Truncated CyaA Vectors for Negatively Charged Antigen Delivery
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Solution Overview
Problem
Existing CyaA vectors face inefficiencies in translocating antigens with acidic amino acid stretches and negatively charged polypeptides across cell membranes, leading to weak or no protective cellular immune responses, which is a drawback for tumor regression and prevention.
Innovation Solution
Development of new CyaA vectors with deleted adenylate cyclase domains and insertion of large antigens or highly negatively charged antigens, which surprisingly enhance translocation efficiency and induce strong CD8+ and CD4+ T-cell responses.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CyaA vectors are used to deliver antigens with acidic amino acid stretches or negatively charged polypeptides, then the antigen delivery function is compromised, but using alternative vectors or methods increases device complexity or reduces productivity
Solution Approach 1:
The invention extracts and removes the adenylate cyclase catalytic domain (residues 184-386) from the CyaA protein, which is the source of the problem. By deleting this domain, the patent eliminates the region that causes acidic charge interference and prevents proper translocation of negatively charged antigens, while retaining the translocation capability of the remaining N-terminal and C-terminal domains
Solution Approach 2:
The invention changes the electrostatic parameters of the CyaA vector by removing the catalytic domain with its acidic residues. This parameter change (deletion of residues 184-386) fundamentally alters the charge distribution of the vector, enabling it to successfully translocate antigens with acidic amino acid stretches that would otherwise be repelled or blocked by the original CyaA structure
2Reliability
If large antigens or highly negatively charged antigens are inserted into traditional CyaA vectors, then translocation efficiency decreases, but the need for tumor regression and prevention requires effective delivery of such antigens
Solution Approach 1:
The patent extracts the problematic catalytic domain (residues 184-386) from CyaA, creating a truncated vector structure that can accommodate large and highly charged antigens without the steric and electrostatic interference caused by the full-length catalytic domain
Solution Approach 2:
The invention segments the CyaA protein by dividing it into three parts: the N-terminal domain (residues 1-183), the deleted catalytic domain (residues 184-386), and the C-terminal domain (residues 387-1706). By reconnecting the N-terminal and C-terminal domains, the patent creates a segmented vector structure that maintains translocation function while removing the interfering middle section
Data Source
Figure 1A~1B
Figure 2
Figure 3A~3B
AI summary
The invention relates to a chimeric protein comprising or consisting of, from N-terminal to C-terminal, (a) a N-terminal part of a Bordetella CyaA protein (b) a heterologous polypeptide comprising antigens originating from different HPVs, and (c) a C-terminal part of a Bordetella CyaA protein. The invention also relates to a polynucleotide encoding this chimeric protein. A composition comprising at least one chimeric protein(s) of the invention and the prophylactic and/or therapeutic uses of said composition are also part of the invention.