The latest Investing Matters Podcast episode featuring Jeremy Skillington, CEO of Poolbeg Pharma has just been released. Listen here.
4.3.3. Development of resistance
Bacteria have the ability to acquire resistance to new methods of inhibition, in ways previously
described in 1.1.2. It is important to consider how the use of Affimer proteins as VIM-1 inhibitors
could exert a selective pressure which may lead to the evolution of resistance, it is possible bacteria
could develop such mechanisms of resistance to any Affimer reagents.
The evolution of resistance can only be tested in longitudinal studies of Gram-negative bacteria by
treating VIM-1 expressing cells with various concentrations of Affimer61 and antibiotics then
passaging over a number of weeks to assess whether any strains were capable of evading Affimer
inhibition by acquiring resistance. An adaptive library evolution, similar to those used in 2014 by
Jahn et al. could be utilised, whereby increasing antibiotic drug concentration in set stages over a
period of two weeks resulted in cells expressing increased resistance to the antibiotics used [109]
.
4.3.4. Immunogenicity
The ability of a substance to provoke an immune response in a host is referred to as
immunogenicity, and this is another important consideration when developing novel reagents such
as the Affimer [110]. Anti-drug antibodies (ADAs) can be expressed in response to a therapeutic, and
bind in a tiered response that can cause immune complexes to be formed, and can result in
erroneous activation of the complement cascade [111]
.
With regards to the immunogenicity of the Affimer structure, Avacta Life Sciences Ltd published
observations using human ex vivo samples. Three different structures of Affimer scaffold were
tested at five-fold the concentration of Avastin after a period of seven days in T-cell cultures, and
analysed by flow cytometry [110]. It was found that all three structures elicited low levels of immune
response, among these the type 2 Affimer structure that was used in this thesis[110]. These results
give hope to the future use of Affimer technology in therapeutics.
4.3.2 Intracellular Delivery
One challenge to overcome for the use of Affimers as therapeutics is the use of a reliable system for
intracellular delivery. Due to their size and polarity, proteins are more challenging to diffuse across a
cell membrane than small molecules. A protein such as an Affimer is around 12 kDa, and is therefore
unlikely to passively diffuse across a membrane in great enough numbers to elicit an efficacious
response [101]. The added complexity of a double-membrane cell envelope in the case of Gram?negative bacteria means it is increasingly difficult. There is evidence to suggest though that MBL’s
like NDM-1 and VIM-1 are localised to the periplasm, such that an inhibitor need only permeate through the outer membrane of a Gram-negative bacterium to be effective thereby indicating a
higher level of target accessibility compared to cytoplasmic targets [101], [102]
.
Cell-penetrating peptides (CPP’s) are water soluble, partly hydrophobic peptides that have the ability
to penetrate a cell membrane without causing significant damage, and have the capacity to deliver
an internalised covalently bound biologically active cargo with low toxicity [103]. In 2004 TP10 CPPs
were shown to deliver SYTOX Green - a nucleic acid stain – preferentially to S. aureus cells when
incubated with both HeLa and non-invasive S. aureus, fluorescence increased in S. aureus without
increasing at the same rate in HeLa cells [104]. In 2015, CPPs were covalently conjugated to peptide
nucleic acids (PNAs) to target intracellular RNA polymerase a subunit (rpoA) expression, and found a
50% reduction in gene expression at 1 µM[105]
.
Polymersomes are lipid based delivery systems that can release their encapsulated molecule under a
variety of conditions such as change in pH, redox potential, ionic strength or instability within the
design of the system[106]. Polymersomes have the ability to encapsulate amphiphilic, hydrophobic,
and hydrophilic molecules making them well suited to carrying proteins [107]. They have been shown
to have the capacity to deliver drug mixtures of doxorubicin (DOX) and paclitaxel (TAX) into tumour
tissues in hours at a low pH and 37°C [108]
.
4.3.1. Affimers as Therapeutics and Diagnostics
As discussed in the introduction (see 1.5.2) there are currently no clinically approved metallo-ß?lactamase inhibitors currently in use, though some are in late-stage clinical trials. This goes to
demonstrate the potential impact a successful inhibitory Affimer could have clinically. If it could be
shown that inhibition is possible through the use of an Affimer reagent against a single MBL, tests
could be performed to identify any potential inhibition of related MBLs. If it was not applicable due
to the binding location varying between tangentially related MBLs, the high variability in binding
potential for the rest of the phage library could be screened in a similar way against a new target or
ranges of targets, in order to try and identify a specific binder – and potential inhibitor – for each.
hydrolyse the nitrocefin reagent
independently or inhibit VIM-1 activity.
4.2. Affimer Protein Expression and Purification
As described in section 3.2.2, for initial screening purposes Affimers were produced in 50 mL of
autoinduction media and purified by nickel affinity chromatography. This provided a sufficient
protein yield to perform nitrocefin assays in triplicate on a single plate. Once selected and subcloned
into pET11a vectors, Affimers were produced in 500 mL culture volumes and purified by nickel
affinity chromatography. This yielded greater protein expression, commonly greater than 10 mg of
protein per culture which was enough for several assays in both biological and technical triplicate
repeats. One advantage of Affimers is their high yield from E. coli cells, which is advantageous over
other technologies such as the production of antibodies and was evident in this study Coupled with these findings, the nickel affinity chromatography as a purification method appeared
to be efficacious, yielding high amounts of protein in pure samples. Bands on SDS-PAGE gels were
clear signifying their expression, with little - to no - protein contamination.
4.3. Affimers as inhibitors of Metallo-ß-lactamases
In this thesis, inhibition of the rate of nitrocefin hydrolysis by VIM-1 was accomplished with
Affimer61, reducing the rate by 47%, which suggests that Affimer reagents can modulate the activity
of VIM-1. Further characterisation must be performed on this and other Affimers, to establish an IC50
and determine the method of inhibition. An IC50 denotes in this case the amount of Affimer required
to inhibit the hydrolysis of nitrocefin by VIM-1 by 50%. This would be established by performing a
series of nitrocefin assays utilising a constant VIM-1 concentration, with an initially high (in order to
establish maximal inhibition, close to 100%) but decreasing concentration of Affimer reagent.
Technical and biological repeats would be necessary as within this study, to establish at what
concentration of Affimer is VIM-1 inhibited by 50%. The lower this value, the better the Affimer
would be since less of the reagent would be required to reach this inhibitory level.
Structural characterisation of the Affimer-target complex would shed light on the binding
interactions and the method of inhibition of Affimer 61. Identification of the binding site of
Affimer61 could also be referenced against known sequences of VIM-variants to establish the
likelihood of whether it could be used as an inhibitor against these also.
The use of alanine Affimers in this project is consistent with other controls used in Affimer projects.
The truncated variable regions, consisting primarily of alanine to allow structure and flexibility
without complex sidechain interactions, allows for establishing during assays that the basic structure
of the Affimer itself is not responsible for perceived effects. In the case of this project, it was to
ensure that the Affimer structure was not able to either hy
Discussion
As the global spread of antimicrobial resistance continues to increase, so too does the need for
novel reagents that are able to detect, bind, and modulate resistance mechanisms. Inhibitors that
target metallo-ß-lactamases are highly sought-after as their function allows them to threaten the
efficacy of our current clinical arsenal of antibiotics.
Affimer reagents have previously been found to inhibit the activity of NDM-1, a metallo-ß-lactamase
(MBL) with a functional similarity to VIM-1. In prior studies at the University of Leeds inhibition of
NDM-1 was found to be up to 85% at five-fold the Affimer. A. Herbert and Dr L. Faveri identified
Affimer 21 which inhibits NDM-1 by a non-competitive mechanism[88][87]. This work investigates the
proposal that Affimer reagents could be found to have a similar inhibitory effect on VIM-1.
4.1. VIM-1 Protein Expression and Purification
Initially VIM-1 proteins were expressed in BL21 Star (DE3) cells, which yielded a good quantity of
protein, around 3 mg per 500 mL culture. Initially VIM-1 was chemically biotinylated through the use
of EZ-link NHS-SS-Biotin, which binds primarily to lysine residues, of which there are six in VIM-1.
Chemical biotinylation is a common method of biotinylating proteins prior to immobilisation on
streptavidin coated surfaces in preparation for phage panning in phage display and phage ELISA[81]
.
In this study chemical biotinylation was found not to be successful (see Results 3.1.2.) An alternate
method of protein biotinylation is to add a biotin acceptor peptide (BAP) tag to the terminus of a
protein sequence. In strains containing the BirA plasmid, an expression plasmid for BirA ligase, the
BAP tag is biotinylated. For this reason, AVB101 cells were used for the expression of biotinylated
protein. Expression in AVB101 cells showed a protein yield of 2.8 mg per 500mL culture. The extent
of biotinylation of the resulting protein was analysed via Western blot and ELISA assays.
It should be noted for future work on preparing biotinylated protein for the purpose of protein
immobilisation, is that the genetic BAP-tag approach results in a lower diversity of orientations of
the immobilised protein compared to that of chemical biotinylation. As such, it may be that though
the results of the phage display were better than when using chemical biotinylation (such as through
NHS-SS-Biotin) it may represent in other cases a reduced ability to find a suitable Affimer or other
binder that would instead have bound to a -now secluded- section of the protein. This should also be
a consideration when proteins such as metallo-ß-lactamases are being used, as covering the active
site of an enzyme could reduce the potential of finding a broad-spectrum Affimer reagent that acts
as an inhibitor
I've skipped through majority as it's too much to take in this morning....
But read last bit
Discussion 4.
https://etheses.whiterose.ac.uk/31089/
https://newsrnd.com/life/2022-07-20-watch--what-innovative-treatments-are-available-for-metastatic-breast-cancer----walla!-health.ByWJm9FBhc.html
Last excerpt -
Concluding remarks :
Affimers and nanobodies are both examples of small probes that are becoming increasingly widely used in imaging. A specific advantage is their use in super-resolution imaging, which overcomes many challenges associated with traditional approaches, including allowing better penetration into samples and reduced linkage error, which has been well demonstrated for both nanobodies and Affimers. Although each of these small probes has their advantages and disadvantages, Affimers have the specific benefits of employing a completely in vitro screen – ease of production and purification from E. coli, excellent stability and straightforward labelling for downstream applications. The ability of both nanobodies and Affimers to recognise protein conformers and specific isoforms is likely to lead to new insights into cell biology, as well as highlight their therapeutic potential, and we expect these probes to become more widely used in the future.
Covid restrictions could RETURN to 'protect the NHS': Free tests and mask wearing could be reintroduced if rising number of cases has impact on the healthcare backlog - as hospital admissions are on brink of hitting 18-MONTH high
https://www.dailymail.co.uk/health/article-11002951/Return-mandatory-masks-Curbs-come-virus-impacts-backlog-health-minister-says.html
And this. ..
Another use for affimers in the future? Published 2021
Piggybacking on the Cholera Toxin: Identification of a CTB-Binding Protein as an Approach for Targeted Delivery of Proteins to Motor Neurons
Matthew R Balmforth et al. Bioconjug Chem. 2021.
Abstract
A significant unmet need exists for the delivery of biologic drugs such as polypeptides or nucleic acids to the central nervous system for the treatment and understanding of neurodegenerative diseases. Naturally occurring bacterial toxins have been considered as tools to meet this need. However, due to the complexity of tethering macromolecular drugs to toxins and the inherent dangers of working with large quantities of recombinant toxins, no such route has been successfully exploited. Developing a method where a bacterial toxin's nontoxic targeting subunit can be assembled with a drug immediately prior to in vivo administration has the potential to circumvent some of these issues. Using a phage-display screen, we identified two antibody mimetics, anticholera toxin Affimer (ACTA)-A2 and ACTA-C6 that noncovalently associate with the nonbinding face of the cholera toxin B-subunit. In a first step toward the development of a nonviral motor neuron drug-delivery vehicle, we show that Affimers can be selectively delivered to motor neurons in vivo.
https://pubmed.ncbi.nlm.nih.gov/34565149/
I posted this yesterday....
Affimers
Affimer Tagged Cubosomes: Targeting of Carcinoembryonic Antigen Expressing Colorectal Cancer Cells Using In Vitro and In Vivo Models
Arindam Pramanik et al. ACS Appl Mater Interfaces. 2022.
Abstract
Nanomedicines, while having been approved for cancer therapy, present many challenges such as low stability, rapid clearance, and nonspecificity leading to off-target toxicity. Cubosomes are porous lyotropic liquid crystalline nanoparticles that have shown great premise as drug delivery vehicles; however, their behavior in vivo is largely underexplored, hindering clinical translation. Here, we have engineered cubosomes based on the space group Im3m that are loaded with copper acetylacetonate as a model drug, and their surfaces are functionalized for the first time with Affimer proteins via copper-free click chemistry to actively target overexpressed carcinoembryonic antigens on LS174T colorectal cancer cells. Unlike nontargeted cubosomes, Affimer tagged cubosomes showed preferential accumulation in cancer cells compared to normal cells not only in vitro (2D monolayer cell culture and 3D spheroid models) but also in vivo in colorectal cancer mouse xenografts, while exhibiting low nonspecific absorption and toxicity in other vital organs. Cancerous spheroids had maximum cell death compared to noncancerous cells upon targeted delivery. Xenografts subjected to targeted drug-loaded cubosomes showed a 5-7-fold higher drug accumulation in the tumor tissue compared to the liver, kidneys, and other vital organs, a significant decrease in tumor growth, and an increased survival rate compared to the nontargeted group. This work encompasses the first thorough preclinical investigation of Affimer targeted cubosomes as a cancer therapeutic.
https://pubmed.ncbi.nlm.nih.gov/35196008/
Affimer Tagged Cubosomes: Targeting of Carcinoembryonic Antigen Expressing Colorectal Cancer Cells Using In Vitro and In Vivo Models
Arindam Pramanik et al. ACS Appl Mater Interfaces. 2022.
Abstract
Nanomedicines, while having been approved for cancer therapy, present many challenges such as low stability, rapid clearance, and nonspecificity leading to off-target toxicity. Cubosomes are porous lyotropic liquid crystalline nanoparticles that have shown great premise as drug delivery vehicles; however, their behavior in vivo is largely underexplored, hindering clinical translation. Here, we have engineered cubosomes based on the space group Im3m that are loaded with copper acetylacetonate as a model drug, and their surfaces are functionalized for the first time with Affimer proteins via copper-free click chemistry to actively target overexpressed carcinoembryonic antigens on LS174T colorectal cancer cells. Unlike nontargeted cubosomes, Affimer tagged cubosomes showed preferential accumulation in cancer cells compared to normal cells not only in vitro (2D monolayer cell culture and 3D spheroid models) but also in vivo in colorectal cancer mouse xenografts, while exhibiting low nonspecific absorption and toxicity in other vital organs. Cancerous spheroids had maximum cell death compared to noncancerous cells upon targeted delivery. Xenografts subjected to targeted drug-loaded cubosomes showed a 5-7-fold higher drug accumulation in the tumor tissue compared to the liver, kidneys, and other vital organs, a significant decrease in tumor growth, and an increased survival rate compared to the nontargeted group. This work encompasses the first thorough preclinical investigation of Affimer targeted cubosomes as a cancer therapeutic.
https://pubmed.ncbi.nlm.nih.gov/35196008/
Piggybacking on the Cholera Toxin: Identification of a CTB-Binding Protein as an Approach for Targeted Delivery of Proteins to Motor Neurons
Matthew R Balmforth et al. Bioconjug Chem. 2021.
Abstract
A significant unmet need exists for the delivery of biologic drugs such as polypeptides or nucleic acids to the central nervous system for the treatment and understanding of neurodegenerative diseases. Naturally occurring bacterial toxins have been considered as tools to meet this need. However, due to the complexity of tethering macromolecular drugs to toxins and the inherent dangers of working with large quantities of recombinant toxins, no such route has been successfully exploited. Developing a method where a bacterial toxin's nontoxic targeting subunit can be assembled with a drug immediately prior to in vivo administration has the potential to circumvent some of these issues. Using a phage-display screen, we identified two antibody mimetics, anticholera toxin Affimer (ACTA)-A2 and ACTA-C6 that noncovalently associate with the nonbinding face of the cholera toxin B-subunit. In a first step toward the development of a nonviral motor neuron drug-delivery vehicle, we show that Affimers can be selectively delivered to motor neurons in vivo.
https://pubmed.ncbi.nlm.nih.gov/34565149/
Are Britain and the US heading for a super-flu outbreak this winter? Australia sees massive surge that's being blamed on lockdowns
Also :-
Rapid antigen test being developed that can detect both Covid and the flu - but not in time for Australia's forecast tough winter
https://www.dailymail.co.uk/news/article-10931319/Rapid-antigen-test-developed-detect-Covid-flu-late-winter.html
https://www.dailymail.co.uk/health/article-10926427/Horror-flu-season-Australia-prompts-warnings-virus-cause-havoc-Britain-winter.html
For reference :
Bristol Myers Squibb and Turning Point Therapeutics have announced a definitive merger agreement under which Bristol Myers Squibb will acquire Turning Point Therapeutics for $76.00 per share.(£61)
https://www.pbiforum.net/mag/featured/bristol-myers-squibb-to-acquire-precision-oncology-company-turning-point-therapeutics/
Centre, NHS Greater Glasgow & Clyde
[Recruiting]
https://clinicaltrials.gov/ct2/history/NCT04969835?A=4&B=5&C=Side-by-Side#StudyPageTop
Part 3
Kyu-seong Lee, vice president of research at Samsung Seoul Hospital and Director of the Future Medicine Research Institute, said, “In order to develop cell/gene therapy for rare and incurable diseases, innovation in the cell/gene field is necessary.” Through the research cooperation of
Yoon-Sil Jang, head of the Cell and Gene Therapy Research Center at Samsung Seoul Hospital, said, “Starting with this collaboration, we expect Apicell Therapeutics to develop innovative cell and gene therapies through excellent research capabilities, mutual feedback and joint research in the clinical field.”
https://newsroom.daewoong.co.kr/archives/13717