Lysine conjugated immunoglobulins
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Publication Number
US-11753669-B2
Publication Date
2023-09-12
Expiration Date
Abstract
Provided herein are conjugated immunoglobulins and methods for generating conjugated immunoglobulins using a microbial transglutaminase.
Core Innovation
The invention provides conjugated immunoglobulins comprising an immunoglobulin, or an antigen-binding portion thereof, and an acyl donor substrate, using microbial transglutaminase-mediated conjugation. The immunoglobulin comprises engineered lysine residues formed by mutating specified natural amino acid residues to lysine residues at EU-numbered heavy-chain and light-chain positions.
The acyl donor substrate comprises a glutamine residue and is defined according to Formula (I) or Formula (II). Z is a carboxylbenzyloxy (CBZ) group or an amino acid residue, each L is independently a straight or branched linker from 1 to 20 carbon atoms with optional atom substitutions or an amino acid residue, and m and n are integers from 0 to 5. Y is a therapeutic agent or a diagnostic agent, including auristatin F in a dependent refinement.
The engineered lysine residue on the immunoglobulin, or antigen-binding portion thereof, is conjugated to the glutamine residue of the acyl donor substrate. The document further refers to immunoglobulin and antigen-binding formats such as IgG1-4 and Fab/Fab′ variants, and to nucleic acid molecules encoding conjugatable immunoglobulins and host cells expressing the conjugatable immunoglobulins.
Claims Coverage
The independent claim coverage centers on a conjugated immunoglobulin or antigen-binding portion with engineered lysine residues at specified EU-numbered positions and a glutamine-containing acyl donor substrate defined by Formula (I) or Formula (II). The claim set includes engineered lysine substitutions, the substrate structure, the conjugation relationship, payload identity, and, in the method claim, microbial transglutaminase that conjugates the engineered lysine residue to the glutamine residue to generate the conjugated immunoglobulin.
Engineered lysine residues at EU-numbered heavy and light chain positions
The immunoglobulin, or antigen-binding portion thereof, comprises an engineered lysine residue formed by mutating selected natural amino acid residues to lysine residues. The selected residues include heavy-chain sites such as T135K, S136K, L193K, D221K, T223K, H224K, T225K, M252K, N297K, and P445K, and light-chain sites such as L201K and S202K on a kappa light chain and E213K on a lambda light chain, numbered according to the EU numbering system.
Glutamine-containing acyl donor substrate of Formula (I) or Formula (II)
The acyl donor substrate comprises a glutamine residue and is according to one of Formula (I) or Formula (II). Z is a CBZ group or an amino acid residue; each L is independently a straight or branched linker from 1 to 20 carbon atoms with optional atom substitutions or an amino acid residue; m and n are integers from 0 to 5; and Y is a therapeutic agent or a diagnostic agent.
Conjugation between engineered lysine and glutamine residue
The engineered lysine residue of the immunoglobulin, or antigen-binding portion thereof, is conjugated to the glutamine residue of the acyl donor substrate.
Microbial transglutaminase conjugation using Formula (I) or Formula (II) acyl donor substrate
A method for generating a conjugated immunoglobulin comprises contacting an immunoglobulin, or antigen-binding portion thereof, with a microbial transglutaminase and an acyl donor substrate, wherein the microbial transglutaminase conjugates the engineered lysine residue to the glutamine residue thereby generating the conjugated immunoglobulin.
Payload agent selection for Y
Y is a therapeutic agent or a diagnostic agent, and in dependent refinement Y is specified as auristatin F.
Constrained functional agent-to-immunoglobulin ratio
In dependent refinements, the ratio of the functional agent to the immunoglobulin, or its antigen-binding portion, is within 1:1 to 100:1 or within 1:1 to 200:1.
Across the covered claim set, the inventive concept is a conjugated immunoglobulin in which EU-numbered engineered lysine residues are conjugated to a glutamine-containing acyl donor substrate defined by Formula (I) or Formula (II), where the conjugate carries a therapeutic or diagnostic agent and may be subject to functional-agent-to-immunoglobulin ratio constraints.
Stated Advantages
The document identifies disadvantages of conventional lysine/cysteine conjugation approaches and states that predictable conjugation can be achieved by engineering specific lysine residues into immunoglobulin or antigen-binding portions.
Engineered multi-site conjugates are described as increasing theoretical DAR.
Documented Applications
Therapeutic agent conjugates.
Diagnostic agent conjugates.
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