Company: CERO
Filing Date: 2025-02-07
Form Type: 424B3
Source: 0001213900-25-011071
Chunk: 165

Company: CERO THERAPEUTICS HOLDINGS, INC.
Filing Date: 2025-02-07
Form: 424B3
Chunk 165
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-4 appears to function as a
tether during phagocytosis to allow intracellular signaling by other transmembrane phagocytic molecules with which it associates, such
as the integrins which are expressed ubiquitously on T cells. Since CER-1236 contains only the ECD of TIM-4, binding to TIM-4-L on tumor
cells recruits the cell-surface phagocytosis machinery, and simultaneously directly activates CER-1236 T cells through the intracellular
CD3ξ and CD28 costimulatory domains. Phagocytosis and cytokine secretion are further enhanced by the TLR2 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.

CER-1236 displays robust, target-specific phagocytic activity

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