Pro-apoptotic Construct Bypassing Serpin B9 Resistance
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Solution Overview
Problem
Current cancer treatments using engineered cytotoxic lymphocytes face challenges in effectively targeting and killing solid tumors due to resistance mechanisms, particularly the expression of Serpin B9, which inhibits granzyme activity, leading to suboptimal clinical efficacy.
Innovation Solution
Development of a nucleic acid molecule encoding a pro-apoptotic protein with a granule-localizing domain and a modified BH3 effector domain, such as NOXA with a BIM BH3 domain, to enhance the killing capacity of engineered immune cells by bypassing Serpin B9 inhibition and targeting multiple pro-survival BCL-2 family proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If engineered cytotoxic lymphocytes use granzyme-perforin pathway to kill tumor cells, then tumor cell killing capacity is improved, but Serpin B9 expression by target cells inhibits granzyme activity and reduces killing efficacy
Solution Approach 1:
The pro-apoptotic protein is segmented into two functional domains: a granule-localizing domain (separately from the BH3 effector domain) that targets the protein to lytic granules, and a modified BH3 effector domain that directly induces apoptosis. This segmentation allows the protein to bypass the granzyme-Serpin B9 interaction by being delivered as a pre-assembled unit to the granule, where it can subsequently transfer to the target cell and induce apoptosis independently of granzyme activity.
Solution Approach 2:
The granule-localizing domain acts as an intermediary that facilitates the delivery of the BH3 effector domain to the lytic granule. This intermediary mechanism enables the pro-apoptotic protein to utilize the existing granzyme-perforin delivery pathway without being subject to Serpin B9 inhibition, as the granule-localizing domain directs the protein to co-pack with granzymes and perforin, and the BH3 domain then mediates the actual apoptotic effect in the target cell.
2Reliability
If engineered CLs are used to treat solid tumors, then immune therapy efficacy is improved, but tumors develop natural resistance mechanisms that reduce killing effectiveness
Solution Approach 1:
The BH3 effector domain is modified by replacing the native BH3 domain of NOXA with the BH3 domain of BIM, creating a chimeric protein with enhanced pro-apoptotic activity. This parameter change in the molecular structure of the pro-apoptotic protein alters its ability to interact with BCL-2 family proteins, enabling it to overcome tumor resistance mechanisms that normally protect against apoptosis, thereby improving immune therapy efficacy against solid tumors.
3Reliability
If pro-apoptotic proteins are delivered through the granzyme-perforin pathway, then tumor cell killing is enhanced, but the complexity of the nucleic acid construct increases
Solution Approach 1:
The nucleic acid construct merges two functional elements into a single polypeptide: the granule-localizing domain and the modified BH3 effector domain. This merging allows the protein to be delivered through the existing granzyme-perforin pathway (utilizing the granule-localizing domain's ability to co-pack with granzymes) while simultaneously providing enhanced pro-apoptotic activity (through the modified BH3 domain), thereby achieving improved tumor cell killing without requiring separate delivery mechanisms.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The strategy effectively induces apoptosis in tumor cells by delivering pro-apoptotic proteins through the natural granzyme-perforin pathway, overcoming resistance and achieving enhanced tumor cell killing with minimal side effects.
Implementation Method 1
a granule-localizing domain, preferably a granzyme, more preferably granzyme B, may act as a chaperone for the cell-specific transfer of a pro-apoptotic protein from an effector cell into a target cell
Implementation Method 2
At the target cell membrane, perforin aggregates to form multimeric, transmembrane pores. This allows the entry of granzyme into cytosol of the target cell
Implementation Method 3
the modified BH3 effector domain, which binds a multidomain BCL-2 family protein and/or a BH3 domain-binding groove of a multidomain BCL-2 family protein
Implementation Method 4
wherein the pro-apoptotic protein is able to induce apoptosis in a target cell, in particular in a cancer cell
Implementation Method 5
it preferentially induces cell apoptosis by promoting mitochondrial outer membrane permeabilization and/or activating caspases, which cleaves many substrates, including caspase-activated DNase to execute cell death
Data Source
AI summary
The current disclosure relates to pro-apoptotic molecules with a B-cell lymphoma 2 (BCL-2) homology 3 (BH3) effector domain. The current disclosure furthermore relates to pro-apoptotic constructs wherein the pro-apoptotic molecules are linked to a granule-localizing domain. The pro-apoptotic construct may be transferred from an effector cell to a target cell to induce apoptosis. The current disclosure also relates to the nucleic acid molecules encoding the pro-apoptotic proteins and the uses thereof in a medical therapy such as cancer therapy, including chimeric antigen receptor cell therapy and the like.