Impact of p53 Knock-Down on T-Cell Proliferation and T-Cell Mediated Cytotoxicity Against AML Cell Lines Mediated By a CD33 Specific BiTE® Antibody Construct
Authors: Pawlowsky Lea , Brauchle Bettina , Kazerani Pasikhani Maryam , Ogrinc Wagner Ana , Bücklein Veit , Lichtenegger Felix S. , Kischel Roman , Metzeler Klaus H. , Spiekermann Karsten , Andreeff Michael , Daver Naval G. , von Bergwelt Michael , Subklewe Marion 
Published: November 2019

Bispecific T-cell engaging (BiTE®) antibody constructs recruit T cells independent of the T-cell receptor specificity. Proof of concept was shown by Blinatumomab, a CD19xCD3 BiTE® antibody construct, for treatment of r/r B-cell precursor ALL. Response rates of 43% led to its approval in 2015, however, the mechanisms of innate and adaptive resistance in non-responders are still incompletely understood. To improve response rates, it is imperative to understand intrinsic and acquired resistance mechanisms. We assessed the relevance of p53 knockdown (kd) in AML cells on cytotoxicity mediated by AMG 330, a CD33 specific BiTE®antibody construct. We initially hypothesized that p53 kd cells would show differential sensitivity to T-cell mediated cytotoxicity.
12 AML cell lines with different p53 status (wt: MV4-11, Molm-13; mut/del: HL60, MONO-MAC 6, K562, Kasumi-1, Kasumi-3, THP-1, SKM-1, NB-4, KG1a) or p53 wt and paired p53 kd cell lines (MV4-11, Molm-13 and OCI-AML3) were cocultured with healthy donor (HD) T cells at an effector: target (E:T) ratio of 1:6 with AMG 330 (5 ng/ml) or a control BiTE®for 3 days. Cytotoxicity was calculated as %specific lysis = 100 × (1 - live CD33+cellsAMG 330/live CD33+cellscBiTE®). To assess T cell proliferation, T cells were stained with the proliferation dye Far Red. For analysis of the costimulatory/coinhibitory expression profile of the paired cell lines, cells were stained for CD80, CD86, CD40L, OX40L, PD-L1, Tim-3 and corresponding isotypes either in steady state or after 72h stimulation with TNFα and IFNγ. To dissect the influence of receptor-ligand interactions versus secretome, we performed transwell experiments. HD T cells and murine Ba/F3 cells (transduced with CD33 and CD86) were cocultured at an E:T ratio of 1:10 in presence of 5 ng/ml AMG 330 or control BiTE®in the lower compartment. In the upper compartment AML cells were cultured, allowing the exchange of soluble factors but no cell-cell contact. Ba/F3 cells were used as a surrogate model to activate T cells and minimize the influence of human checkpoint molecules. To further delineate the influence of p53 kd on T-cell function, we performed transcriptome analysis of the paired AML cell lines.
First, 12 AML cell lines were analyzed for AMG 330-mediated cytotoxicity upon coculture with T cells. No difference based on p53 status was observed (p53 wt vs p53 mut (65.86% vs 53.92%; n=27). However, phenotypic and genetic heterogeneity of AML cell lines could mask the influence of p53 mutation on the pharmacodynamic activity of AMG 330. To exclude these cofounding covariables, we performed cytotoxicity assays using p53 wt and paired p53 kd cell lines. AMG 330-mediated cytotoxicity was significantly higher for p53 wt cell lines compared to p53 kd cell lines (wt vs kd: MV4-11: 89.8% vs 72.3%, p<0.03; Molm-13: 90.0% vs 85.2%, p<0.03; OCI-AML3: 89.5% vs 81.2%, p<0.03). Surprisingly, we observed significantly reduced T-cell proliferation in co-cultures with p53 kd cell lines vs p53 wt cell lines (wt vs kd: MV4-11: 74.0% vs 61.6%, p<0.03; Molm-13: 65.7% vs 56.4%, p<0.03; OCI-AML3: 34.9% vs 26.4%, p<0.03). We further demonstrated that the difference is not due to different expression levels of the target antigen CD33 or the costimulatory/coinhibitory molecules CD80, CD86, CD40L, OX40L, PD-L1 and Tim-3 (n= 4). In transwell experiments, T cells proliferated at a lower rate in the presence of p53 kd AML cells in the upper comportment compared to those in the presence of p53 wt AML cells (MV4-11 p53 wt vs p53 kd: 67.9% vs 50.9%, p<0.03) leading to the conclusion that the inhibition of T-cell proliferation is mediated by soluble factors secreted by AML cells. Bulk RNA sequencing of p53 kd AML cell lines compared to p53 wt revealed downregulation of a co-stimulatory ligand 4-1BBL with log2 fold change of - 0.33, -0.30, -0.39 in Molm-13, MV4-11 and OCI-AML3, respectively.
In summary, we have observed lower activity of AMG 330 against p53 kd cell lines compared to p53 wt AML cell lines. This correlated with a decrease in T-cell proliferation indicating that the genetic profile of the AML cells modulates T-cell function. We hypothesize that p53 kd AML cells suppress T-cell proliferation, thereby reducing the E:T ratio and redirected cytotoxicity. In a next step, we will try to identify possible secreted mediators of T-cell modulation by mass. We will also characterize primary patient samples for their T-cell-inhibiting capacities, e.g. p53-mutated disease.

Disclosures
Lichtenegger: Roche: Employment. Kischel:AMGEN Research (Munich) GmbH: Employment, Equity Ownership. Metzeler:Celgene: Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Otsuka: Honoraria. Andreeff:NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; CPRIT: Research Funding; Breast Cancer Research Foundation: Research Funding; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy. Daver:Celgene: Consultancy; Otsuka: Consultancy; Jazz: Consultancy; Daiichi Sankyo: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Immunogen: Consultancy, Research Funding; Forty-Seven: Consultancy; Novartis: Consultancy, Research Funding; Agios: Consultancy; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Servier: Research Funding; Incyte: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; Karyopharm: Consultancy, Research Funding; Glycomimetics: Research Funding; Hanmi Pharm Co., Ltd.: Research Funding; Astellas: Consultancy; NOHLA: Research Funding. Subklewe:Pfizer: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Morphosys: Research Funding; Janssen: Consultancy; Oxford Biotherapeutics: Research Funding; Celgene: Consultancy, Honoraria; AMGEN: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Research Funding; Miltenyi: Research Funding.