Myeloid and dendritic cells enhance therapeutics-induced cytokine release syndrome features in humanized BRGSF-HIS preclinical model.

Objectives: Despite their efficacy, some immunotherapies have been shown to induce immune-related adverse events, including the potentially life-threatening cytokine release syndrome (CRS), calling for reliable and translational preclinical models to predict potential safety issues and investigate their rescue. Here, we tested the reliability of humanized BRGSF mice for the assessment of therapeutics-induced CRS features in preclinical settings. Methods: BRGSF mice reconstituted with human umbilical cord blood CD34+ cells (BRGSF-CBC) were injected with anti-CD3 antibody (OKT3), anti-CD3/CD19 bispecific T-cell engager Blinatumomab, or VISTA-targeting antibody. Human myeloid and dendritic cells' contribution was investigated in hFlt3L-boosted BRGSF-CBC mice. OKT3 treatment was also tested in human PBMC-reconstituted BRGSF mice (BRGSF-PBMC). Cytokine release, immune cell distribution, and clinical signs were followed. Results: OKT3 injection in BRGSF-CBC mice induced hallmark features of CRS, specifically inflammatory cytokines release, modifications of immune cell distribution and activation, body weight loss, and temperature drop. hFlt3L-boosted BRGSF-CBC mice displayed enhanced CRS features, revealing a significant role of myeloid and dendritic cells in this process. Clinical CRS-managing treatment Infliximab efficiently attenuated OKT3-induced toxicity. Comparison of OKT3 treatment's effect on BRGSF-CBC and BRGSF-PBMC mice showed broadened CRS features in BRGSF-CBC mice. CRS-associated features were also observed in hFlt3L-boosted BRGSF-CBC mice upon treatment with other T-cell or myeloid-targeting compounds. Conclusions: These data show that BRGSF-CBC mice represent a relevant model for the preclinical assessment of CRS and CRS-managing therapies. They also confirm a significant role of myeloid and dendritic cells in CRS development and exhibit the versatility of this model for therapeutics-induced safety assessment.

The future is on the way: ß cell function preservation for type 1 diabetes.

This phase 3 trial of PROTECT confirms the beneficial effects of teplizumab (anti-CD3 treatment) in type 1 diabetes and expands the indications from diabetes prevention (stage 2) to early treatment (stage 3). More efforts are needed to confirm the effects in the general population, with a higher dose, and with repeated dosing.

EGF-Receptor against Amphiregulin (AREG) Influences Costimulatory Molecules on Monocytes and T Cells and Modulates T-Cell Responses.

Amphiregulin (AREG) is a ligand of the epidermal growth factor receptor (EGFR) and has been shown to regulate the phagocytosis-induced cell death of monocytes in peripheral blood. AREG-dependent apoptotic signaling engages factors of the intrinsic and extrinsic apoptotic pathway, such as BCL-2, BCL-XL, and death ligand/receptor CD95/CD95L. Here, we tested the hypothesis that AREG influences costimulatory monocyte functions, which are crucial for T-cell responses. We found a stronger expression of AREG and EGFR in monocytes compared to lymphocytes. As a novel function of AREG, we observed reduced T-cell proliferation following polyclonal T-cell stimulation with OKT3. This reduction of proliferation occurred in the presence of monocytes as well as in their absence, monocyte signaling being replaced by crosslinking of OKT3. Increasing concentrations of AREG down-modulated the concentration of costimulatory B7 molecules (CD80/CD86) and HLA-DR on monocytes. In proliferation assays, CD28 expression on T cells was down-modulated on the application of OKT3 but unaltered by AREG. LcK activation, following OKT3-stimulation, was reduced in T cells that had been coincubated with AREG. The effects of AREG on T-cell phenotypes were also present when monocytes were depleted and OKT3 was crosslinked. The rearranged expression of immunological synapse proteins was accompanied by an alteration of T-cell polarization. Although the proportion of regulatory T cells was not shifted by AREG, IL-17-expressing T cells were significantly enhanced, with a bias toward TH1-polarization. Taken together, these results suggest that AREG acts as an immunoregulatory molecule at the interface between antigen-presenting cells and T cells.

Ex-vivo CS1-OKT3 dual specific bivalent antibody-armed effector T cells mediate cellular immunity against multiple myeloma.

Bispecific T cell engaging antibodies (bsAbs) have emerged as novel and powerful therapeutic agents for redirecting T cells towards antigen-specific tumor killing. The cell surface glycoprotein and SLAM family member, CS1, exhibits stable and high-level expression on malignant plasma cells including multiple myeloma, which is indicative of an ideal target for bsAb therapy. Here, we developed a CS1 bsAb (CS1-dbBiTE) using Click chemistry to conjugate intact anti-CS1 antibody (Elotuzumab) and anti-huOKT3 antibody at their respective hinge regions. Using a cellular therapy approach, human T cells were armed ex-vivo with CS1-dbBiTE prior to examining effector activity. Our data indicates that arming T cells with CS1-dbBiTE induced T cell activation and expansion and subsequent cytotoxic activity against CS1-bearing MM tumors, demonstrated by significant CD107a expression as well as inflammatory cytokine secretion. As expected, CS1-dbBiTE armed T cells showed significantly reduced effector activity in the absence of CS1 expression. Similarly, in MM mouse xenograft studies, armed T cells exhibited effective anti-tumor efficacy highlighted by reduced tumor burden in MM.1S tumor-bearing mice compared to controls. On the basis of these findings, the rationale for CS1 targeting by human T cells armed with CS1-dbBiTE presents a potentially effective therapeutic approach for targeting MM.

A multi-functional drug delivery nanosystem release of TLR-7 immunostimulant and OKT3 induced efficient cancer immunotherapy.

Immunotherapy has some shortcomings such as off-target toxicity, treatment time and poor immunogenicity, which limit its therapeutic effect. Nanomaterials are particularly attractive in immunotherapy due to their drug delivery capabilities. Nano drug delivery system loaded with Toll-like receptor (TLR) agonist imiquimod (IMQ) and CD3 immune antibody OKT3 is constructed by using polydopamine (PDA) and CaCO3. While PDA-IMQ@CaCO3-OKT3 (PICO NPs) drug delivery system has the advantages of high biocompatibility, low toxicity, degradability. Antitumor studies in vitro and in vivo have shown that the system can effectively inhibit the proliferation of mouse breast cancer cells and the activity of Regulatory T Cells (Tregs), activate immunogenic cell death (ICD), and enhance the activity of antigen-presenting cells (APCs). Effectively eliminate tumor immunosuppression and fully activate immune function.

Anti-CD79b/CD3 bispecific antibody combined with CAR19-T cells for B-cell lymphoma treatment.

CD19 CAR-T (chimeric antigen receptor-T) cell immunotherapy achieves a remission rate of approximately 70% in recurrent and refractory lymphoma treatment. However, the loss or reduction of CD19 antigen on the surface of lymphoma cells results in the escape of tumor cells from the immune killing of CD19 CAR-T cells (CAR19-T). Therefore, novel therapeutic strategies are urgently required. In this study, an anti-CD79b/CD3 bispecific antibody (BV28-OKT3) was constructed and combined with CAR19-T cells for B-cell lymphoma treatment. When the CD19 antigen was lost or reduced, BV28-OKT3 redirected CAR19-T cells to CD79b+ CD19- lymphoma cells; therefore, BV28-OKT3 overcomes the escape of CD79b+ CD19- lymphoma cells by the killing action of CAR19-T cells in vitro and in vivo. Furthermore, BV28-OKT3 triggered the antitumor function of CAR- T cells in the infusion product and boosted the antitumor immune response of bystander T cells, markedly improving the cytotoxicity of CAR19-T cells to lymphoma cells in vitro and in vivo. In addition, BV28-OKT3 elicited the cytotoxicity of donor-derived T cells toward lymphoma cells in vitro, which depended on the presence of tumor cells. Therefore, our findings provide a new clinical treatment strategy for recurrent and refractory B-cell lymphoma by combining CD79b/CD3 BsAb with CAR19-T cells.

Novel anti-CD30/CD3 bispecific antibodies activate human T cells and mediate potent anti-tumor activity.

CD30 is expressed on Hodgkin lymphomas (HL), many non-Hodgkin lymphomas (NHLs), and non-lymphoid malignancies in children and adults. Tumor expression, combined with restricted expression in healthy tissues, identifies CD30 as a promising immunotherapy target. An anti-CD30 antibody-drug conjugate (ADC) has been approved by the FDA for HL. While anti-CD30 ADCs and chimeric antigen receptors (CARs) have shown promise, their shortcomings and toxicities suggest that alternative treatments are needed. We developed novel anti-CD30 x anti-CD3 bispecific antibodies (biAbs) to coat activated patient T cells (ATCs) ex vivo prior to autologous re-infusions. Our goal is to harness the dual specificity of the biAb, the power of cellular therapy, and the safety of non-genetically modified autologous T cell infusions. We present a comprehensive characterization of the CD30 binding and tumor cell killing properties of these biAbs. Five unique murine monoclonal antibodies (mAbs) were generated against the extracellular domain of human CD30. Resultant anti-CD30 mAbs were purified and screened for binding specificity, affinity, and epitope recognition. Two lead mAb candidates with unique sequences and CD30 binding clusters that differ from the ADC in clinical use were identified. These mAbs were chemically conjugated with OKT3 (an anti-CD3 mAb). ATCs were armed and evaluated in vitro for binding, cytokine production, and cytotoxicity against tumor lines and then in vivo for tumor cell killing. Our lead mAb was subcloned to make a Master Cell Bank (MCB) and screened for binding against a library of human cell surface proteins. Only huCD30 was bound. These studies support a clinical trial in development employing ex vivo-loading of autologous T cells with this novel biAb.

Genetic code expansion in E. coli enables production of a functional 'ready-to-click' T cell receptor-specific scFv.

Antibody-based cancer therapies have been evolving at a rapid pace in the pharmaceutical market. Bispecific antibody-drug conjugates that engage immune cells to target and kill cancer cells with precision have inspired the development of immunotherapy. Miniaturized antibody fragments such as diabodies, nanobodies, or single-chain variable fragments (scFvs) hold great promise as antibody-drug conjugates as they specifically target tumor tissue and can penetrate it. Here, we optimized the soluble periplasmic expression of the scFv OKT3 comprising the variable VH and VL domains of the mouse anti-human CD3 antibody muromonab-CD3 (trade name Orthoclone OKT3) in E. coli. By an expansion of the genetic code, we site-specifically incorporated the reactive non-canonical amino acid Nε-((2-azidoethoxy)carbonyl)-L-lysine (AzK) into scFv OKT3 using an orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair. To confirm the AzK incorporation and to demonstrate the accessibility of the reactive azide group, we conjugated a fluorophore to scFv OKT3 AzK variants by copper-free strain-promoted alkyne-azide cycloaddition ('click chemistry'). The scFv OKT3 wild type and the AzK variants bound T cells at nanomolar concentrations. In this study, a 'ready-to-click' scFv OKT3 was successfully developed for future applications, e.g. as controlled anti-T cell antibody-drug conjugate or bispecific T cell engager and for imaging immune T cell migration in cancers.

Structure-based engineering of a novel CD3ε-targeting antibody for reduced polyreactivity.

Bispecific antibodies continue to represent a growth area for antibody therapeutics, with roughly a third of molecules in clinical development being T-cell engagers that use an anti-CD3 binding arm. CD3 antibodies possessing cross-reactivity with cynomolgus monkey typically recognize a highly electronegative linear epitope at the extreme N-terminus of CD3 epsilon (CD3ε). Such antibodies have high isoelectric points and display problematic polyreactivity (correlated with poor pharmacokinetics for monospecific antibodies). Using insights from the crystal structure of anti-Hu/Cy CD3 antibody ADI-26906 in complex with CD3ε and antibody engineering using a yeast-based platform, we have derived high-affinity CD3 antibody variants with very low polyreactivity and significantly improved biophysical developability. Comparison of these variants with CD3 antibodies in the clinic (as part of bi- or multi-specifics) shows that affinity for CD3 is correlated with polyreactivity. Our engineered CD3 antibodies break this correlation, forming a broad affinity range with no to low polyreactivity. Such antibodies will enable bispecifics with improved pharmacokinetic and safety profiles and suggest engineering solutions that will benefit the large and growing sector of T-cell engagers.

Nasal administration of anti-CD3 monoclonal antibody modulates effector CD8+ T cell function and induces a regulatory response in T cells in human subjects.

Background: Parenteral anti-CD3 Mab (OKT3) has been used to treat transplant rejection and parental administration of a humanized anti-CD3 Mab (Teplizumab) showed positive effects in diabetes. Nasal administration of anti-CD3 Mab has not been carried out in humans. Nasal anti-CD3 Mab suppresses autoimmune diseases and central nervous system (CNS) inflammation in animal models. We investigated the safety and immune effects of a fully humanized, previously uncharacterized nasal anti-CD3 Mab (Foralumab) in humans and its in vitro stimulatory properties. Methods: In vitro, Foralumab were compared to UCHT1 anti-human CD3 mAb. For human administration, 27 healthy volunteers (9 per group) received nasal Foralumab or placebo at a dose of 10ug, 50ug, or 250ug daily for 5 days. Safety was assessed and immune parameters measured on day 1 (pre-treatment), 7, 14, and 30 by FACS and by scRNAseq. Results: In vitro, Foralumab preferentially induced CD8+ T cell stimulation, reduced CD4+ T cell proliferation and lowered expression of IFNg, IL-17 and TNFa. Foralumab induced LAP, TIGIT, and KLRG1 immune checkpoint molecules on CD8+ and CD4+ T cells in a mechanism independent of CD8 T cells. In vivo, nasal Foralumab did not modulate CD3 from the T cell surface at any dose. Immune effects were primarily observed at the 50ug dose and consisted of reduction of CD8+ effector memory cells, an increase in naive CD8+ and CD4+ T cells, and reduced CD8+ T cell granzyme B and perforin expression. Differentially expressed genes observed by scRNAseq in CD8+ and CD4+ populations promoted survival and were anti-inflammatory. In the CD8+ TEMRA population there was induction of TIGIT, TGFB1 and KIR3DL2, indicative of a regulatory phenotype. In the memory CD4+ population, there was induction of CTLA4, KLRG1, and TGFB whereas there was an induction of TGF-B1 in naïve CD4+ T cells. In monocytes, there was induction of genes (HLA-DP, HLA-DQ) that promote a less inflammatory immune response. No side effects were observed, and no subjects developed human anti-mouse antibodies. Conclusion: These findings demonstrate that nasal Foralumab is safe and immunologically active in humans and presents a new avenue for the treatment of autoimmune and CNS diseases.

Acute exercise mobilizes NKT-like cells with a cytotoxic transcriptomic profile but does not augment the potency of cytokine-induced killer (CIK) cells.

CD3+/CD56+ Natural killer (NK) cell-like T-cells (NKT-like cells) represent <5% of blood lymphocytes, display a cytotoxic phenotype, and can kill various cancers. NKT-like cells can be expanded ex vivo into cytokine-induced killer (CIK) cells, however this therapeutic cell product has had mixed results against hematological malignancies in clinical trials. The aim of this study was to determine if NKT-like cells mobilized during acute cycling exercise could be used to generate more potent anti-tumor CIK cells from healthy donors. An acute exercise bout increased NKT-like cell numbers in blood 2-fold. Single cell RNA sequencing revealed that exercise mobilized NKT-like cells have an upregulation of genes and transcriptomic programs associated with enhanced anti-tumor activity, including cytotoxicity, cytokine responsiveness, and migration. Exercise, however, did not augment the ex vivo expansion of CIK cells or alter their surface phenotypes after 21-days of culture. CIK cells expanded at rest, during exercise (at 60% and 80% VO2max) or after (1h post) were equally capable of killing leukemia, lymphoma, and multiple myeloma target cells with and without cytokine (IL-2) and antibody (OKT3) priming in vitro. We conclude that acute exercise in healthy donors mobilizes NKT-like cells with an upregulation of transcriptomic programs involved in anti-tumor activity, but does not augment the ex vivo expansion of CIK cells.

Optimizing rAAV6 transduction of primary T cells for the generation of anti-CD19 AAV-CAR-T cells.

Recombinant Adeno-associated virus(rAAV) is currently the most widely used gene delivery vector and has been successfully used in various disease models, benefiting from its low immunogenicity, almost no toxicity, and no reported pathogenicity in humans. However, its low transduction efficiency for primary cells, especially for T lymphocytes, limits its further application in the field of cell therapy. In this study, we optimized the protocol for rAAV6 transduction of primary T cells, significantly improved the expression efficiency of the rAAV6 delivered CAR gene, and successfully generated rAAV6-based CAR-T cells (AAV-CAR-T). The gene expression intensity (mean fluorescence intensity, MFI) of rAAV6 transduced T cells treated with the tyrosine kinase inhibitor, Genistein, was increased 1-3-fold. Moreover, our results showed that rAAV6 efficiently transduced T cells stimulated with OKT3 and the gene expression could be enhanced 3-fold with an OKT3 concentration of 50 ng/mL in the medium. The gene expression intensity of T cells treated with OKT3 together with genistein could be augmented by 7-fold. Based on the above-optimized method, CAR-T cells prepared with rAAV6 showed evident anti-tumor ability both in vitro and in vivo. Our findings established an efficient method for the AAV transduction of T cells and would provide an alternative way for the preparation of CAR-T cells.

Development of a flow cytometry-based potency assay for prediction of cytokine storms induced by biosimilar monoclonal antibodies.

Cytokine release syndrome (CRS) is an undesired immune reaction that may cause dangerous side effects after the administration of novel biological therapies. In vitro cytokine release assays (CRA) are used for preclinical safety assessment prior to first-in-man dose administration of therapeutic monoclonal antibodies (mAbs). A variety of CRA platforms has been developed where the analysis of secreted cytokines is performed. Analysis of T cell activation markers is not performed routinely in CRA platforms and few studies have described intracellular cytokine levels after stimulation with therapeutic mAbs. In the present study, we performed a CRA using intracellular cytokine staining and assessment of extracellular T cell activation markers by flow cytometry. We used commercially available reference mAbs for the stimulation of peripheral blood mononuclear cells (PBMCs). We found that stimulation using solid phase (SP) dry coating with two different CD28 antibodies and muromonab-CD3 increased the percentage of IFN-É£ + CD4+ and CD8+ T cells as well as of CD3-CD56+ NK cells compared to stimulation with antibodies in aqueous phase (AP). Expression of the T cell activation markers CD25 and CD69 on CD4+ and CD8+ T cells was also increased upon SP muromonab-CD3 stimulation. Using multiplex cytokine assessment, we showed that stimulation in AP using ANC28.1, CD28.2 and muromonab-CD3 led to an increase of IFN-É£, GM-CSF, TNF-α, and IL-2 secretion. Stimulation of PBMCs preincubated at high-density culture led to an increase in IFN-É£ production but not in the expression of activation markers compared to low-density culture. Our findings demonstrated that flow cytometry analyses for assessing relevant T cell and NK cell markers may be used as a supplement to multiplex cytokine analysis in CRAs. The approach may be a valuable addition that enables a more precise description of the mechanisms leading to CRS.

SIRPγ-CD47 Interaction Positively Regulates the Activation of Human T Cells in Situation of Chronic Stimulation.

A numerous number of positive and negative signals via various molecules modulate T-cell activation. Within the various transmembrane proteins, SIRPγ is of interest since it is not expressed in rodents. SIRPγ interaction with CD47 is reevaluated in this study. Indeed, we show that the anti-SIRPγ mAb clone LSB2.20 previously used by others has not been appropriately characterized. We reveal that the anti-SIRPα clone KWAR23 is a Pan anti-SIRP mAb which efficiently blocks SIRPα and SIRPγ interactions with CD47. We show that SIRPγ expression on T cells varies with their differentiation and while being expressed on Tregs, is not implicated in their suppressive functions. SIRPγ spatial reorganization at the immune synapse is independent of its interaction with CD47. In vitro SIRPα-γ/CD47 blockade with KWAR23 impairs IFN-γ secretion by chronically activated T cells. In vivo in a xeno-GvHD model in NSG mice, the SIRPγ/CD47 blockade with the KWAR23 significantly delays the onset of the xeno-GvHD and deeply impairs human chimerism. In conclusion, we have shown that T-cell interaction with CD47 is of importance notably in chronic stimulation.

Versatile and rapid microfluidics-assisted antibody discovery.

Recent years have seen unparalleled development of microfluidic applications for antibody discovery in both academic and pharmaceutical research. Microfluidics can support native chain-paired library generation as well as direct screening of antibody secreting cells obtained by rodent immunization or from the human peripheral blood. While broad diversities of neutralizing antibodies against infectious diseases such as HIV, Ebola, or COVID-19 have been identified from convalescent individuals, microfluidics can expedite therapeutic antibody discovery for cancer or immunological disease indications. In this study, a commercially available microfluidic device, Cyto-Mine, was used for the rapid identification of natively paired antibodies from rodents or human donors screened for specific binding to recombinant antigens, for direct screening with cells expressing the target of interest, and, to our knowledge for the first time, for direct broad functional IgG antibody screening in droplets. The process time from cell preparation to confirmed recombinant antibodies was four weeks. Application of this or similar microfluidic devices and methodologies can accelerate and enhance pharmaceutical antibody hit discovery.

Phosphatidylserine-Targeting Monoclonal Antibodies Exhibit Distinct Biochemical and Cellular Effects on Anti-CD3/CD28-Stimulated T Cell IFN-γ and TNF-α Production.

Phosphatidylserine (PS)-targeting monoclonal Abs (mAbs) that directly target PS and target PS via ß2-gp1 (ß2GP1) have been in preclinical and clinical development for over 10 y for the treatment of infectious diseases and cancer. Although the intended targets of PS-binding mAbs have traditionally included pathogens as well as stressed tumor cells and its associated vasculature in oncology, the effects of PS-targeting mAbs on activated immune cells, notably T cells, which externalize PS upon Ag stimulation, is not well understood. Using human T cells from healthy donor PBMCs activated with an anti-CD3 + anti-CD28 Ab mixture (anti-CD3/CD28) as a model for TCR-mediated PS externalization and T cell stimulation, we investigated effects of two different PS-targeting mAbs, 11.31 and bavituximab (Bavi), on TCR activation and TCR-mediated cytokine production in an ex vivo paradigm. Although 11.31 and Bavi bind selectivity to anti-CD3/28 activated T cells in a PS-dependent manner, surprisingly, they display distinct functional activities in their effect on IFN-γ and TNF-ɑ production, whereby 11.31, but not Bavi, suppressed cytokine production. This inhibitory effect on anti-CD3/28 activated T cells was observed on both CD4+ and CD8+ cells and independently of monocytes, suggesting the effects of 11.31 were directly mediated by binding to externalized PS on activated T cells. Imaging showed 11.31 and Bavi bind at distinct focal depots on the cell membrane. Collectively, our findings indicate that PS-targeting mAb 11.31 suppresses cytokine production by anti-CD3/28 activated T cells.

Nanovesicles released by OKT3 hybridoma express fully active antibodies.

Recent findings have shown that nanovesicles preparations from either primary immune cells culture supernatants or plasma contain immunoglobulins, suggesting that a natural way of antibody production may be through exosome release. To verify this hypothesis, we used the OKT3 hybridoma clone, which produces a murine IgG2a monoclonal antibody used to reduce rejection in patients undergoing organ transplantation. We showed exosome-associated immunoglobulins in hybridoma supernatants, by Western blot, nanoscale flow cytometry and immunocapture-based ELISA. The OKT3-exo was also being able to trigger cytokines production in both CD4 and CD8 T cells. These results show that nanovesicles contain immunoglobulin and could be used for immunotherapy. These data could lead to a new approach to improve the effectiveness of therapeutic antibodies by exploiting their natural property to be expressed on nanovesicle membrane, that probably render them more stable and as a consequence more capable to interact with their specific ligand in the best way.

Developing an Arrayed CRISPR-Cas9 Co-Culture Screen for Immuno-Oncology Target ID.

Immunotherapies including PD-L1 blockade have shown remarkable increases in the T cell-directed antitumor response; however, efficacy is seen only in a minority of patients. Recently, pooled CRISPR-Cas9 knockout (CRISPRn) screens in tumor/immune co-culture systems have identified a number of genes that confer resistance to T cell killing in pathways including antigen presentation and cytokine signaling, providing insight into tumor mechanisms that cause resistance to immunotherapies. The development of an arrayed CRISPRn screen in a tumor/immune co-culture system would allow the identification of novel targets for immuno-oncology, characterization of hits from pooled screens, and multiple assay endpoints to be measured per gene. Here, a small-scale arrayed CRISPRn screen was successfully developed to investigate the effects on a co-culture of T cells and Cas9-expressing PC9 lung adenocarcinoma cells modified to express anti-CD3 antibody on the cell surface (PC9-OKT3 T cell system). A focused CRISPRn library was designed to target genes involved in known resistance mechanisms (including antigen presentation, cytokine signaling, and apoptosis) as well as genes involved in immune synapse interactions. The viability of PC9 cells was assessed in two-dimensional adherent co-cultures via longitudinal imaging analysis. Knockout of epidermal growth factor receptor (EGFR) and PLK1 in tumor cells cultured alone or with T cells resulted in increased tumor cell death, as expected, whereas knockout of the test gene ICAM1 showed subtle donor-specific resistance to T cell killing. Taken together, these data provide proof of concept for arrayed CRISPRn screens in tumor/immune co-culture systems and warrant further investigation of in vitro co-culture models.