Figure 4.3.6: Nicotiana benthamiana part of the Nicotine plant family with fluorescent spots of green fluorescent protein (GFP) indicating the presence of plantibodies ^{91, 92}.

Newer plant transformation technologies have since come about to strengthen protein production in plants. This includes plastid engineering where the gene is put into a plastid rather than the genome as plastids are more abundant which enables increased expression ⁸⁷. Agroinfiltration is a method of inducing transient expression with the use of agrobacterium as the vector for delivery with vacuum infiltration ⁹³. Magnifection, is also a transient expression system which uses agrobacterium to deliver viral vectors for high level expression and deconstructed viral vectors can be used to prevent movement from cell to cell ⁸⁷.

Suppliers: Planet Biotechnology, Leaf Expression Systems, Icon Genetics

There are other expression vectors being tested for recombinant antibodies including insects such as Drosophila melanogaster, gram positive bacteria such as Bacillus subtilis, fungi such as Aspergillus, and protozoa that can perform both N- and O-glycosylation ⁹⁴.

5 Advantages of Animal-Free Antibodies (AFAs)

Animal-free antibodies have several advantages over traditional animal-produced antibodies. They play a key role in the third R of the 3R system; replacement. By replacing animals as the expression vectors, with phage, yeast, or single B cells, for antibody discovery, and mammalian cell lines for production, not only reduces the number of animals used in science but also increases the specificity and reduces the immunogenicity of antibodies.

Hybridomas can be unreliable. They are subject to contamination, genetic drift and their instability can lead to loss of antibody expression or additional expression of L chains ²⁷. Hybridomas are dependent on animal-derived growth factors and the switch to serum-free growth was not compatible. To reduce and replace animals in research we need to steer clear of hybridoma reliant methods ⁹⁵. AFAs on the other hand have little batch-to-batch variation and are more reproducible making them an excellent alternative ³¹.

Inclusion of competitor molecules in the production of AFAs has enabled direct selection of specific targets ²⁶. The affinity of antibodies produced in this way is much higher than immunization and allows control over both selection and screening. Otherwise conserved antibody targets are accessible through the use of in vitro methods, this was previously not feasible due to immunological tolerance. In vitro methods do not need additional steps to overcome this tolerance whereas immunization requires extra steps to overcome their immunogenicity lengthening an already long process.

As the antibody sequence is known and can be cloned it has made the process more reproducible. It has also allowed the manipulation and editing of genes to produce new antibodies and to select for the production of only certain fragments. Antibodies can also be selected for certain characteristics through the inclusion of competitor molecules or agents to induce certain conformations. Recombinant antibodies are valuable tools in the lab as they can be converted to species compatible forms by changing the C domain to the native species’ ²⁸.

Hybridomas were quickly established as a valid method of antibody production and with high uptake, uptake of phage display on the other hand was slower. This lag was mainly due to patents, many of these patents have started to expire in recent years seeing an increase in the use of phage technologies which they expect to rival the uptake levels seen for hybridomas ⁷⁶. AFAs have been well established in the therapeutic sector but uptake in other sectors hasn’t matched up ⁹⁶.

Phage display has the advantage that it requires less antigen to locate a matching antibody than immunization, while also using controlled selection conditions that allow for smaller fragment production along with allowing the swapping of fragments ²⁶.

In vitro technologies can identify differences in protein sequences and structural changes in the small molecules they target. Phage display is uniquely good at identifying tyrosine sulfation, a common PTM that is responsible, in part, for protein-protein interactions and other functions in the body. It is generally hard to target but phage display allows the selection of antibodies specific enough to recognize them ^{76, 97}.

One big advantage of AFAs over animal-derived is the ability to increase affinity through the use of more than one library, creating smaller and smaller clonotypes at the end of each new library, leaving the highest affinity antibodies to choose from in the last clonotype.

Through in vitro methods ELISA positive antibodies can be generated in as little as 2 weeks with an average production time taking 8 weeks. The average time for hybridoma-generated antibodies is 4 months ²⁸. In addition to this, large-scale production of rAbs is much cheaper making them more accessible to researchers ⁹⁸.

These are constantly evolving technologies and over the years previous limitations have been overcome such as, the production of unglycosylated or incorrect glycosylation of antibodies from bacterial or yeast cultures which were swapped out for human cell lines producing fully human antibodies in vitro.

These advantages make a very convincing argument, and the EU parliament agreed. In 2010 a law (2010/63/EU) ⁹⁹ was passed that prohibits the production of antibodies through animal methods where alternative methods with equal or better quality exist. This is just one part of the EU’s commitment to the 3R process of animal use, another part includes the ban on cosmetic testing on animals. This quickly spread to other countries due to the inclusion of imported cosmetics under this law. The 2010/63/EU law also discourages the use of imported animal-derived antibodies ⁹⁶, which has seen talks overseas in the Organization for Economic Co-operation and Development (OECD) and the US National Institutes of Health (NIH) as to what they can do to prevent a trade stop ¹⁰⁰.

This law has been a long time coming after the EURL ECVAM safety advisory (ESAC) advised in 1998 that the in vivo production of mAbs in animals, specifically through the ascites method was no longer needed as its alternative, phage display produced antibodies, provided as good as, if not better quality reagents than the ascites method. This had the added advantage of also reducing cruelty associated with traditional production methods ¹⁰¹. A minimum of 2 rabbits or guinea pigs and between 5-10 rats or mice are used in polyclonal production methods and approximately 5 mice are immunized for every one hybridoma produced. These all make up a sizable chunk of the 2.7 million animals used for science and medicine in the EU. Under this law, production or testing via animal methods requires a very valid and robust justification that has to be supplied in the application and approved by the authorities before proceeding ¹⁰⁰.

There is no scientific justification for the use of animals to produce antibodies. Phage displayed antibodies don’t just look like in vivo antibodies, they also have the same functional biological mechanisms as the in vivo adaptive immune system. They are identical to in vivo antibodies in both structure and function so meet the requirements to support the 2010/63/EU directive ¹⁰⁰.

We have found ourselves in a reproducibility crisis, in one such study a mere 6 of 53 studies were reproducible and in a separate study they found that less than 3000 of some 6000 commercial antibodies only bound their specified target, meaning more than half of those antibodies bound additional off-target epitopes or didn’t bind their specified target at all. Many commercially produced antibodies are not validated and as a result many antibodies can have low specificity for their target antigen. Most large pharmaceutical companies have departments dedicated to validation but the same cannot be said for research antibodies. In most research papers, the source of antibodies is not stated, and when they do, purchasing the same antibodies does not guarantee the same batch and target specificity. As mentioned before batch variation is a big reproducibility issue with animal derived antibodies, each new immunization will create a new mix of antibodies so it is impossible to get the same batch twice unless from the same animal or by recombinant animal-free methods ¹⁰².

In this report they recommend two ways to make antibodies more reproducible. The first is by defining antibodies by sequence and producing them recombinantly. Phasing out of hybridoma-derived antibodies is possible by sequencing the popular antibodies and making them recombinantly, preventing the future immunization of animals. The second recommendation was to use methods such as the display methods or single B-cell technology which use controlled protocols ¹⁰².

More advantages can be found on the People for Ethical Treatment of Animals (PETA) website here, including ethical and economic reasons for changing to AFAs.

6 Next-Generation Antibody Generation:

Antibody discovery and production is an ever-evolving process with a near-constant flow of new techniques and technologies to advance antibody production. In this section, we will cover some of these advances including the incorporation of artificial intelligence (AI) and the production of bispecific antibodies.

6.1 Artificial Intelligence

AI has been employed for both discovery and generation through the use of its various types. Machine learning comes up frequently due to the impressive increase in affinity associated with its use. Long short-term memory (LSTM) specifically has been used in this application, identifying higher affinity antibody sequences for antibody affinity maturation. It was trained with next-generation sequencing (NGS) binding sequences and applied to anti-hapten antibody screening (also known as anti-target metabolite antibodies) to make better affinity antibodies. Compared with the anti-hapten screening on its own, the antibodies produced had 100-fold better affinity through exploration of virtual sequences beyond what is found in phage libraries ¹⁰³.

Panning enrichment is an application of the convolutional neural network (CNN)-based regression model. It was noted that sequences from machine learning had higher affinity than panning methods ¹⁰⁴. Through the use of both CNN and LSTM, they have been able to train models to distinguish between non-binder and binders for a clustered regularly interspaced short palindromic repeat (CRISPR) induced homology-directed repair (HDR) mechanism. This method has a hypothesized use in the optimization and engineering of therapeutic antibodies ¹⁰⁵.

Deep learning, on the other hand, learns the long-range sequence dependencies. These dependencies in antibodies present as two amino acids separated by a long intermediate sequence but in 3D space are very close together. This makes it a very good platform for the identification of antibodies with correct binding sites and properties, as binding is generally based on shape. It is challenged by the size of the sets used for training, these are smaller than the sets they aim to build ¹⁰⁶.

These are not the only applications, combinations of phage/yeast display followed by high throughput screening (HTS) and machine learning can create focused and antigen-specific clonotypes giving a choice of antibody affinity for the target antigen ²⁷. In general, machine learning in the early stages of antibody discovery can be very useful for screening of function and affinity ¹⁰⁶. AI has the potential to combine both in vivo and in vitro methods of production solving both ethical and scientific issues through generation with their vast libraries of antibodies including data on their structure, sequence, and binding affinity ¹⁰⁷. Advances can bypass antibody libraries and move discovery to in silico models.

6.2 Bispecific Antibodies

Bispecific antibodies are antibodies that have different H chain and different L chain pairs. They can be symmetric or asymmetric in structure and are classified based on this and their composition ²¹. Within the asymmetric and symmetric structure categories, there are also the Fc (IgG-like) and non-Fc (non-IgG) containing bispecifics. These categories determine the properties and applications of the antibody, for example, non-Fc containing bispecifics won’t be able to contribute to Fc-mediated effector functions such as ADCC, complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP) but are smaller in size so will have enhanced penetration powers and reduced nonspecific recruitment ^{21, 108}. They consist of two fragments, usually scFvs, held together by a peptide linker, disulfide bond, or non-covalent inter-domain interactions ¹⁰⁹. There are also more than 100 other different bispecific antibody formats ¹⁰⁹.

They can target either two different antigens at a time or two different epitopes of the one antigen which makes for a more specific, tailored treatment compared to natural antibodies. This can come in the form of, for example, simultaneously binding tumor cell receptors while also recruiting cytotoxic immune cells ¹⁹.

There is no single standard production method, rather a variety of them that can produce the 100s of different bispecific antibody formats. Generation can be through genetic, biochemical, or molecular means. Production of first-generation bispecifics started in the 60s ¹¹⁰ through chemical conjugation of mAbs or by fusion of hybridomas. Chemical conjugation uses catalytic antibodies to join the reactive bispecific peptides of two different antibodies together, either whole Igs or only Fab fragments ¹¹¹. Hybrid hybridomas (also known as quadromas) are created through somatic hybridization of two hybridomas ¹¹¹. They generate cell lines that make two different H and L chains in a single cell. It wasn’t until the 90s that bispecifics entered their first clinical trials ¹⁰⁸. Bispecifics at that time had high immunogenicity due to production using mouse antibodies which also induced a HAMA response reducing efficacy and inducing severe adverse effects ^{108, 111}.

Advances over the years especially in rAb generation have been transferred to bispecific manufacturing of second-generation antibodies with defined properties and structure. This allowed the use of only the binding fragments of the antibody in bispecific production ¹¹¹. Bispecifics are useful as therapeutics, initially only involved in effector cell retargeting but now also involved in dual targeting and pre-targeting, half-life extension, and delivery through cellular barriers among others ²¹. Large-scale production of bispecific antibodies has been established with the use of different engineering technologies such as asymmetric re-engineering technology (ART)-Ig ¹¹². To create bispecifics with specific targets, engineering must enable the inclusion, for example, of the cluster of differentiation (CD)64 protein for the recruitment of macrophage and monocytes or CD16 to target NK cells and macrophage ¹⁰⁸.

Bispecific antibodies can increase binding, selectivity, and anti-tumor activity to name but a few ¹¹¹. As of 2018, there were only two bispecific antibodies approved for the treatment of cancer. An Fc-containing one, catumaxomab (now withdrawn in the EU ¹¹³), and a non-Fc containing one, blinatumomab. These are second-generation antibodies with either a combination of an anti-CD3 domain or an anti-Fc binding domain with anti-tumor binding domains. These bispecifics recruit immune effector cells for immunotherapy ¹⁰⁸. In 2021 another was approved for use by the FDA for non-small cell lung cancer (NSCLC) called amivantamab ¹¹⁴. Only one other bispecific antibody has been approved for use, which is emicizumab for the treatment of hemophilia-A in 2018 ¹¹⁵, but 100s of others are in development ¹¹⁶.

Both bispecific antibodies and antibody-drug conjugates (ADC) were created to overcome the resistance that has developed for normal therapeutic antibodies. ADCs consist of an antibody to target the antigens present on cancer cells and a potent cytotoxic payload that is connected by a chemical linker. After antibody binding, the ADC is endocytosed into the cell where the antibody or linker is degraded releasing the drug. This method has allowed the directed delivery of the drug into the cancer cell itself while sparing the normal healthy tissue ¹⁰⁸.

7 Conclusion:

Antibodies are hugely beneficial in science and medicine, from their participation in assays to their use as treatments for the likes of cancer, rheumatoid arthritis, hemophilia, etc. These applications have benefited from advances in recent years that have enabled quicker production and production with specific advantages such as increased affinity or reduced immunogenicity making more specific antibodies with less chance of adverse reactions. These advances have also created choices for researchers with many variations of antibodies available from chimeric to fully human, covering all aspects of research and development. They have overcome many limitations over the years from immunogenicity and resistance to poor therapeutic targeting and efficacy. The creation of increased valency antibodies has enabled targeting to multiple epitopes ensuring the correct antigen is bound. Animal-derived methods of production are outdated and very rarely needed, AFAs are the way forward, they are generated in a fraction of the time, have the same if not better efficacy, reduced immunogenicity, and are ethically produced. There is the potential and the means to completely replace animals in the production and testing of antibodies and antibody products, which in years to come is what will be seen thanks to the cooperation of participating countries in the 3R (Reduction, Replacement, and Refinement) project for animal use in science, and the advances in science enabling this move.

8 Providers:

The following providers either provide exclusively non-animal produced antibodies or animal free antibodies in addition to animal-derived antibodies.

Information provided courtesy of companies’ websites. Tissue For Research Ltd is not responsible for any information about, or services provided by, external companies.

Abcalis – By using our sequence defined recombinant antibodies, you can obtain superior quality of results with animal-free reagents. It is our mission and aspiration every single day to achieve this goal, both for a sustainable future and medical safety of this and every future generation to come.

Abcam – They have a selection of animal-free antibodies and proteins generated in E.coli, yeast, synthetic, human embryonic kidney (HEK), or Chinese hamster ovary (CHO) cell expression systems ranging from fragments to full-length proteins. They no longer use the ascites method but instead use tissue culture and expression vectors. The antibodies they provide are for research purposes only but they can also be used commercially through a partnership with Abcam.

Absolute Antibody – Absolute Antibody provides Antibody Sequencing, Engineering & Recombinant Expression.

Adimab – Discovery of antibodies is via a yeast system with either synthetic or natural libraries incorporated. This system allows the selection of properties in real-time. They can also use phage display libraries and can optimize almost any property. They also offer the ability to create bispecifics based on IgG, scFv, and CD3.

AdipoGen Life Sciences – Through the use of human naïve phage display libraries they produce animal-free monoclonal antibodies. Their antibodies focus on functional activation/blocking of their targets and they also employ a selection strategy that allows the selection of antibodies based on custom functions such as targeting a specific conformational state antigen or identification of multi-protein complexes. They are available via AdipoGen’s custom services for research, pre-clinical and therapeutic applications.

Aptagen – Aptagen is a leading world-wide biotechnology company offering aptamer (synthetic antibody) products and services as research reagents, diagnostic and biomarker discovery tools, as well as for use in drug discovery and targeted delivery for therapeutics, and bioindustrial applications. We have a combined 25+ years of experience in creating aptamers that bind a wide variety of targets against small molecules, proteins, biomarkers and cells. Aptagen has performed this service for more than 50 companies, organizations, and institutions worldwide.

AvantGen – AvantGen has constructed 30 human antibody libraries (Germliner™) with multiple human antibody germline frameworks. These were selected based on their high stability, solubility and expression level. Diversity is focused in the CDR regions and mimics the diversity of the natural human antibody repertoire for that variable region family. The framework sequences are left as fully human germline sequences. AvantGen’s proprietary libraries are comprised of >100 billion members, are well expressed and displayed with AvantGen’s yeast display system allowing efficient human antibody discovery.

BioRad – These have produced thousands of completely animal-free antibodies. They mainly use phage display with a fully synthetic, naïve human combinatorial antibody library. Recently they have employed a new technique of antibody production called SpyTag that makes antibodies by protein ligation and site-directed conjugation within 8 weeks. With each antibody webpage, they have included a list of applications tested in-house and through information in peer-reviewed journals.

Cell Signalling Technology (CST) – They also dropped the ascites method in favor of cDNA and phage libraries expressed in vectors such as CHO cells, HEK cells, Pichia pastoris, etc. CST only provides recombinant antibodies for research and development use.

Geneva Antibody Facility – Providing recombinant antibodies to the academic community, not-for-profit. It is also known as the ABCD database, each antibody within this database is linked to the UniProt website for more information as well as its own ABCD detailed summary which includes all known research applications, this is constantly being updated as new information is published. It is also linked to a manufacturing company that can make these antibodies on demand. It consists of over 23,000 antibodies and 4000 antigens.

GenScript – GenScript offers one non-animal expression method using bacterial vectors and a variety of recombinant antibodies for research use. Their BacPower system uses E.coli as the expression vector and can produce proteins in an average of 4-6 weeks. They also offer another alternative to animal use in their insect expression system. This system infects baculovirus-infected cells with the recombinant plasmid to produce either virus or recombinant antibodies.

Human Antibody Core Facility – One of the few laboratories in the world that produces fully-human, full-length, antigen-specific antibodies for use in studying human immune responses. The Core has achieved breakthroughs in antibody technology and produced hundreds of high affinity protective antibodies to influenza, anthrax lethal toxin, and various S. pneumonia polysaccharides. The Core aims to support investigators by helping quantify the antibody secreting cell responses after vaccination and by generating human monoclonal antibodies to be characterized. Pathogen-specific human monoclonal antibodies are also available for licensing agreements and other forms of commercial development.

HYBRiBODY – They produce antibodies through phage display offering selection of recombinant antibodies and nanobodies, that are ELISA validated, for basic research and diagnostics. They also offer additional services to test the validated antibodies including cell imaging and can create ‘minibodies’ with the species-specific Fc region you require. They can employ Yeast two Hybrid technology after successive rounds of phage display to screen for intrabodies, which are antibodies that work inside living cells. Intrabodies are very useful for live cell imaging and in vivo applications.

ProMab – They provide a library of human Fab fragments only, isolated from 120 donors. These antibodies are present in their general recombinant and natural catalogs but more specific antibodies can be obtained from their custom services too. They pan the library against your antigen, perform ELISA on them in E.coli, sequence the selected clones out of this, then cloning, transfection, and confirmation tests are completed before at least 5 positive clone sequences are sent via email.

Prellis – Their EXIS platform uses healthy donor B cells to make organoids which they then immunize with the antigen of interest. They can then get the antibody of interest from this immunized organoid. It boasts that it can reduce discovery time from 6-12 months in animals, to merely 3 weeks with EXIS, and also reduces costs.

Specifica – They use next-generation sequencing to produce antibody libraries in yeast or phage formats, as scFv or Fab libraries or common or variable light chains. Through the use of AI machine learning, they can split the libraries into clonotypes providing a whole host of antibodies against a variety of epitopes. They do initial screening with the Generation 3 phage display library (has natural CDR sequences, reducing liabilities and ensuring correct folding) then they use the yeast displayed library to narrow it down to fully binding antibodies. Each library they produce is unique to help avoid competitors choosing and developing the same therapeutic.

Tresars – Through Tresars use of display technology they provide fully reproducible and recombinant antibodies with serum-free methods, endeavouring to overcome the antibody reproducibility crisis without the use of animals.

YUMAB – YUMAB provides individually designed contract research projects for the discovery and the development of fully human antibodies, as well as R&D services for the engineering of antibodies.

10% discount on your next human biospecimen order from Tissue For Research (Accio BioBank Online) when you forward proof of purchase, or in-house generation, of animal-free recombinant antibodies. Additional discounts are available when ordering from our partners- please get in touch at info@biobankonline.com for more details of how you can benefit from this.

 9 Abbreviations

ADC – Antibody Drug Conjugate
ADCC – Antibody-Dependent Cellular Cytotoxic
ADCP – Antibody-Dependent Cellular Phagocytosis
AFA – Animal-Free Antibody
AI – Artificial Intelligence
ART-Ig – Asymmetric Re-engineering Technology – Immunoglobulin
ATD – Amino Terminal Domain
ATG16L – Autophagy Related 16 Like 1
C – Constant
C1q – complement Component 1q
CDC – Complement-Dependent Cytotoxicity
CDR – Complementarity Determining Region
CD – Cluster of Differentiation
CHO – Chinese Hamster Ovary cells
CNN – Convolutional Neural Network
CRISPR – Clustered Regularly Interspaced Short Palindromic Repeats
CST – Cell Signalling Technologies
E – Envelope protein
EBV – EBola Virus
EGFR – Epidermal Growth Factor Receptor
ELISA – Enzyme-Linked Immunosorbent Assay
ESAC – European Union Reference Laboratory for alternatives to animal testing (EURL ECVAM) Safety Advisory
Fab – Fragment antigen-binding
FACS – Fluorescent Activated Cell Sorting
Fc – Fragment crystallizable
FcεR – Fc epsilon Receptor
FDA – US Food and Drug Administration
Fv – Variable Fragment
GFP – Green Fluorescent Protein
GI – GastroIntestinal
H – Heavy
HAMA – Human Anti-Mouse Antibody
HDR – Homology Directed Repair
HEK – Human Embryonic Kidney cells
HIV – Human Immunodeficiency Virus
HTS – High Throughput Screening
Ig – Immunoglobulin
IHC – ImmunoHistoChemical
INN – International Nonproprietary Names
L – Light
LGI1 – Leucine-rich glioma-inactivated 1
LPS –  LipoPolySaccharide
LSTM – Long Short Term Memory
mAb – monoclonal Antibody
MACS – Magnetic- Activated Cell Sorting
NGS – Next Generation Sequencing
NMDA – N-methyl-D-aspartate
NSCLC – Non-Small Cell Lung Cancer
OECD – the Organisation for Economic Co-operation and Development
pAb – polyclonal Antibody
PCR – Polymerase Chain Reaction
PETA – People for Ethical Treatment of Animals
PPS – Pneumococcal polysaccharides
PRM – Protein-Ribosome-mRNA
PTM – Post Translational Modification
rAb – recombinant Antibody
RT-PCR – Reverse Transcriptase Polymerase Chain Reaction
scFv – single-chain Variable Fragment
TNF⍺ – Tumor Necrosis Factor-alpha
US NIH – US National Institutes of Health
V – Variable
ZIKV – ZIKa Virus

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