SignificanceEx vivo manipulation of primary cells is critical to the success of this emerging generation of cell-based therapies, such as chimeric antigen receptor T cells for the treatment of cancer and CRISPR for the correction of developmental diseases. However, the limitations of existing delivery approaches may dramatically restrict the impact of genetic engineering to study and treat disease. In this paper, we compared electroporation to a microfluidic membrane deformation technique termed “squeezing” and found that squeezed cells had dramatically fewer side effects than electroporation and gene expression profiles similar to those of unmanipulated cells. The significant differences in outcomes from the two techniques underscores the importance of understanding the impact of intracellular delivery methods on cell function for research and clinical applications.

The presence of T regulatory (Treg) cells in the tumor microenvironment is associated with poor prognosis and resistance to therapies aimed at reactivating anti-tumor immune responses. Therefore, depletion of tumor-infiltrating Tregs is a potential approach to overcome resistance to immunotherapy. However, identifying Treg-specific targets to drive such selective depletion is challenging. CCR8 has recently emerged as one of these potential targets. Here, we describe GS-1811, a novel therapeutic monoclonal antibody that specifically binds to human CCR8 and is designed to selectively deplete tumor-infiltrating Tregs. We validate previous findings showing restricted expression of CCR8 on tumor Tregs, and precisely quantify CCR8 receptor densities on tumor and normal tissue T cell subsets, demonstrating a window for selective depletion of Tregs in the tumor. Importantly, we show that GS-1811 depleting activity is limited to cells expressing CCR8 at levels comparable to tumor-infiltrating Tregs. Targeting CCR8 in mouse tumor models results in robust anti-tumor efficacy, which is dependent on Treg depleting activity, and synergizes with PD-1 inhibition to promote anti-tumor responses in PD-1 resistant models. Our data support clinical development of GS-1811 to target CCR8 in cancer and drive tumor Treg depletion in order to promote anti-tumor immunity.

Introduction: Immune checkpoint blockade (ICB) has revolutionized the treatment of many cancers, still most patients do not respond to PD-1 or CTLA-4 inhibitors. Thus, new Immuno-Oncology (IO) therapies that could potentially benefit non-responding patients are greatly needed. Jounce has generated cell type-specific gene signatures as a means of probing The Cancer Genome Atlas and other large datasets for novel IO targets. Regulatory T cells (Tregs) are one attractive cell type for targeting as they may contribute to resistance to ICB. While Tregs are critical for immune homeostasis and preventing tissue damage, they are often present in large numbers within tumors where they may suppress anti-tumor immunity. Therapeutic strategies that specifically deplete tumor-infiltrating Tregs (TITRs) while sparing peripheral and normal tissue Tregs are highly desirable. Using a Treg gene signature, we have a found a strong correlation with TITRs and CCR8 (C-C motif chemokine receptor 8) across multiple tumor types. CCR8 may be differentiated from other known Treg targets in this regard, as its expression was found to be highly selective to TITRs. Methods and Results: We first assessed CCR8 levels on TITRs across multiple tumor types and compared expression to Tregs in normal colon tissue or peripheral blood. On average, peripheral blood Tregs had nearly undetectable CCR8 expression and normal colon tissue Tregs showed 4 to 5-fold lower levels of CCR8 than TITRs. We then generated a panel of monoclonal antibodies (mAbs) that bind specifically to CCR8, but not other family members, and block CCR8 signaling induced by its ligand CCL1. The ability of these mAbs to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) of target cells expressing CCR8 was tested. When target cells expressed CCR8 at levels equivalent to normal tissue Tregs no ADCC activity was observed. In contrast, when cells expressed CCR8 at levels equivalent to TITRs, robust ADCC was observed, but only using antibodies in which the human IgG1 Fc was afucosylated. Thus, afucosylated anti-CCR8 antibodies demonstrated a therapeutic window whereby TITRs but not normal tissue Tregs could be depleted. An Fc competent, mouse-specific, anti-CCR8 antibody showed single agent tumor growth inhibition across several murine tumor models - including models in which anti-PD-1 was ineffective. Anti-CCR8 was a potent combination partner with anti-PD-1 resulting in 50% complete tumor regressions in PD-1 resistant models. Conclusions: Based on these pre-clinical data JTX-1811, a high affinity CCR8-specific humanized monoclonal antibody with enhanced ADCC activity, is being developed for the selective depletion of tumor-infiltrating Tregs. JTX-1811 may be useful in PD-1 resistant settings and may restore the activity of PD-1 inhibitors in the setting of primary or acquired resistance to ICB. Citation Format: Fabien Dépis, Changyun Hu, Jessica Weaver, Lara McGrath, Boris Klebanov, Joshua Buggé, Ben Umiker, Christine Fregeau, Dhruvkumar Upadhyay, Anirudh Singh, Chang-Ai Xu, Vikki Spaulding, Michelle Priess, Masie Wong, Seema Naheed, Yan Zhang, Kristin Legendre, Edward C. Stack, Alessandro Mora, Margaret Willer, Kristan Meetze, Monica Gostissa, Michael A. Meehl, Donald R. Shaffer. Preclinical evaluation of JTX-1811, an anti-CCR8 antibody with enhanced ADCC activity, for preferential depletion of tumor-infiltrating regulatory T cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4532.

An electroporation-mediated genetic transformation or the marine diatom Thalassiosira pseudonana was developed. Using a sorbitol-based buffer, T. pseudonana cells were successfully transformed with the Tpfcp/nat plasmid at an efficiency of 2820 per 10 8 cells. This represents a six-fold improvement compared with previously published methods.

While the ex vivo manipulation of primary cells has signaled a new era in the application of cell-based therapies, common methods to manipulate primary cells have limitations. To overcome the limitations associated with conventional cell delivery and engineering systems, we have developed an approach to delivery where cells are mechanically deformed as they pass through a constriction. This cellular deformation results in the diffusion of material from the surrounding buffer directly into the cytosol. This system has demonstrated efficacy in patient-derived cells, such as stem cells and immune cells, with a variety of target molecules that are difficult to address with alternative methods. Moreover, by eliminating the need for electrical fields or exogenous materials such as viral vectors and plasmids, it minimizes the potential for cell toxicity and off-target effects. Here, we present evidence detailing our ability to deliver functional material to primary human CD34+ cells via cell deformation with little detectable perturbation in baseline gene expression, cell function, and viability. To determine effect of cell deformation on gene expression and to compare to other delivery systems, human CD34+ cells (n = 3 donors) were subjected to cell deformation or electroporation and gene expression changes were compared to unmanipulated control cells using microarray analysis. Differential gene expression with respect to both methods of delivery was assessed by performing t tests on the coefficient of a linear mixed-effects model that treated delivery method as a fixed effect and donor as a random effect. Electroporation produced substantially more changes in gene expression (5,285 genes with FDR q < 0.25) than cell deformation (no genes with FDR q < 0.25) as compared to untreated controls. Subsequently, we designed a series of experiments to manipulate gene expression with the CRISPR-CAS9 system using cell deformation to deliver CAS9 ribonucleoproteins (RNPs; recombinant CAS9 protein complexed with a single-guide RNA) designed to edit a model locus, the C-C chemokine receptor type 5 (CCR5). Here, we show that the delivery of the CRISPR-CAS9 system via cell deformation results in significant CCR5 mutagenesis. Furthermore, CD34+ cells subjected to cell deformation proliferate and differentiate at rates similar to unmanipulated control cells, as determined by the Colony-Forming Cell (CFC) assay. Disruption of the CCR5 locus was observed in individual BFU-E colonies by performing Sanger sequencing. These data suggest that cell deformation is a viable delivery method for genetic engineering of primary human CD34+ cells with little impact on baseline gene expression or the ability of hematopoietic progenitors to proliferate and differentiate. The ability to deliver structurally diverse materials to difficult-to-transfect primary CD34+ cells indicate that this method could potentially enable many novel clinical applications. Disclosures Bridgen: SQZ Biotechnologies: Employment, Equity Ownership. DiTommaso:SQZ Biotechnologies: Employment, Equity Ownership. Buggé:SQZ Biotechnologies: Employment, Equity Ownership. Gilbert:SQZ Biotechnologies: Employment, Equity Ownership. Bernstein:SQZ Biotechnologies: Employment, Equity Ownership. Sharei:SQZ Biotechnologies: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.