Overcoming lung cancer immunotherapy resistance by combining nontoxic variants of IL-12 and IL-2.

Engineered cytokine-based approaches for immunotherapy of cancer are poised to enter the clinic, with IL-12 being at the forefront. However, little is known about potential mechanisms of resistance to cytokine therapies. We found that orthotopic murine lung tumors were resistant to systemically delivered IL-12 fused to murine serum albumin (MSA, IL12-MSA) because of low IL-12 receptor (IL-12R) expression on tumor-reactive CD8+ T cells. IL2-MSA increased binding of IL12-MSA by tumor-reactive CD8+ T cells, and combined administration of IL12-MSA and IL2-MSA led to enhanced tumor-reactive CD8+ T cell effector differentiation, decreased numbers of tumor-infiltrating CD4+ regulatory T cells, and increased survival of lung tumor-bearing mice. Predictably, the combination of IL-2 and IL-12 at therapeutic doses led to significant dose-limiting toxicity. Administering IL-12 and IL-2 analogs with preferential binding to cells expressing Il12rb1 and CD25, respectively, led to a significant extension of survival in mice with lung tumors while abrogating dose-limiting toxicity. These findings suggest that IL-12 and IL-2 represent a rational approach to combination cytokine therapy whose dose-limiting toxicity can be overcome with engineered cytokine variants.

Investigative needle core biopsies for multi-omics in Glioblastoma.

Glioblastoma (GBM) is a primary brain cancer with an abysmal prognosis and few effective therapies. The ability to investigate the tumor microenvironment before and during treatment would greatly enhance both understanding of disease response and progression, as well as the delivery and impact of therapeutics. Stereotactic biopsies are a routine surgical procedure performed primarily for diagnostic histopathologic purposes. The role of investigative biopsies - tissue sampling for the purpose of understanding tumor microenvironmental responses to treatment using integrated multi-modal molecular analyses ('Multi-omics") has yet to be defined. Secondly, it is unknown whether comparatively small tissue samples from brain biopsies can yield sufficient information with such methods. Here we adapt stereotactic needle core biopsy tissue in two separate patients. In the first patient with recurrent GBM we performed highly resolved multi-omics analysis methods including single cell RNA sequencing, spatial-transcriptomics, metabolomics, proteomics, phosphoproteomics, T-cell clonotype analysis, and MHC Class I immunopeptidomics from biopsy tissue that was obtained from a single procedure. In a second patient we analyzed multi-regional core biopsies to decipher spatial and genomic variance. We also investigated the utility of stereotactic biopsies as a method for generating patient derived xenograft models in a separate patient cohort. Dataset integration across modalities showed good correspondence between spatial modalities, highlighted immune cell associated metabolic pathways and revealed poor correlation between RNA expression and the tumor MHC Class I immunopeptidome. In conclusion, stereotactic needle biopsy cores are of sufficient quality to generate multi-omics data, provide data rich insight into a patient's disease process and tumor immune microenvironment and can be of value in evaluating treatment responses. One sentence summary: Integrative multi-omics analysis of stereotactic needle core biopsies in glioblastoma.

Identification and quantitation of hinge cysteinylation on endogenous IgG2 antibodies from human serum.

Cysteinylation is a post-translational modification (PTM) that occurs when a cysteine residue on a protein forms a disulfide bond with a terminal cysteine molecule. This PTM has been found in the hinge region of several recombinant therapeutic IgG2 antibodies, but the impact of cysteinylation on the safety and immunogenicity of therapeutics remains unclear. In this study, we characterized recombinant and endogenous IgG2 antibodies to quantify their levels of hinge cysteinylation, if present. To the best of our knowledge, this is the first study to identify and quantify hinge cysteinylation in endogenous IgG2 antibodies from healthy human serum. We used anti-IgG2 immunopurification of human serum to specifically enrich for endogenous IgG2 antibodies, and then subjected the resulting samples to Lys-C peptide mapping coupled with targeted mass spectrometry techniques. Using this analytical workflow, we found that all healthy human serum samples tested (N = 10) contained quantifiable levels of hinge cysteinylation (0.8 ± 0.3%) in their endogenous human IgG2s (IgG2-A isoform). These findings demonstrate that hinge cysteinylation in therapeutic IgG2s, at least up to a certain level, is well tolerated in humans and pose minimal safety or immunogenicity risks.

Engineering of Chimeric Polyketide Synthases Using SYNZIP Docking Domains.

Engineering of assembly line polyketide synthases (PKSs) to produce novel bioactive compounds has been a goal for over 20 years. The apparent modularity of PKSs has inspired many engineering attempts in which entire modules or single domains were exchanged. In recent years, it has become evident that certain domain-domain interactions are evolutionarily optimized and, if disrupted, cause a decrease of the overall turnover rate of the chimeric PKS. In this study, we compared different types of chimeric PKSs in order to define the least invasive interface and to expand the toolbox for PKS engineering. We generated bimodular chimeric PKSs in which entire modules were exchanged, while either retaining a covalent linker between heterologous modules or introducing a noncovalent docking domain, or SYNZIP domain, mediated interface. These chimeric systems exhibited non-native domain-domain interactions during intermodular polyketide chain translocation. They were compared to otherwise equivalent bimodular PKSs in which a noncovalent interface was introduced between the condensing and processing parts of a module, resulting in non-native domain interactions during the extender unit acylation and polyketide chain elongation steps of their catalytic cycles. We show that the natural PKS docking domains can be efficiently substituted with SYNZIP domains and that the newly introduced noncovalent interface between the condensing and processing parts of a module can be harnessed for PKS engineering. Additionally, we established SYNZIP domains as a new tool for engineering PKSs by efficiently bridging non-native interfaces without perturbing PKS activity.

R-loop generation during transcription: Formation, processing and cellular outcomes.

R-loops are structures consisting of an RNA-DNA duplex and an unpaired DNA strand. They can form during transcription upon nascent RNA "threadback" invasion into the DNA duplex to displace the non-template strand. Although R-loops occur naturally in all kingdoms of life and serve regulatory roles, they are often deleterious and can cause genomic instability. Of particular importance are the disastrous consequences when replication forks or transcription complexes collide with R-loops. The appropriate processing of R-loops is essential to avoid a number of human neurodegenerative and other clinical disorders. We provide a perspective on mechanistic aspects of R-loop formation and their resolution learned from studies in model systems. This should contribute to improved understanding of R-loop biological functions and enable their practical applications. We propose the novel employment of artificially-generated stable R-loops to selectively inactivate tumor cells.

A novel mode for transcription inhibition mediated by PNA-induced R-loops with a model in vitro system.

The selective inhibition of transcription of a chosen gene by an artificial agent has numerous applications. Usually, these agents are designed to bind a specific nucleotide sequence in the promoter or within the transcribed region of the chosen gene. However, since optimal binding sites might not exist within the gene, it is of interest to explore the possibility of transcription inhibition when the agent is designed to bind at other locations. One of these possibilities arises when an additional transcription initiation site (e.g. secondary promoter) is present upstream from the primary promoter of the target gene. In this case, transcription inhibition might be achieved by inducing the formation of an RNA-DNA hybrid (R-loop) upon transcription from the secondary promoter. The R-loop could extend into the region of the primary promoter, to interfere with promoter recognition by RNA polymerase and thereby inhibit transcription. As a sequence-specific R-loop-inducing agent, a peptide nucleic acid (PNA) could be designed to facilitate R-loop formation by sequestering the non-template DNA strand. To investigate this mode for transcription inhibition, we have employed a model system in which a PNA binding site is localized between the T3 and T7 phage RNA polymerase promoters, which respectively assume the roles of primary and secondary promoters. In accord with our model, we have demonstrated that with PNA-bound DNA substrates, transcription from the T7 promoter reduces transcription from the T3 promoter by 30-fold, while in the absence of PNA binding there is no significant effect of T7 transcription upon T3 transcription.