Company: CERO
Filing Date: 2025-04-15
Form Type: 10-K
Source: 0001213900-25-032134
Chunk: 112

Company: CERO THERAPEUTICS HOLDINGS, INC.
Filing Date: 2025-04-15
Form: 10-K
Item: Item 1
Chunk 112
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LR2 intracellular signaling domain.

In preclinical studies, CER-1236 empowers T
cells with phagocytic and cytotoxic potency

In an in vitro evaluation
of the phagocytic potential of CER-1236, CER-transduced T cells demonstrated robust phagocytosis of TIM-4-L. CER-1236 T cells were produced
by transducing donor T cells using a lentiviral vector encoding for the chimeric receptor CER-1236, yielding a high percentage of T cells
expressing the TIM-4 receptor, in similar CD4:CD8 ratios to untransduced cells. CER-1251 T cells, which express matching intracellular
signaling domains but are unable to bind to TIM-4-L due to a mutation in the gene encoding for the TIM-4 binding site, were also produced
as a negative control.

TIM-4-L-coated agarose beads
were prelabeled with pHrodo red, a pH-sensitive dye which displays limited fluorescence at neutral pH but generates significant fluorescence
in acidic pH. The post-phagocytic fusion of phagosomes and lysosomes leads to a drop in pH which can be detected by pH-sensitive dyes.
As is illustrated in the graphic below, CER-1236 T cells co-cultured with TIM-4-L-coated beads displayed significant phagocytic activity
with up to 60% of CER-T cells acquiring a pHrodo red signal, indicative of bead capture and internalization. By contrast, untransduced
T cells and CER-1251 T cells, with a mutation in the TIM-4 binding site, demonstrated minimal phagocytosis.

9

CER-1236 displays robust, target-specific phagocytic activity

Gene expression patterns
demonstrate the combined cytotoxic and phagocytic functions of CER-1236 T cells. RNA-sequencing enables the interrogation of the transcriptional
profile of CER-1236 T cells after stimulation with TIM-4-L, with defined separation between the CER-1236 activated cells and the untransduced
and CER-1251 control T cells. As shown in the gene expression profile below, over 1,700 genes were noted to be differentially expressed
in CER-1236 stimulated T cells in comparison to CER-1251 stimulated T cells. Among these genes were those related to pathways with