CD3-Binding Polypeptide Constructs for T Cell Engagers
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Solution Overview
Problem
Current T cell engagers (TCEs) face challenges in maintaining stability and effectiveness due to the weak hydrophobic interaction between VH and VL domains, leading to instability and reduced activity, particularly in in vitro and in vivo applications.
Innovation Solution
A polypeptide construct with specific CDR sequences in the VH and VL regions, including amino acid substitutions such as X24V, D15E, H1A, X12E, F4I, and N6S, enhances the interaction between VH and VL domains, increasing thermal stability and maintaining cytotoxic activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If conventional T cell engagers use standard VH-VL domain interactions, then the basic binding function is achieved, but thermal stability is insufficient and high molecular weight aggregates form
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions at defined positions in the CDR sequences (e.g., X24V, D15E, H1A, X12E, F4I, N6S) to modify the hydrophobic interaction strength between VH and VL domains. These targeted mutations change the chemical parameters of the protein domains to enhance stability without altering the overall protein structure or function.
Solution Approach 2:
The patent applies local quality by making specific modifications only in the CDR (complementarity determining region) sequences of the VH and VL domains, while leaving the rest of the protein structure unchanged. The mutations are localized to specific positions (e.g., position 24 in CDR-H1, position 15 in CDR-H2) to enhance local hydrophobic interactions without affecting global protein properties.
2Reliability
If T cell engagers are designed for high cytotoxic activity, then effective target cell killing is achieved, but stability and shelf life are reduced
Solution Approach 1:
The patent modifies the biochemical parameters of the T cell engager by introducing specific amino acid substitutions that enhance the hydrophobic interaction between VH and VL domains. This changes the stability parameter of the molecule, allowing it to maintain its cytotoxic activity over extended periods and improving shelf life without compromising the reliability of target cell killing.
3Ease of operation
If T cell engagers use standard VH-VL domain coupling, then basic binding function is maintained, but side effects increase due to instability
Solution Approach 1:
The patent applies local quality by making targeted mutations only in the CDR sequences of the VH and VL domains, specifically at positions that affect hydrophobic interactions. This localized modification enhances stability and reduces side effects while preserving the essential binding function, as the framework regions and overall protein structure remain unchanged.
Data Source
AI summary
The invention relates to a polypeptide or polypeptide construct comprising: a binding domain binding to an extracellular epitope of the human CD3s chain comprising or consisting of a VH region and a VL region, wherein i) the VH region comprises: a CDR-H1 sequence of X1YAX2N, where X1 is K, V, S, G, R, T, or I; and X2 is M or I; a CDR-H2 sequence of RIRSKYNNYATYYADX1VKX2, where X1 is S or Q; and X2 is D, G, K, S, or E; and a CDR-H3 sequence of HX1NFGNSYX2SX3X4AY, where X1 is G, R, or A; X2 is I, L, V, or T; X3 is Y, W or F; and X4 is W, F or Y; and ii) wherein the VL region comprises: a CDR-L1 sequence of X1 SSTGAVTX2X3X4YX5N, where X1 is G, R, or A; X2 is S or T; X3 is G or S; X4 is N or Y and X5 is P or A; a CDR-L2 sequence of X1TX2X3X4X5X6; where X1 is G or A; X2 is K, D, or N; X3 is F, M or K; X4 is L or R; X5 is A, P, or V; and X6 is P or S; and a CDR-L3 sequence of X1LWYSNX2VW, where X1 is V, A, or T; and X2 is R or L; and iii) wherein one or more of CDR sequences of the VH region of i) and/or of the VL region of ii) comprise one amino acid substitution or a combination thereof selected from X24V and X24F in CDR-H1; D15, and X116A in CDR-H2; H1, X12E, F4, and N6 in CDR-H3; and X11L and W3 in CDR-L3. The invention also relates to a polynucleotide encoding the polypeptide or polypeptide construct of the invention, a vector comprising said polynucleotide and a host cell transformed or transfected with said polynucleotide or with said vector. Moreover, the invention also provides for a process for the production of said polypeptide or polypeptide construct and a pharmaceutical composition comprising said polypeptide or polypeptide construct of the invention. Furthermore, the invention relates to medical uses of said polypeptide or polypeptide construct and kits comprising said polypeptide or polypeptide construct.