Hydra Linker

Facilitated by the advanced “DrugLnk” organic synthesis platform, Creative Biolabs offers a unique customized design and synthesis service to prepare branched linkers for antibody-drug conjugates (ADCs). The resulted “hydra linkers” open a new avenue for the development of ADCs possessing a higher drug-to-antibody ratio (DAR).

Why Branched Linkers?

DAR is one of the most crucial attributes that impacts significantly on the bio-distribution as well as pharmacokinetic properties of an ADC. ADCs with higher DAR (bearing more payload per antibody) often show greater potency in vitro. However, high DAR value appears to have a negative impact on ADC in vivo efficacy, presumably due to the faster plasma clearance of highly loaded antibodies. This observation is the basis for a controlled DAR range of 2~4 in modern ADC developments via different conjugation chemistries through the native cysteine or lysine residues, which produce a distribution of heterogeneous drug-loading species. In a recent effort, a homogeneous ADC with a DAR of 8 was produced via cysteine conjugation and this ADC also suffers an accelerated plasma clearance due to its unique drug-linker complex and the heavy modification to the antibody.

Hydra Linker

The concept of an “ideal” ADC is to deliver the maximum payload into the targeted cells. Thus, an elaborate balance between the number of payload and the extent of antibody modification should be explored and established to approach a high-DAR ADC with extensive plasma retention time. Branched linkers, or “hydra linkers”, may just be the solution to this long-lasting problem in ADC development.

What are Branched Linkers?

Branched linkers (Hydra Linkers) are featured by their multiple payload loading capacity. They are bi-functional linkers that contain a main stem with a functional group at one terminus and a “branching point” at the other end. The functional group on the main stem reacts with conjugation sites on the antibody while multiple braches, each bearing a second functional group that reacts with the payload, are installed at the branching point. Hydra linker is a multi-loading approach that allows an increased DAR on an ADC with reduced chemical or enzymatic modification to the antibody structure. Hydra linkers promote conjugation efficiency, require minimal destabilization of the antibody structure, and thus, increase ADC efficacy.

Branched Linker Formats

  • Polyethylene glycol (PEG) Branched Linkers

The potency of most ADC payload drugs resides in their hydrophobic moieties. To increase the solubility of high-DAR ADCs and to prevent aggregation caused by non-specific hydrophobic interactions between payload molecules, Creative Biolabs has developed an approach to mask the inherent hydrophobicity of payloads with PEG. In this method, PEG is inserted as a spacer between the payload and the antibody, shieling the payload with a tether of hydrophilic entities from the environment to reduce the potential for nonspecific hydrophobic interactions. A unique feature with our design is that the payload is constrained to a close proximity of the antibody by a PEG24 tether between the antibody and a cleavable linker. In PEG-based branched hydra linkers, PEG24 tethers are incorporated as branches, each followed by a cleavable linker as well as the payload of choice.

Using this method, ADCs with DAR values as high as 8 can be prepared that exert improved homogeneity and enhanced in vivo potencies comparing to lower DAR ADCs.

Hydra Linker Fig 2. Structures of branched glucuronide-MMAE drug linkers. (Lyon, R. P., 2015)

  • Dual-cysteine Multiplexing Carrier

This approach creates a multiplexing drug carrier that is installed onto native antibody intra-chain disulfides via maleimide chemistry. The drug carrier contains two orthogonally protected cysteine residues that are sequentially unmasked after antibody conjugation for the coupling with the same or different payloads/payload-linker complexes. Using a PEG24 stretcher to facilitate high drug loading and to reduce hydrophobicity-induced ADC aggregation as well as a self-stabilizing maleimide (mDPR) to minimize drug-linker dissociation in vivo, the hydra linkers generated by this approach is highly advantageous. Homogeneous ADCs with a DAR value at 16 have been generated with the dual-cysteine multiplexing carrier.

Hydra Linker Fig 3. Schematic representation of a dual-cysteine multiplexing carrier for ADC preparation. Part B shows a multiplexing drug carrier bearing Cys(SiPr) and Cys(Acm) groups that are unmasked under orthogonal conditions. Part C shows the homogeneous dual-drug ADCs prepared using this carrier that bears 16 total drugs, split evenly (8+8) between the two component drugs. (Levengood, M. R., 2017)

  • Other Types of Branched Linkers

Creative Biolabs has also developed several other formats of branched linkers, including the cathepsin B-cleavable valine-citrulline (Val-Cit) branched linkers and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers branched linkers.

The Val-Cit branched linker is comprised of a lysine scaffold with the branching point, several PEG spacers, a primary amine for MTGase-mediated antibody-linker conjugation, and two azide groups for payload installation by azide–alkyne click reaction. The HPMA branched linker is constructed based on the soluble polymer poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA) backbone, which improves the physicochemical and pharmacokinetic properties of the polymer-drug complexes by increasing circulating half-life as well as the solubility of hydrophobic drugs.

Hydra Linker Fig 4. Schematic diagram of MTGase-mediated antibody–drug conjugation using linear or branched linkers.  (Anami, Y., 2017)

Features of Hydra Linkers

  • Higher DAR
  • Enhanced in vitro and in vivo potency
  • Improved conjugation efficiency and ADC homogeneity
  • Reduced chemical or enzymatic modification to the antibody structure
  • Efficient ADC construction and enhanced ADC efficacy

Creative Biolabs is committed to provide customized branched linkers and high quality chemical synthesis services to promote the progress of ADC development projects. Please contact us for more information and a detailed quote.


  1. Levengood, M. R., (2017). “Orthogonal Cysteine Protection Enables Homogeneous Multi‐Drug Antibody–Drug Conjugates.” Angewandte Chemie International Edition, 56(3), 733-737.
  2. Anami, Y., (2017). “Enzymatic conjugation using branched linkers for constructing homogeneous antibody–drug conjugates with high potency.” Organic & Biomolecular Chemistry, 15(26), 5635-5642.
  3. Lyon, R. P., (2015). “Reducing hydrophobicity of homogeneous antibody-drug conjugates improves pharmacokinetics and therapeutic index.” Nature biotechnology, 33(7), 733-735.

For lab research use only, not for any in vivo human use.

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