mRNA–LNP Bispecific Antibodies: Demonstrating T-Cell Activation and Targeted Tumor Killing

Demonstrating T-Cell Activation and Tumor Cell Killing using mRNA–LNP-Delivered Bispecific Antibodies

Bispecific antibodies (bsAbs) have emerged as powerful tools for redirecting T-cells toward tumor cells by simultaneously engaging a tumor-associated antigen and CD3 on T-cells. However, conventional recombinant bsAb production can be time-consuming and lacks flexibility for rapid screening or iterative optimization.

mRNA–LNP technology offers an alternative approach by enabling transient expression of bispecific antibodies following mRNA–LNP delivery. This facilitates streamlined design–test cycles while preserving functional activity.

To evaluate this approach, mRNA encoding a HER2–CD3-Fc bispecific antibody (T-cell engager format) delivered via LNPs was assessed in both in vitro and in vivo preclinical models, focusing on T-cell activation, tumor cell killing, and overall therapeutic efficacy.

In Vitro Validation

T-Cell Activation via mRNA–LNP-Encoded bsAb

HER2–CD3-Fc bsAb encoded by mRNA–LNP induces robust, dose-dependent T-cell activation, as demonstrated by increased secretion of key effector cytokines, including IFN-γ, TNF-α, and Granzyme B.

This activation is dependent on HER2 target engagement, confirming the specificity of the bispecific construct

Figure 1: Dose-Dependent T-Cell Activation Induced by HER2–CD3-Fc mRNA–LNP

Targeted Tumor Killing with mRNA–LNP Delivery

mRNA–LNP-delivered HER2–CD3-Fc bsAb mediates efficient cytotoxicity against HER2-positive tumor cell lines (e.g., NCI-H460, SKOV-3, A549, A1847), with minimal activity observed in HER2-negative controls.

Real-time cell analysis confirms dose-dependent tumor cell killing, demonstrating functional T-cell redirection.

Figure 2: Selective Tumor Cell Killing Mediated by mRNA–LNP-Encoded HER2–CD3-Fc bsAb

In Vivo Efficacy

Tumor Growth Suppression

To assess therapeutic efficacy in vivo, HER2–CD3-Fc bsAb mRNA–LNPs were evaluated in tumor-bearing models.

mRNA–LNP delivery of bispecific antibodies results in robust T-cell–mediated anti-tumor activity:

Significant reduction in tumor volume over time
Decrease in final tumor weight compared to control groups
Sustained anti-tumor response following treatment
Evidence of effective immune-mediated tumor clearance

HER2–CD3-Fc mRNA–LNP treatment induces significant tumor growth inhibition in vivo, as shown by reduced tumor volume and weight compared to controls.

The results confirm effective T-cell–mediated anti-tumor activity following mRNA–LNP-mediated expression of the bispecific antibody.

Figure 3: In Vivo Tumor Growth Inhibition Following HER2–CD3-Fc mRNA–LNP Treatment

Functional Comparison to Protein-Based Bispecific Antibodies

To further assess functional performance, mRNA–LNP-encoded bispecific antibodies were evaluated for their ability to induce tumor cell killing and immune activation, and compared to recombinant protein-based bispecific antibodies.

Figure 4: Dose-Dependent Tumor Cell Killing by mRNA–LNP-Encoded HER2–CD3 bsAb

Real-time cell analysis demonstrates that mRNA–LNP-encoded HER2–CD3 bispecific antibodies induce efficient and dose-dependent tumor cell killing in HER2⁺ cell lines (A549, A1847), with activity comparable to recombinant protein-based bsAbs.

Figure 5: Cytokine Secretion Induced by mRNA–LNP-Encoded HER2–CD3 bsAb

Cytokine profiling shows robust immune activation following mRNA–LNP delivery, with increased secretion of key effector molecules, including IFN-γ and Granzyme B, confirming effective T-cell engagement across tumor models.

Together, these results demonstrate that mRNA–LNP delivery enables functional activity comparable to protein-based bispecific antibodies, supporting its use for preclinical evaluation of bispecific formats.

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