic and immune cell characterization of cerebrospinal fluid through CSF-BAM

Patients who have radiographically detectable lesions in their brain or other symptoms compatible with brain tumors pose challenges for diagnosis. The only definitive way to diagnose such patients is through brain biopsy, an obviously invasive and dangerous procedure. Here we present a new workflow termed “CSF-BAM” that simultaneously identifies B cell or T cell receptor rearrangements, A neuploidy, and M utations using PCR-mediated amplification of both strands of the DNA from CSF samples. We first describe the details of the molecular genetic assessments and then establish thresholds for positivity using training sets of libraries from patients with or without cancer. We then applied CSF-BAM to an independent set of 206 DNA samples from patients with common, aggressive cancer types as well as other forms of brain cancers. Among the 126 samples from patients with the most common aggressive cancer types (high grade gliomas, medulloblastomas, or metastatic cancers to the brain), the sensitivity of detection was >81%. None of 33 CSF-BAM assays (100% specificity, 90% to 100% credible interval) were positive in CSF samples from patients without brain cancers. The sensitivity of CSF-BAM was considerably higher than that achieved with cytology. CSF-BAM provides an integrated multi-analyte approach to identify neoplasia in the central nervous system, provides information about the immune environment in patients with or without cancer, and has the potential to inform the subsequent management of such patients. Statement of significance There is a paucity of technologies beyond surgical biopsy that can accurately diagnose central nervous system neoplasms. We developed a novel, sensitive and highly specific assay that can detect brain cancers by comprehensively identifying somatic mutations, chromosomal copy number changes, and adaptive immunoreceptor repertoires from samples of cerebrospinal fluid.

NXPE1 alters the sialoglycome by acetylating sialic acids in the human colon

Mild periodic acid Schiff staining (mPAS) of human colonic tissue has been used to answer a variety of fundamental questions in germline and somatic genetics. mPAS staining is known to reflect sialic acid O-acetylation, but a full accounting of the genes contributing to sialoglycome diversity is incomplete. Using haplotypes derived from whole genome sequencing, we identified a region on chromosome 11 that is associated with inherited differences in mPAS staining. Of the genes in this region, only NXPE1 haplotypes correlated perfectly with mPAS staining in the original cohort used for whole genome sequencing, as well as in a validation cohort. Transcriptomic analysis suggested that one common allele of NXPE1, with a single base pair substitution in its promoter region, greatly reduces expression of the gene. Genetic manipulation of NXPE1 expression confirmed this conclusion and caused changes to modified sialic acid levels. Finally, high-performance liquid chromatography (HPLC) confirms that enzymatically active NXPE1 is capable of transferring an acetyl group from acetyl coenzyme A to sialic acid in vitro. These findings suggest that NXPE1 is the long-sought gene responsible for differences in colon mPAS staining and may be the prototype of a new family of sialic acid O-acetylation-modifying genes.

ME3BP-7 is a targeted cytotoxic agent that rapidly kills pancreatic cancer cells expressing high levels of monocarboxylate transporter MCT1

Nearly 30% of Pancreatic ductal adenocarcinoma (PDAC)s exhibit a marked overexpression of Monocarboxylate Transporter 1 (MCT1) offering a unique opportunity for therapy. However, biochemical inhibitors of MCT1 have proven unsuccessful in clinical trials. In this study we present an alternative approach using 3-Bromopyruvate (3BP) to target MCT1 overexpressing PDACs. 3BP is a cytotoxic agent that is known to be transported into cells via MCT1, but its clinical usefulness has been hampered by difficulties in delivering the drug systemically. We describe here a novel microencapsulated formulation of 3BP (ME3BP-7), that is effective against a variety of PDAC cells in vitro and remains stable in serum. Furthermore, systemically administered ME3BP-7 significantly reduces pancreatic cancer growth and metastatic spread in multiple orthotopic models of pancreatic cancer with manageable toxicity. ME3BP-7 is, therefore, a prototype of a promising new drug, in which the targeting moiety and the cytotoxic moiety are both contained within the same single small molecule.

Figure S7 from The origin of highly elevated cell-free DNA in healthy individuals and patients with pancreatic, colorectal, lung, or ovarian cancer

Supplemental Figure 7. The amount of total cfDNA before and after surgery in patients with pancreatic cancer.

Figure S2 from The origin of highly elevated cell-free DNA in healthy individuals and patients with pancreatic, colorectal, lung, or ovarian cancer

Supplemental Figure 2. The concentration of plasma cfDNA in previous described patients with cancer according to stage. (A) The concentration of cfDNA in patients of all cancer types (9). The concentration of cfDNA in patients with Stage III cancers was significantly higher than that of patients with Stage I cancers (p < 0.01). (B) The concentration of cfDNA in individual cancer types. Stage was classified according to AJCC 7th edition. The concentration of cfDNA in patients with Stage III breast cancer was significantly higher than that of patients with Stage I breast cancer (p < 0.05). For both panels, data were derived from the previously published CancerSEEK study (9).

Figure S10 from The origin of highly elevated cell-free DNA in healthy individuals and patients with pancreatic, colorectal, lung, or ovarian cancer

Supplemental Figure 10. In silico mixing experiments (N = 10) of buffy coat bisulfite sequencing data with liver (A), lung (B), colon epithelial cell (C), and left atrium (D) bisulfite sequencing data deconvoluted using the Moss et al. (43) reference matrix and quadratic programming shows excellent agreement between predicted and actual fractional contribution.

Supplementary Notes 1-5 from The origin of highly elevated cell-free DNA in healthy individuals and patients with pancreatic, colorectal, lung, or ovarian cancer

Supplementary Notes 1-5. Supplementary Note 1: Origins of cell-free DNA. Supplementary Note 2: Leukocyte Lysis. Supplementary Note 3: Reference datasets and deconvolution algorithms used to interpret whole genome bisulfite sequencing data. Supplementary Note 4: Turnover rates. Supplementary Note 5: Relationships between ctDNA and tissue specific cfDNA in cancer patients.