Use CDD Vault to register and annotate antibodies and ADCs with their complete chemical structure, and benefit from the biological visual representation of the molecules.

In this article we will describe the best practices for ADC imports and how to reach the desired representation with the correct annotations by importing information on both the antibody and conjugated drugs.

The process used to import ADCs very closely resembles the process for antibody registration, therefore in this article we will highlight the main aspects related to the conjugation of drugs to the antibody structure. Please refer to this article for the best practices in importing the antibody structure: Antibody registration

Step 1: formatting the ADCs sequences

To import ADCs, you will need to prepare a spreadsheet with information about the heavy and light chains of the antibody, as well as additional column(s) with either the molecular structure or the Vault ID for the ADC’s payload(s). 

For best practices, we suggest independently registering the drugs within CDD Vault, and use their Molecule-Batch-ID to load them as payloads in ADCs.

Alternatively, if the payload is not available yet inside the Vault, a new structure has to be registered and you can provide it either as SMILES or MOL format within the column.

In the case of a single drug as a payload, the datasheet may look like the following example where we describe the heavy and light chain structure, as well as the payload ID:

If your ADC has multiple unique payloads, one column for each drug has to be added to the datasheet as in the example below:

Please note: Each unique structure should be in a single column. DAR can be noted later. There is no need to add as many columns as payloads, since the process will ask later how many units of a single payload are needed.

Accepted antibody/drug ratios and combinations

Currently CDD Vault supports the loading and conjugation of up to 4 different drugs to multiple sites on the antibody.

If the chemical and visual representation of the conjugation is not of interest, a combination of antibody and a single drug with a defined ratio is possible without upper limits of equivalents.

Conjugation sites annotation

To fully represent and store the chemical information of the conjugation, the antibody chains have to be correctly annotated with the information regarding the amino acids involved in the conjugation reaction.

As for domains, hinge region, and disulfide bridges, this annotation also needs to be defined within brackets, as in this example below:

Heavy → …{H:219-233}{@223:1}{@229:2}{@232:3}{$106:1}{$386:1}

• The symbol $ identifies the annotation for the drug conjugation site on the antibody chain.
• The first number defines the amino acid involved in the conjugation reaction.
• The second number defines the ID of conjugation. 
  • There should be as many IDs as unique drugs, so for two different drugs, both 1 and 2 should be used. Up to 4 different conjugation sites can be defined.

The resulting table with all of the described annotations will look like this for a single loaded entity (annotations for the antibody structure included and highlighted in grey):

If multiple drugs are to be loaded in different sites, the annotations and the table should reflect it as in the example below, where the two amino acids are annotated with a different ID ({$106:1}{$386:2}) and two columns are present for the two different drugs:

Please note: Use {H: to identify the hinge region to allow CDD to best render your antibody.

Chemical requirements for conjugation

When annotating the amino acids corresponding to the conjugation site, or declaring the drug that will be attached, we need to make sure that they are fit for the conjugation reaction taking place within CDD Vault to create the chemically aware ADC.

Conjugation site chemistry

The amino acids available as attachment sites for conjugation are those that in CDD Vault are declared with a connection point. This means that their structure within CDD Vault is already defined with an R3 group. The following list covers the natural amino acids that are set up in CDD Vault with an R3 group, but many more non-natural amino acids are already available for conjugation or can be defined as new custom amino acids (Bulk peptide import including custom modification):

• Cysteine - C
• Aspartic acid - D
• Glutamic acid - E
• Glutamine - G
• Histidine - H
• Lysine - K
• Asparagine - N
• Arginine - R
• Serine - S
• Threonine - T
• Tryptophan - W
• Tyrosine - Y

Drug structure definition

In order for drugs to be allowed as conjugates in CDD Vault, they have to be registered with a connection point as part of the structure. This means that in the structure an R1 group must be added to identify the conjugation site for that molecule to the antibody structure.

If the drug is already defined without the conjugation site, a new structure entity has to be created. To connect the two structures, it is always possible to link the batches via a Batch link with the appropriate field in the Batch level.

Step 2: importing the ADC in CDD Vault

Following the same first steps of the antibody import, we will arrive at the following interface where we can define heavy and light chains. Likewise, we can define the payload column as “Drug conjugate” clicking on the corresponding “Chain” option:

Each payload has to be defined as a “Drug conjugate” in order to be attached to the desired conjugation site on the antibody’s chains.

The following options allow you to define the attachment chemistry:

• “Source” lets you define whether the structure is already present in the Vault (Molecule name) or if the structure is novel, and therefore part of the row content. If you add the chemical structure here, you will need to specify whether the format is SMILES or Molfile.
• “Attach” defines which of the conjugation’s IDs has to be used for the selected payload. If more payloads are present, each payload can be attached to only one site. 
• “Condensation” is used to register the molecule with the correct leaving group, and therefore correctly estimate the molecular weight of the ADC.
• “Equiv” defines the ratio drug/antibody if the antibody chains are imported without any annotation. If annotations are present, this field can be ignored.

Once all of the payloads have been correctly attached to the antibody, a preview of the representation will be available and the “Process File” button will be active to proceed with the import of the ADC within CDD Vault.

As a next step, you will proceed to import the structure as well as mapping potential content in other columns to other fields within your Vault. Remember to map the newly created JSON column as an Antibody as in the default option.

Once uploaded correctly, you will be able to find your ADC in your Vault categorized as Mixture, and with the various information pertaining to the different components stored in the Antibodies section in the Molecule field. The antibody, the drugs, and the ADC are all linked to each other in the Vault, therefore you will be able to track each drug to which antibody it has been conjugated via the drug’s Links tab in the Referencing Mixtures section.