New Results
, Chinh Tran-To Su, Wai-Heng Lua, Jun-Jie Poh, Yuen-Ling Ng, Anil Wipat, Samuel Ken-En Gan
Summary Boosting the production of recombinant therapeutic antibodies is crucial in both academic and industry settings. In this work, we investigated the usage of varying signal peptides by antibody genes and their roles in recombinant transient production. Comparing myeloma and the native signal peptides of both heavy and light chains in 168 antibody permutation variants, we performed a systematic analysis, finding amino acids counts to be involved in antibody production to construct a model for predicting co-transfection transient recombinant antibody production rates using the HEK293 system. The findings also provide insights into the usage of the large repertoire of antibody signal peptides.
The signal peptide (SP) of a protein is a short tag of amino acids at the N- or C-terminal that predestinates the protein location extracellularly or within the cell to the organelles. Known organelle targeting SPs include the nucleus localization or export signal, mitochondria signals, endoplasmic reticulum (ER) secretion or retention signal, and peroxisome signals.
Secreted by plasma B cells, antibodies are tagged with the ER secretion signal/SP at the N-terminal and translocated into the ER lumen, before being passed to the Golgi apparatus and sorted into secretory vesicles for extracellular secretion. The SP is unique to the protein and generally contains a positively charged N-terminal, followed by a hydrophobic region and a neutral polar C-terminal. Depending on its location at the N or C terminal of the protein, there is a cleavage site separating the SP from the protein that would be recognized by a signal peptidase. While secretory SPs are involved in the co-translational co-translocation pathway with the primary function to export proteins, it remains enigmatic to why antibodies utilize a large repertoire of SPs (e.g. Vκ1 family with 22 SPs, VH3 family with 50 SPs, retrieved from the IMGT database at the point of writing), hinting of possible roles in antibody production.
Modifying SPs for better recombinant protein production has been largely successful in many studies, however, the underlying mechanism of such effects remains enigmatic. With recent studies demonstrating cross-talks of antibody elements, where the constant regions, variable regions and their pairings affect antibody production and function, there is increasing evidence that antibodies ought to be investigated holistically, especially as therapeutics. Through the inclusion of antibody SP in the analysis, deeper insights into antibody V-region pairing effects in transient recombinant production can be further holistically considered. In this work, we unraveled the role of total amino acid usage that may underlie the usage of the diverse repertoire of antibody SPs in compensating for V-region hypervariability to overcome production bottlenecks.
Antibody SPs were initially named by the constant region isotypes but were recently re-classified in IMGT by the V-region family. With exception to the wild-type IgE signal peptide: Humighae 1, termed ‘IgE’ SP, references to the SPs in this research follows the IMGT convention. Given the large number of antibody germline SPs, we selected consensus/dominant representatives of each VH and Vκ family as representative ‘native’ SPs.
VH and Vκ variants with Pertuzumab and Trastuzumab CDRs were paired with their respective SPs (i.e. VH1 SP to VH1 framework (FWR), Vκ1 SP to Vκ1 FWR) and production levels compared to utilizing only the IgE SP from our previous work. Given the variability in transient co-transfections, recombinant Pertuzumab and Trastuzumab Vκ1 VH3 with the IgE SP were used for normalization (100%) to facilitate comparisons. Given the high CDR similarity of both Trastuzumab and Pertuzumab, this allows an investigation of minute CDR difference effects on protein production.
Within the Pertuzumab variants, production levels with the IgE SP tend to be higher than those using native SPs (e.g. highest producing pair using the IgE SP: Vκ4|VH3 at 125%, while the highest producing pair utilizing their respective SP, were Vκ3|VH7 at 80%) with exceptions (Vκ1|VH1, Vκ2|VH1, Vκ1|VH4, Vκ2|VH4, Vκ1|VH7, and Vκ2|VH7). Regardless of the SP, antibodies paired with Vκ5 family constructs gave poor yields.
Figure 1. Production rates (%) of recombinant antibodies containing various heavy and light chain pairings. (A & B) Recombinant Pertuzumab (A) and Trastuzumab (B) variants with the various SPs grouped according to VH families. (C & D) Recombinant Pertuzumab and Trastuzumab variants with the various SPs grouped according to Vκ families. Recombinant Pertuzumab and Trastuzumab variants produced with native (blue) or the ‘IgE’ (orange) SPs respectively, are shown. In all experiments, the recombinant wild-type Pertuzumab (POK PG1) or Trastuzumab (HOK HG1) was used (shown in the first column). The production rate of the variants utilizing the ‘IgE’ SP was used as the reference for the production rate.
Of the Trastuzumab variants, there is general agreement to the trends observed in the Pertuzumab dataset where IgE SP variants generally having higher production rates than native SP variants. The highest producing antibody with IgE SP is Vκ4|VH3 at 235%, with its corresponding counterparts with native SPs at 94%. The only exceptions where the native SPs had higher productions were the Vκ1 |VH5 and Vκ1 |VH7 pairs.
In the Pertuzumab dataset, the Vκ5 family is the sole poor producing family, whereas the low production families in the recombinant Trastuzumab model dataset extended to VH1, 2, 4 and 6 (light chains that paired with these VHs had lower yields). One notable exception was the Vκ5|VH3 pair that was produced at higher levels compared to other Vκ5 family permutations in the Trastuzumab dataset.
VH1 and VH7 genes shared the same SP amino acid sequence but with different codons. While this was normalized through codon optimization, there were still distinct different productions between the two VH families, with VH7 being the better producing partner (average production of VH7 with its light chain partners at 48% when using native SPs and at 58% when using IgE SP) compared to VH1 (at 37% when using native SPs and at 48% when using IgE SP) in the Pertuzumab dataset. The effect between VH1 and VH7 was even more pronounced in the Trastuzumab dataset, where VH1 recombinant production level were significantly reduced (at 7% when using native SPs, and 20% when using IgE SP) as compared to VH7 (at 37% when using native SPs, and 110% when using the IgE SP).
To investigate the role of light chain SPs, Vκ1 SP was grafted onto the Pertuzumab and Trastuzumab VH3 FWR and compared to the IgE and native SPs pairings after normalization with the respective Trastuzumab/Pertuzumab variants with the IgE SP. The respective heavy and light chains of Pertuzumab/Trastuzumab with the IgE SP are termed IgESP-Vκ1 (light chain) and IgESP-VH3 (heavy chain) in Figure 2. The native SPs (Vκ1 SP-Vκ1, VH3SP-VH3) had the lowest recombinant production at 48% and 87% for the Pertuzumab and Trastuzumab models, respectively. The Vκ1 SP (Vκ1SP-Vκ1, Vκ1SP-VH3) had the highest production at 112% and 240% in the Pertuzumab and Trastuzumab models, respectively.
Figure 2. Transient recombinant antibody production rates of wild-type Vκ1|VH3 Pertuzumab (blue) and Trastuzumab (orange) using the IgE, Vκ1 and native SPs in %.
Figure 3. Recombinant protein production (%) with IgE, Vκ1 and mutated Vκ1 SPs, (Vκ1 P18R and Vκ1 P18S) against the wild-type Vκ1 |VH3 Pertuzumab (blue) and Trastuzumab (orange) models.
To study the effect of myeloma SPs on improving production levels, we performed single amino acid mutagenesis (P18R and P18S) on the Vκ1 SP. The mutated SPs were generated based on a previous reported myeloma Vκ1 SP associated Fanconi’s syndrome SP, and the IgE SP sequence, also from a myeloma pat
