I'm Manuele Martinelli, a chemist with almost 10 years of experience as a senior R&D scientist (focused on protein production and characterization). During my time in the lab I've had the opportunity to test a lot of instruments, procedures and reagents, and I'd like to share some Cool Protein ProtoCols (ProteCool!)with you in the hope that they help you with your experiments.Since I was born strabic, I always had a different point of view, which I want to share with you! :-)
Humans and mice share five primary antibody (immunoglobulin) isotypes: IgM, IgD, IgG, IgA, and IgE. While they share identical foundational functions, they differ notably in structural subclasses, serum concentrations, and specific immunological functions
Antibiotic resistance genes (e.g Ampicillin, Kanamycin) are the most commonly used markers for plamisd selection in DNA production and recombinant protein expression processes.
Adding an antibiotic resistance gene to the plasmid solves 2 problems at once: It allows a scientist to easily select the plasmid-containing bacteria when the cells are grown on selective media and at the same time provides those bacteria with a pressure to keep your plasmid.
For the reason the most of commercial vectors suitable for recombinant protein expression in E.coli as pET, pQE, pMal, pCold, pGEx carry Ampicillin, Kanamicin selection marker. .
Even if this strategy work very well in R&D setting, on the contrary it has several drawbacks for biological manufacturing.
The spreading of antibiotics in the environment and consequent emergence of multi-resistant pathogenic bacterial strains has become a general promise to even further increasome categories of biological products such as DNA vaccines where potential issue of allergic reesponses to some classes of antibiotics is evoked and the necessity to document the trace amount of antibiotics.
Apart from the use of the antibiotic itself, there is another emerging limitation in the use the antibiotic resistance gene used as a selection marker due to the potential risk of horizontal transfer of antibiotic resistance gene to environmental microbes
Behind the regulatory issues, the use of antibiotic resistance gene marker imposes a significant metabolic burden on the cells and may. also impact the process yield.
At least but not last,antibiotics themselves are expemsive and therefore often omitted in fermentations, leading to plasmid loss and a corresponding loss in product yield.
In this context novel strategies to efficiently replace antibiotic-based selection are required.
To date, several systems have been developed based on different principles, each presenting advantages and drawbacks.
The most common way to achieve selection in the absence of antibiotics is the complementation of an essential gene making use of an expression vector in a strain with a defect or inhibited expression of the same essential gene.
Several examples are reported in literature where the plasmid selection is achieved through the complementation of amino acid auxotrophy (e,g Proline; Glycine) and more recently of QAPRTase, an enzyme implied in de novo nicotinamide adenine dinucleotide biosynthesis.
Many different E.coli auxothopic strains were already generated but to ensure the proper selective pressure, the auxotrophy complementation systems reported so far require the use of chemically defined media since the standard complex media contain variable amounts of aminoacids as well as other catabolites that covercome the need of biosynthetic pathway and as a consequence the loss of selective pressure for the complementation plasmid maintenance.
About 10 years ago I was involved in a project aimed to developed a novel antibiotic free plasmid selection approach for protein selection in E.coli.
Some experiences gained in previous work activities such as:
1) The positive effect that the supplementation δ-ALA (δ-aminolevulinic acid) has on recombinant Human cyt c expression in E.coli (CERM)
2) The use of 5-aminolevulinic acid (ALA) as a prodrug to stimulate intracellular Heme biosynthesis to produce the natural photosensitizer (PS) Protoporphyrin IX (PpIX) in antimicrobial photodinamic therapy (Molteni Therapeutics)
lead me tot think that the complementation of hemA deficient bacteria by a vector carrying a functional hemA gene, as a selection marker, which confers to the transformed bacteria the ability to grow and maintain the vector in any medium that does not support growth of hemA deficient bacteria of a mutation in heme biosynthetic pathway and at the same time the complementation of empty cells with 5-ALA allow to easly propagate and prepare the empty competent cells.
After several months of work we produced an interesting data package supporting our hipotesis and a patent application was filled and submitted (WO2015165841 - AN ANTIBIOTIC-FREE METHOD FOR SELECTWO2015165841 - AN ANTIBIOTIC-FREE METHOD FOR SELECTION OF TRANSFORMED BACTERIA)
Even if the patent was not accepted since the examing authority do not recognize its inventive steps since the HemA was already show to work as selectable marker in Aspergillus Oryzae, i still think that it is work very well in E.coli since we was able to show that the Delta HemA E.coli show negligible growth in both chemical defined and complex media but the growth it readly restored when there are complemented with 5-ala or plasmid carryng (see Fig2a,2b and Fig3a,3b of the WO2015165841 patent application) the HemA gene and that the expression was mantained after a several different passages.
E. coli BL21(DE3) DeltaHemA/pet21-BFP and E. coli BL21(DE3)/pet21-BFP were cultivated without antibiotic selection for many generations by diluting a 12h culture 1:100 in fresh medium for several times. After 1,2,5,10,15 passages the recombinant protein expression was analyzed (1G ≈ 7-8 duplications) and as reported in the Fig5 of the WO2015165841 patent apprication, the BFP production in HemA mutant strain complemented with BFPpet24-HemA is stable after 15 cultivation cycles without antibiotic while in the wild type strain transformed with standard BFPpet24, it dramatically decreases after 5 cultivation cycles without selection.
I would like to Thanks to
Maria Giuliani
that performed the most of the experimental work
and provide an essential contribute in experiment desing
Full length human IgG antibodies are composed of 2 regions, the N-terminal region of each chain corresponding to the Fab (fragment for antigen binding which contain the variable regions and the CDRs) and the C-terminal region corresponding to the Fc (crystallizable fragment)
Until some years ago the scientist
focus mainly their efforts in the optimization of the Fab regions to develop
antibodies with highest antigen affinity and lower immunogenicity.
In the last years, especially with the
emergence of bispecific antibodies, e.g T-cell engagers there has
been a growing interest in modifying the Fc region to modulate
(enhance or remove) the antibody effector function.
The Fc region is able to bind with high
affinity the Fc gamma receptors (FcγRs), and the neonatal Fc receptor
(FcRn) expressed in the surface of the immune system cells (Reference) by triggering different effector functions such as
antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent
cellular phagocytosis (ADCP) and complement- dependent cytotoxicity (CDC) responses (Reference)
Among the four subclasses of human IgG (IgG1, IgG2, IgG3, IgG4), which differ in their constant regions, particularly in their hinges and CH2 domains, IgG1 has the highest FcγR-binding affinity, followed by IgG3, IgG2, and IgG4. As a result, different subclasses modulate in different way the different responses ADCC, ADCP and CDC. (Reference)
In in the first generation of
monoclonal antibodies the Fc activity was modulated by producing of the
antibody in different Fc format on the basis of the antigen properties and the MoA
(mechanism of action) that they would confer to the antibody:
Blocking antibodies that do not have to activate the immune response where produced in the IgG4 format
For example Nivolumab which binds to the PD-1 receptor located on the cell membrane and inhibits its interaction with its ligands, PD-L1 and PD-L2. Since PD-1 is expressed on activated T cells, NK cells, regulatory T cells and B cells. Nivolumab has been desinged to block the interaction with the PD-L1 and PD-L2 and therefore releases immune cells from pathological immune suppression, but not activate the immune-response against these immune-cells otherwise we will obtain the unwanted depletion of effector cells inducing serious adverse events..
On the contrary, mabs targeting antigen expressed in the surface of cancer cells (e.g Cetuximab targeting EGFR) were produced in igG1 form to activate the immune-response against those cells and induce cell clearance. In fact, it has been demonstrated in preclinical models and ex vivo studies that target-bound cetuximab IgG1 isotype mAbs stimulate natural killer (NK) cell–driven cytotoxicity against tumor cells coated in mAbs via the interaction of the constant region and the CD16 receptor on NK cells
More recently igG1-Fc sequence engineering were largely exploited to further improve the modulation of Fc function:engineering as well as CHO cell line
Fc Function enhancement:
It is well-known that this effector function is modulated by the N-linked glycosylation in the Fc region of the antibody (N297 of IgG1).
In particular, absence of core
fucose on the Fc N-glycan has been shown to increase IgG1 Fc binding affinity
to the FcγRIIIa present on immune effector cells such as natural killer cells
and lead to enhanced ADCC activity.
As I reported in the ProteoCool Pills #10, the simple supplementation of the culture media with 2F-Peracetyl-Fucose allow to produce low fucosilated monoclonal antibodies in mg scale to be used for R&D screening, on the other hand, several strategies (as the GlymaxX® and POTELLIGENT® have been developed to build stable clones for large scale production based of afucosylated antibodies with improved therapeutic potency.
Since those cell lines may show some drawbacks in terms of
growth rate and mab productivity at the same time other scientists focus also
in the development of igG1 mutant (eg S239D/1332E named SDIE and G236A/S239D/A330L/I332E named GASDALIE – US patentUS20230057150A1 those similarly to afucosilated mab show improved affinity for the FcγRs even
in produced in the standard CHO cell lines.
In the past I had the opportunity to do some trials comparing
the ADCC activity (using the Promega ADCC assay, both F
and V versions) with using the igG1 WT afucosilated and igG1 SDIE mutant
(produced with standard fucosilation) for the same clone and interestingly (data not shown) it seems that their effect
could be combined at least from the results of this in vitro assay.
B) Fc Function silencing:
The
most recent alternative to the use of IgG1, which allow to point mutations in
the linker region between hinge and Fc domains were described to reduce or
fully abrogate Fcγ binding affinity and downstream effector cell activation
In
the following Table are reported some of the most known and used mutations:
From literature
the STR (recently developed by MAbSolve seems to be the only mutation that completely
abolish all the ADCC, ADCP and CDC activities (Reference) and it seems that STR do not alter the
antibody developability profile.
Even in this case I had the opportunity to do some R&D trials comparing the igG1 human wild type, LALAPG and STR mutants and I was not able to was not able to reveal meaningful differences between LALAPG and STR with the Promega ADCC assay (complete abolishment in both cases – data not shown) but this can be due to the fact that as shown in seems that the advantage of STR vs LALAPG is in the lower affinity of STR for the FcγRI which is probably more involved in trigger the ADCP than ADCC response or that the sensitivity of those kind of assay is not enough to detect so small differences (see Table3) the authors do not reveal any differences even in ACDP assays
I also performed antibody aggregation propensity detected
performing SEC on 10mg/ml samples subjected to different incubations (4°C,
37°C, Freeze/Thaw) and in this case the LALAPG seems to be slightly better
(data not show)
However, this post does not would provide any ranking or evaluation of the different mutants but just inform you about the possibility to test them to achieve the wanted MoA for your new monoclonal antibody.
A Bispecific antibodies (BsAb) is a single molecule that comprise two single binding entities that are physically connected, which enables simultaneous binding to two different epitopes on two different antigens or even in some cases two different epitopes on the same antigens (named biparatopic antibodies).
The ability of BsAbs to bind to different antigens was largelly exploited in immunotherapy to act as a linker beetween a cytotoxic cell and a target (a tumour cell) to be killed.
For example T-cell-redirecting bispecific antibodies (named T-cell engagers) are specifically designed to bind to tumor-associated antigens, thereby engaging with CD3 on the T cell receptor. This linkage between tumor cells and T cells actively triggers T cell activation and initiates targeted killing of the identified tumor cells. These antibodies have emerged as one of the most promising avenues within tumor immunotherapy.
The main issue associated
with asymmetric BsAbs is mispairing of polypeptide chains leading to
product-related impurities. In fact BsAbs assemble require co-expression of 4 different chains (2 different heavy chains and 2 different light chains), each heavy chain can
form either homo- or heterodimers, which in turn can associate with the two
light chains. From a stochastic view point, this leads to 16 chain arrangements
with 10 unique combinations, including only one, that represent only the 10% of
the total mixture, that is the desidered BsAb where heavy chain exhibit the
correct heterodimer and pairing with distinct light chains and this low
probability impairs the manufacturability of bsAbs
Protein engineering was largelly applied in the last 20 years to solve this issue.
First of all, complementary mutations that favors heavy chain (HC) heterodimerization while disfavouring formation of HC homodimers were reported. The first reported, and most widely used, platform is the knob-into-hole (KiH) which introduces a large bulky tryptophan in one HC and smaller sterically complementary residues in the other HC. The KiH strategy is widely implemented because it is highly effective in suppressing the most of HC homodimers and because its patent has expired
While the correct heavy chain heterodimerization can be enabled using the knobs-into-holes (KiH) approach, the correct association of generic light chains has remained a problem, in fact even in presence of KiH there are still 4 possible random combinations that limit the fraction of desidered form only to about 25%
To solve this issue the KiH could be coupled with the CrossMAb technology that was described and patented by Roche in 2011 (it is stil protected by IP). which is able to enforce correct light chain association in bispecific heterodimeric IgG antibodies.
This technology
could be applied to any existing antibody pair using domain crossover, without
the need for the identification of common light chains, post-translational
processing/in vitro chemical assembly or the introduction of a set of mutations
enforcing correct light chain association.
In the past I had the opportunity to produce for R&D purpose 2 different bispecific containing both KiH and Crossmab and in both cases the antibodies were produced fron CHO with high yields (transient trasfection) and the an very good rate of HC-LC correct coupling (>90%) was detected in both cases by mass-spectrometry.
Chain stores with furniture, DIY, decoupage hobbies as
IKEA, OBI, Leroy Merlin, Bricoman
can be a great source for funny and cheap items useful for improve the laboratory organization
Here i report just some examples:
1) SANDVIVIA OVEN MIT
made in silicone which provides a firm grip and is heat-insulating.
It allow to manipulate and move the hot agarose gel and/or hot culture media removed from microwaves or autoclave.
Avaialble from IKEA (Italy: 3euro - US: 4 dollars )
-----------------------------------------------------------------------------------------------------------------------------
2) VEKSEN CART
It fits in the smallest of laboratory, but there’s plenty of space on the shelves for all for all those staff that you need to have on hand when you work on the laminar hood
or in a laboratory with low space
Assemble the trolley quickly and easily by clicking the parts together without any specific tools.
Available from IKEA (Italy: 10 euro; US: 9 dollars)
----------------------------------------------------------------------------------------------------------------------------
TIKSEN Hook with suction cup.
Alternative clothes hanger solution
Available from IKEA (Italy: 7euro/4p; US: 5dollars/2p)
......................................................................................................................................................................
BEVARA Sealing clip.
Cheap clips for use with Dialysis Tubing
Available from IKEA (Italy 1,5euro/10p: US: 2 dollars/10p )
DSF
(Differential scanning fluorimetry previously named also Thermofluor)
is a very simple tecniques that exploit the ability of a fluorescente probe, typically either SYPRO Orange or 1-anilino-8-naphthalenesulfonate (ANS) – that is quenched in an aqueous environment but becomes strongly fluorescent when bound to exposed hydrophobic groups of a protein. Therefore, the thermal unfolding of a protein of interest in the presence of such a dye, can be followed spectrophotometrically.
DSF is a 96 well based assay that could be performed on a RT-PCR thermocycler, it is inexpensive, fast, and require relatively little sample. All these advantages have made this approach attractive for screening applications in drug discovery and also for protein stability formulation
In the ProteoCool n°24 in suggest as in my opinion the DSF may become an essential assay todetermine lot to lot concistenciy in QC department of company producing recombinant proteins (e.g recombinant vaccine and/or enzymes)
In this post i would like to share with you 2 examples that show as DSF may play an important role also in antibody screening, optimization and developability assesment.
DSF thanks to its excellent throughput and minimal material consumption (about 50ul of antibody at 0,3 mg/ml for each point) allow to readily compare the thermal stability of:
- different antibodies in the same buffer:
- different Fab mutants or Fc mutants in the same buffer;
- different antibody formulations; (optimize pH, salt concentration, excipients)
Example 1:
Comparision of thermal stability of different mabs
Full length human IgG1-Clk mabs in PBS may show 1 or 2 separate transitions on the basis of the Fab stability.
Mabs with lower Fab stability show in general a single transition while mabs with high stability show 2 separate transitions, 1 at about 69°C for the Fc and 1 at higher temperature for the Fab.
In recent years the enhancement or elimination of the Fc-effector function has led to a growing interest in Fc-engineering, to give antibodies specific mechanisms of action and therapeutic properties.
Even if several Fc modification, as:
- LALANA, LALAPG, LALAGR mutations to remove effector function;
- SDIE, GAALIE mutations as well as afucosilation to enanche the effector function
were already reported, few data about developability of those solutions are still available and there is a lot work to do to select an enanched Fc combining the best effector function and good manufacturability, stability and pharmacokinetics.
Those are just 2 examples that show as in short time and using a limited amount od samples, thanks to DSF we can obtain essential informations to guide our decision in antibody during the screening, desing, formulation phases.
Of course, thermal stability itself do not Guarantee that the selected antibody has good developability profile but some of the following other tecniques as:
- DLS;
- Self-interaction determined by BLI;
those provide informations about antibody aggregation propensity
- Hydrofobic interaction chromatografy (HIC)
those provide informations about antibody hydrofobicity
have to be peformed to complement the DSF data.
A great thanks to
Luca Sorrentino
In the Video Section of ProteoCool are actually available more than 30 different video tutorials about molecular biology, protein expression and characterization.
All those video can be easily downloaded using the Video DownloadHelper tool available on Mozilla Firefox browser. No cost or restriction are associated to the ProteoCool video use or sharing.
I just ask you to cite, ProteoCool, in case you will share or use part of it for build new material.
In this post I would like to show you, how video download from a Blog (not only PRoteoCool but all the blogger.com blogs is possible using the Mozilla browser
How yu can do it:
- Open Mozilla Firefox browser;
- Check if the Video DownloadHelper is already installed in your browser
The presence of the Video DownloadHelper is identified by the presence of an Icon that shows 3 balloons (in gray scale) located on the Firefox toolbar.
If Video DownloadHelper is already installed, you can choose your video and download it in few minutes following few steps:
1. Access to the blog from which you would like to download the video (e.g. PRoteoCool)
- Click on the VideoDownload helper icon and proceed with the installation by
following the different instructions.
- Once the 3 balloon icon will appear on the top of your browser, Downloading videos
from the Blogger site is now possible.
Go to the step 1
