Method of producing an immunoligand/payload conjugate
Inventors
Grawunder, Ulf • BEERLI, Roger Renzo
Assignees
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Publication Number
US-9872923-B2
Publication Date
2018-01-23
Expiration Date
Abstract
A method of producing an immunoligand/payload conjugate can encompass conjugating a payload to an immunoligand by means of a sequence-specific transpeptidase, or a catalytic domain thereof.
Core Innovation
The document describes a method of producing an immunoligand/payload conjugate by enzymatically conjugating at least one payload to an immunoligand using a sequence-specific sortase or a catalytic domain thereof. The payload is a toxin having a molecular weight not exceeding 2,500 Dalton, and the conjugate is produced such that either the immunoligand comprises a sortase recognition motif and the toxin is modified with a Gly_n-modification, wherein n>1, or the toxin comprises a sortase recognition motif and the immunoligand is modified with a Gly_n-modification, wherein n>1.
An immunoligand is selected to include an antibody, an antibody-based binding protein containing at least one antibody-derived V_H, V_L, or C_H immunoglobulin domain, an antibody fragment binding to a receptor, antigen, growth factor, cytokine and/or hormone, or an antibody mimetic selected from specified classes including DARPins and aptamers. The document grounds the conjugation concept in examples involving an anti-human CD19 antibody hBU12 with sortase A sequence-specific mechanisms.
The disclosed system further supports production of site- and stoichiometrically controlled conjugates, including homogeneous products with defined drug-to-antibody ratios, and is compatible with crude CHO supernatant. It also describes removable affinity/detection tags for enriching fully conjugated products and multi-payload conjugation using different transpeptidases and motifs.
Claims Coverage
The provided independent claim is clm-00001. It covers producing an immunoligand/payload conjugate using sequence-specific sortase-mediated, Gly_n-modified toxin conjugation, with either the immunoligand or the toxin carrying the sortase recognition motif, and constrains the toxin to a molecular weight not exceeding 2,500 Dalton. The claim coverage also includes the stated immunoligand selections.
Sequence-specific sortase conjugation of a toxin payload
Enzymatically conjugating at least one payload to the immunoligand by a sequence-specific sortase or a catalytic domain thereof, wherein the payload is a toxin having a molecular weight not exceeding 2,500 Dalton.
Mutual presence of sortase recognition motif and Gly_n modification (n>1)
Conjugation where either the immunoligand comprises a sortase recognition motif and the toxin is modified with a Gly_n-modification, wherein n>1, or the toxin comprises a sortase recognition motif and the immunoligand is modified with a Gly_n-modification, wherein n>1.
Immunoligand selection spanning antibodies, fragments, antibody-derived domain proteins, and mimetics
Providing an immunoligand selected from an antibody, an antibody-based binding protein containing at least one antibody-derived V_H, V_L, or C_H immunoglobulin domain, an antibody fragment binding to a receptor, antigen, growth factor, cytokine and/or hormone, or an antibody mimetic selected from specified classes including DARPins and aptamers.
Overall, the claim coverage centers on a sortase-driven, sequence-specific production of immunoligand/payload conjugates, with a toxin payload constrained by molecular weight and with conjugation achieved through the coordinated presence of a sortase recognition motif and Gly_n-modification (n>1) on either the immunoligand or the toxin.
Stated Advantages
Produces homogeneous immunoligand/payload conjugate products.
Provides a defined drug-to-antibody ratio (DAR).
Improves reproducibility compared with stochastic chemical linker conjugation.
Is compatible with crude cell culture supernatant.
Documented Applications
Producing immunoligand/payload conjugates in the context of antibody-drug conjugates (ADCs), using site- and stoichiometrically controlled conjugation.
Conjugating small-molecular-weight toxins (≤2,500 Dalton) to immunoligands for targeted binding to receptors, antigens, growth factors, cytokines, and hormones.
Multi-payload conjugation using different transpeptidases and motifs, and enrichment of fully conjugated products using removable affinity/detection tags.
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