Engineered Probiotics Target Tumors Prior to CAR-T Immunotherapy

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Engineered Probiotics Target Tumors Prior to CAR-T Immunotherapy

Engineered Probiotics Target Tumors Prior to CAR-T Immunotherapy

In a breakthrough published in the journal “Science,” scientists have unveiled a novel cancer therapy that uses engineered probiotics to target tumors and recruit CAR-T cells.

While CAR-T therapies have proven highly effective against blood cancers, they have faced challenges when dealing with solid tumors.

This is primarily due to the insufficient specificity of tumor-associated antigens (TAAs) within solid tumors, leading to off-target toxicity and incomplete cancer cell clearance. One approach to overcoming this bottleneck has been to enhance CAR-T cells’ targeting capabilities by introducing multiple target points.

A recent study published in “Science” proposes an intriguing alternative strategy. It involves using engineered bacteria to tag tumors, paving the way for CAR-T cells. The researchers refer to this system as the Probiotic-Guided CAR-T System (ProCARs).

Engineered Probiotics Target Tumors Prior to CAR-T Immunotherapy

This concept is not entirely new. Solid tumors naturally harbor specific microbial populations that seem to thrive in the hypoxic and necrotic tumor microenvironment (TME), allowing them to selectively colonize the tumor core.

Scientists have previously harnessed this microbial trait to develop a range of engineered bacteria, transforming them into precise delivery vehicles for anti-cancer drugs. While clinical achievements are yet to be realized, current trial data suggests the safety of engineered bacteria.

Researchers have now considered utilizing engineered bacteria to guide CAR-T cells.

The guiding bacteria they designed are based on the probiotic Escherichia coli Nissle 1917 (EcN) and contain a synchronized lysis circuit (SLIC) capable of delivering synthesized CAR targets directly to the tumor core.

SLIC allows bacteria to grow within the tumor to a critical density and then synchronously rupture, releasing therapeutic compounds produced by the bacteria. This leverages the advantage of bacterial quorum sensing, preventing uncontrolled growth outside of the tumor.

The tags used for CAR-T cell guidance are designed as superfolder green fluorescent protein (sfGFP) dimers (diGFP) connected to the placental growth factor-2 heparin-binding domain (PlGF-2123-144). DiGFP has previously been shown to mediate CAR-T cell responses.

Engineered Probiotics Target Tumors Prior to CAR-T Immunotherapy

ProCARs mechanism of action

PlGF-2123-144 can bind to components abundant in the dense extracellular matrix of tumors, such as collagen and fibronectin. This design has two advantages: it restricts the system’s action range to stay within the tumor’s boundaries, thus enhancing safety, and it promotes CAR polarization, enhancing anti-cancer activity.

Engineered Probiotics Target Tumors Prior to CAR-T Immunotherapy

The presence of tag significantly enhances the anti-tumor effect of CAR-T

The researchers tested the ProCARs system’s anti-cancer activity in mice. Initially, they injected engineered bacteria at a concentration of 1×105 CFU into subcutaneous tumor-bearing mice (Naml6) and, 48 hours later, injected 2.5×106 GFP28z+ CAR-T cells into the tumor.

Notably, the presence of tags significantly enhanced CAR-T’s anti-cancer efficacy, with diGFP alone proving ineffective.

The specificity of the ProCARs system was excellent. Researchers examined GFP levels in various organs outside the tumor and found no trace of the engineered bacteria.

The researchers also tested multiple cancer types, including triple-negative breast cancer (MDA-MB-468) and colorectal cancer (CT26/MC38), and observed anti-cancer effects in all cases.

Remarkably, in the experiments, treating only one side of the tumor also slowed the growth of the untreated tumor on the opposite side. This suggests that ProCARs activate an adaptive immune response, providing overall anti-cancer benefits.

Furthermore, the researchers designed a strain that releases a mutant CXCL16 variant (CXCL16K42A) to enhance CAR-T cell recruitment, further strengthening the ProCARs system’s anti-cancer effectiveness.

Finally, the researchers attempted intravenous injection of ProCARs with CXCL16 and tags, which still yielded noticeable therapeutic effects without off-target toxicity.

The ProCARs system demonstrated therapeutic effectiveness in various human and mouse cancer models with different genetic backgrounds, indicating its potential application. In the context of patients often experiencing cancer recurrence due to antigen loss, this treatment method for antigen-unknown tumors holds great promise.

However, compared to mice, humans are more sensitive to endotoxins, and systemic administration of gram-negative bacteria may have potential toxicity in humans. This is a challenge that must be addressed for clinical translation.