Additional Vectors for the GS Xceed® System

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  • Introduction

    To support expression of next generation therapeutic candidates, the GS Xceed® toolbox has been expanded to include new vector systems. The new systems can be evaluated through Research Evaluation Agreement (REA) packages in your lab. Alternatively, the vectors can be evaluated as part of early proof of concept protein expression through Applied Protein Services.   

    Additional vectors include Constant Region Vectors that facilitate the generation of antibody libraries and antibody isotype reformatting. Together with the Site Specific Conjugation Constant Region Vectors, this enables users to access and test conjugation enabled antibody formats with 2 or 4 pre-selected conjugation sites.  

    pXC Multigene vectors expand gene assembly capabilities to incorporate 3 or 4 genes of interest in a single GS Xceed® expression vector. This enables co-expression of helper factors/proteins or simultaneous expression of up to four chains of more complex proteins such as bispecifics.

  • pXC Multigene Vectors

    The pXC Multigene vectors, part of the GS Xceed® System, are an easy way to assemble a GS expression vector for a multi-chain (3 or 4) protein such as a bispecific antibody. The GS Xceed® expression vectors were modified to allow the easy, single-vessel assembly of either a triple product gene or quadruple product gene expression vector (TGV and QGV, respectively) using type IIs restriction enzymes.

    These vectors can be accessed through an REA where studies can be performed within your own facility.  Alternatively, we can build expression vectors and perform early proof of concept expression through Applied Protein Services on your behalf. Vectors from these studies can later be transferred to you under an REA for further in house research.    

    Benefits include:  

    • Easy, single-vessel assembly of either a TGV or QGV
    • Flexible system that allows the protein product genes to be inserted into the expression vector in any order
    • Robust, highly efficient assembly of a multigene expression vector   

     

    The table below identifies the constituents of the pXC Multigene vector system. 

     Part Vectors  Destination Vectors
     pXC - Part A  pXC - TGV  pXC - QGV
     pXC - Part B
     pXC - Part C
     pXC - Part D  

    NB. To produce a TGV the constituent product genes are first individually inserted into Part Vectors A to C (pXC-PartA etc.), which are then combined into the destination vector, pXC-TGV. For a QGV, the product genes are individually inserted into Part Vectors A to D, which are then combined into the destination vector, pXC-QGV.


     

     

    Figure: Schematic of vector assembly for a triple-gene (TGV) or quadruple-gene vector. Genes of interest can be inserted in a controlled way in the desired order as selected by the user.

  • pCon Plus Constant Region Vectors

    The pCon Plus vectors are an easy way to re-express whole antibodies. The GS Xceed® expression vectors were modified to contain the constant region of a range of immunoglobulins (Ig) (see table below for full details). Along with the genetic elements of the Ig variable regions, these pCon Plus vectors offer easy construction of a library of full length antibodies with human constant regions.

    Benefits include:  

    • Easy sub-cloning of variable regions with a single ligation step
    • Fast construction of chimaeras or fully humanized mAbs
    • Wide range of available isotypes and allotypes  
    • Gene optimized DNA sequences
     Constant Region
     Heavy chain  Light Chain
     Isotype Allotype  Isotype  Allotype
     IgG1  f, za and zax  Lambda2  n/a
     IgG2  n-  Kappa I Km(3)
     IgG3  g    
     IgG4  n/a*    

     

    *more stable hinge mutant available

    NB: IgG1, IgG2 and IgG4 constant region genes are also available where the codon for the C-terminal lysine has been removed

     

     

     

     

     

  • Site Specific Conjugation Constant Region Vectors

    The Site Specific Conjugation (SSC) vectors are an easy way to produce conjugation-competent antibodies. pCon Plus Constant Region Vectors, part of the GS Xceed System®, were modified to contain point mutations to introduce cysteine residues at defined positions in the constant region of an immunoglobulin heavy or light chain (Figure 1).   Mutations are introduced as a single point mutation at a single site in either the heavy or light chain.  The table below identifies options including six variants in the heavy chain and two variants in the light chain.  

    Combining these vectors with the genetic elements of the Ig variable regions offers easy construction of a library of full-length antibodies with human constant regions including a defined number and location of bioconjugate attachment points. The conjugation sites have been assessed for aggregation and immunogenicity impact. In conjunction with our pCon Plus vectors, antibody libraries with fine-tuned conjugation capabilities and drug-to-antibody ratios (DAR) of 0, 2 and 4 can be constructed using standard thiol chemistry under mild conditions.  Applications include antibody drug conjugates (ADCs), half-life extension and imaging.  

    Benefits include:

    • Easy subcloning of variable regions with a single ligation step
    • Fast construction of conjugation enabled chimeric or fully humanized mAbs
    • Gene optimized DNA sequences  

     

     

      Figure 1. Available conjugation sites are highlighted in red (heavy chain) and green (light chain)

    Available Heavy and Light Chain Variants

     Site Specific Conjugation Vectors
     Heavy Chain  Light Chain
     Isotype / Allotype Number of variants Isotype / Allotype Number of variants
     IgG1f 6 Kappa (Km(3)) 2