Method of producing an immunoligand/payload conjugate
Inventors
Grawunder, Ulf • BEERLI, Roger Renzo
Assignees
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Publication Number
US-11986535-B2
Publication Date
2024-05-21
Expiration Date
Abstract
The present invention relates to a method of producing an immunoligand/payload conjugate, which method encompasses conjugating a payload to an immunoligand by means of a sequence-specific transpeptidase, or a catalytic domain thereof (Fig. 6b).
Core Innovation
The invention relates to an immunoligand/payload conjugate, in particular an antibody drug conjugate, produced using sequence-specific transpeptidase chemistry for site-specific and sequence-specific conjugation. The conjugate comprises an immunoligand and at least one payload connected through a linker that contains a sortase recognition motif with its C-terminal amino acid replaced with Gly, with n>1.
The payload has a molecular weight not exceeding 2,500 Dalton and includes drugs, markers, and small-molecule toxins. The payload is modified to serve as a substrate for enzymatic attachment using short glycine stretches or short intein recognition sequences that match the engineered recognition motifs on the immunoligand.
The immunoligand is selected from antibodies, antibody-based binding proteins containing at least one antibody-derived VH, VL, or CH immunoglobulin domain, antibody fragments binding to receptors, antigens, growth factors, cytokines, or hormones, or antibody mimetics from a specified list. The disclosure includes engineered immunoligands bearing corresponding sortase tag motifs or split-intein domains, and strategies to enrich for fully conjugated products and enable multiple payloads using sequential conjugations tied to heavy/light chain structure.
The document additionally discloses construction and sequence disclosure of pcDNA3.1-hygro(+) expression vectors encoding N-intein-tagged hBU12 anti-human CD19 IgH and IgL, with Ssp GyrB S11 split intein fused to C-termini and paired with C-terminal affinity tags. Further sequence and construct details are provided for additional sortase A enzyme sequence and construct components and disclosed sequence identifiers.
Claims Coverage
The independent claim coverage is defined by three core elements: a specified class of immunoligand, a payload of molecular weight not exceeding 2,500 Dalton, and a linker having a sortase recognition motif with its C-terminal amino acid replaced with Gly (n>1).
Immunoligand selection and payload-conjugate structure
An immunoligand/payload conjugate comprising an immunoligand selected from an antibody, an antibody-based binding protein containing at least one antibody-derived VH, VL, or CH immunoglobulin domain, an antibody fragment binding to a receptor, antigen, growth factor, cytokine, or hormone, and an antibody mimetic selected from a specified group.
Low-molecular-weight payload
At least one payload having a molecular weight not exceeding 2,500 Dalton.
Sortase recognition motif linker with Gly C-terminal replacement
A linker conjugating the immunoligand or a subunit thereof to the payload, wherein the linker comprises a sortase recognition motif having its C-terminal amino acid replaced with Gly, where n>1.
The claim coverage centers on combining a selected immunoligand class with a payload not exceeding 2,500 Dalton, linked via a sortase recognition motif where the motif’s C-terminal amino acid is replaced with Gly (n>1).
Stated Advantages
Site-specific and sequence-specific conjugation.
Generation of homogeneous ADCs via affinity enrichment.
Conjugation efficiency and in vitro/in vivo activity comparisons are provided.
Strategies to enrich for fully conjugated products and enable multiple payloads using sequential conjugations tied to heavy/light chain structure.
Documented Applications
Site-specific conjugation of glycine-modified toxins to antibody constructs.
Generation of homogeneous ADCs via affinity enrichment.
In vitro/in vivo activity comparisons.
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