Production of nanobodies in Andean camelids and their most common applications: A general review in the medical field

: The heavy chain fraction present in Camelidae antibodies is so-called nanobodies. They have different characteristics when compared to immunoglobulin G, like more diminutive size, higher affinity, shorter half-life in serum, etc. These proteins are codified by B lymphocytes cDNAs and can be produced in different hosts like Escherichia Coli, Pichia Pastoris , plant cells and even insect cells. Andean camelids have been mainly used in the Andean region of South America as transport means and source of raw materials like fibers and meat, then being of great economic importance. However, in Ecuador, the potential of these animals as a source of biomedical products has not been investigated or exploited yet. Due to the scarce information related to these molecules and their industrial production in the country, this review aims to remark on the most common medical application of nanobodies produced from Andean camelids; also, industrial applications are described.


Introduction
Camelid produces antibodies composed of heavy chain constant domains and a single variable domain; this variable domain is known as nanobody (Nb). It possesses a single antigen-binding domain called VHH 1,2 . Antibodies with a single variable chain, known as vNARs, have also been reported in the fish subclass Elasmobranchii to which sharks and rays belong [3][4][5] . Phylogenetic studies have shown that antibodies from Camelidae and Elasmobranchii evolved independently 6,7 . In recent years, Nb has been used as therapeutic agents, in diagnostic tests and as research tools 8-10 . Camelidae and Elasmobranchii possess different kinds of antibodies 6 , but only the antibodies that lack the light chains are of interest in this review.
Camelidae is a family that includes the old-world camelids: -Camelus dromedarius (dromedaries), and C. bactrianus (camels)-and the new-world camelids [11][12][13] . There are four species of new-world camelids in South America 14,15 : Lama glama (llamas), L. guanicoe (guanacos), Vicugna vicugna (vicuñas) and V. pacos (alpacas). All of them represent a genetic resource of great importance, not only from a scientific point of view 16-18 , since they could represent an opportunity for economic, social and technological development in Ecuador.
Nbs were first discovered and described by the Hamers-Casterman team 19 at the Free University of Brussels while analyzing C. dromedarius serum. The potential use of Nbs in the medical field is focused on the prevention, diagnosis and disease treatments 20,21 . Additionally, several preclinical and clinical studies regarding their use in phases 1 and 2 22,23 . The development of a drug at the laboratory scale until its sanitary registration takes about 10 years 24-26 . In fact, in 2019, the Food and Drug Administration (FDA) approved for the first time the therapeutic use of a humanized Nb 27 . Due to the great potential of Nbs, Steeland et al. 28 concluded that these molecules could be positioned as pharmaceuticals in a short period for daily use.
The main objective of this review is to describe the most common medical application of nanobodies produced from Andean camelids to demonstrate the potential of these molecules as a medical product. In addition, a little overview of its recombinant production in different biotechnological hosts and other industrial fields is described.

Immunoglobulin G VS Nanobodies
Structurally, immunoglobulins G possess 2 light and 4 heavy chains. Heavy chains contain 3 constant domains and 1 variable domain (VH), while the light chains contain 1 constant and 1 variable domain (VL) 29,30 . However, Camelidae antibodies lack the light chain 31 , and their 3 chains are composed of 2 constant domains and 1 variable domain (Nb) 32,33 . In addition, Camelidae-like shark antibodies (which lack a light chain and possess 7 chains) are called New Antigen Receptors (NAR) antibodies 34 . They comprise 5 constant domains and 1 variable domain (vNAR) 6,35 . Figure 1 shows the structural comparison between mammalian IgG and the IgG-like antibodies from Elasmobranchii and Camelidae. Figure 2 shows the alignment of Elasmobranchii vNAR, Camelidae Nb and a Mus musculus heavy chain antibody sequences.
The figure shows the amino acid variation between vNAR, VHH and mAb heavy chains. In parenthesis protein name according to DATA bank information.
Despite the biotechnological importance of immunoglo-bulin G, Nbs have certain differences over IgG, which are summarized in Table 1.
The main Nbs disadvantage is their low half-life in human blood serum because they are filtered by the kidney in 26 to 60 minutes [46][47][48] . However, they have low toxicity and can be eliminated from the human body by filtration by the kidneys 49 . Nbs have a low immunogenicity risk profile, which drives their use to develop potential clinical applications 50 . Using Nbs conjugated with small molecules like drugs, toxins, enzymes and imaging agents allows an approach to target sites with low systemic toxicity 51 . In addition, Nbs do not present the solubility and aggregation problems typical of conventional antibodies due to their hydrophilicity 36, 52 .
The size of a camelid antibody is around 95 kDA 53-55 due to the absence of the light chain; Elasmobranchii antibodies size is about 175 kDA 56 while the variable antigen-binding domains (Nbs and vNAR) measure between 12 and 15 kDA 46,56,57 . There is a vast difference between the IgG medical development and the research performed on Nbs and vNAR molecules 58 . Nevertheless, Nb and vNAR have similar advantages to the IgG (more specificity, smaller capacity to bind crypto antigens, more stability) 59,60 . Figure 3 represents the 3D structure of a Nb, a vNAR and a Mus musculus monoclonal antibody (mAb). This figure shows that the Nb and vNAR structures are smaller and more straightforward than the M. musculus mAb, facilitating their production 58 .

How are Nanobodies produced?
The development of Nb libraries can be achieved from a naive or synthetic source or by immunization. All of them are great ways to get the appropriate Nb that best meets the final research goal. For this reason, Nbs are expressed and produced at a large scale in the chosen host 64 . Libraries obtained by immunization are the most widely used and consist in injecting the animal with the correct antigen 65 . Native libraries are developed from non-immunized camelids blood 66 , and synthetic libraries are developed from a determined sequence framework 67,68 or the target antigen 69 , from which randomization of oligonucleotides in the hypervariable regions is performed 70 .nThe yields in the production of Nbs from immune libraries and synthetic libraries can vary significantly 71 . Figure 1 shows a general diagram for Nb production by immunization. This process starts with the inoculation of a young-adult camelid with 50 to 200 μg of the chosen antigen 72 . After the first immunization, the animal must be injected from 4 to 8 times during 2 months 64 . Once the immunization process is completed, the camelid's blood must be extracted and B lymphocytes purified. Further, B lymphocyte mRNA is isolated, purified and recovered to finally get the total complementary cDNA using polymerase chain reaction (PCR) techniques 73,74 .
Then, a Nb library is generated from the above-obtai- ned cDNA, for which the VHH regions are amplified using specific primers 73,75 . Muyldermans (2021) research suggests that the amplification of the VHH regions should be performed using mainly the nested PCR technique 64 . From the PCR products, a gene library is generated 67 ; then the fragments must be chosen by a display technique; the most common technique is the phage display 64 . The phage display library consists of bacteriophages with the antigenic molecule (Nb) in their coat; then, all the library is exposed to the specific antigen 76 . Finally, the selected fragments are screened and then chosen those that produce specific Nbs for the particular antigen 64,73,75,77 .
The selected VHH sequences are cloned at the industrial level in appropriate hosts like bacteria such as Escherichia coli 64,78,79 or yeasts such as Pichia pastoris 75 . Mammalian cells 73 , insects or plants 71 are also excellent options as industrial production hosts 80 . Table 2 exposes the kind of expression and yields of the different host cells. Finally, the proteins are extracted and purified, generally using the ammonium sulfate precipitation method in conjunction with other types of chromatography 73 as shown in Figure 1.

Applications of camelid nanobodies in Human Health
The small Nb size, joined with their high stability, solubility and a great capacity to recognize crypto-antigens place them as excellent alternatives for the prevention 86 , diagnosis 87,88 and human diseases treatment 46,89,90 .
Disease diagnosis using Nbs points to:1-the generation of images from tissues and organs 57,91 ; and 2-the performance of immunoassays to detect pathogen antigens 89,92,93 . Meanwhile, nanobodies as a therapeutic agents seek to attack an antigen, mainly to block virus replication 94,95 or bacteria growth 96 .
Another novel application is their use in antiphonic serums as neutralizers and blockers of toxins present in different types of poisons [97][98][99] .
Finally, a search was made for medical applications of nanobodies developed in Andean camelids. About 518 articles were found using the "Publish or perish" program where Andean camelids were used. The reported applications belonged mainly to the health branch and articles published in the last 5 years including 50 scientific publications that were not review articles.
Using descriptive statistics and frequency histograms, it Production of nanobodies in Andean camelids and their most common applications: A general review in the medical field   was determined that the most common application of Nbs is the treatment and diagnosis of infectious diseases ( Figure  3). However, the condition for which the largest number of Nbs have been developed was cancer (Figure 4). The 2020 pandemic greatly spurred the development of Nbs and other pharmaceuticals for the treatment and/or diagnosis of COVID-19 100,101 . However, Nbs development focused on diagnosis, as shown in table 3; cancer treatment has been developed since before 2020.
Cancer is a disease in which uncontrolled cell multiplication can spread to other parts of the body 102 . Alternatives for cancer prevention have existed since the 1700s 103 , and their treatments include surgeries, chemotherapies, radiotherapies, immunotherapies and hormone therapies 104 . Nbs have a good synergy with these treatments 105 , so they could be used together, although the full range of Nbs applications in cancer has not yet been explored thoroughly 106,107 .
On the other hand, COVID-19 caused by SARS-CoV-2 generates affections on the respiratory system 108 . On March 11 of 2020, the World Health Organization (WHO) declared a world pandemic for this infection 109 . There is still no defined treatment for this disease, although monoclonal antibodies 100,110 and nanobodies seem to be the best option to fight the virus [111][112][113] . The future evolution of the virus must be awaited as it could generate less infectious and virulent variants or more aggressive strains resistant to the current preventive treatments 114 .

Applications of camelid nanobodies in different branches of industry
The main focuses of Nbs in plants are to protect crops by providing immunity against pathogens 72,85 and rapid . Production process of recombinant nanobodies. The first step is to immunize the camelide. Then, the B lymphocytes must be extracted and purified to extract m-RNA. The VHH sequences are obtained by PCR. Thereafter, the nanobody sequences are selected by phage display (or any display method). Finally, the chosen nanobodies are produced recombinantly and purified.

Table 2. Comparison between conventional antibodies and nanobodies.
Production of nanobodies in Andean camelids and their most common applications: A general review in the medical field   detection of toxins that may affect food safety 115 . Passive immunity is the use of external antibodies to protect a patient [116][117][118] ; this concept can be extrapolated to plants, where antibodies would protect them against pathogens 85,119,120 .
On the other hand, Nbs can identify toxins in absorbance, fluorescence or Enzyme-Linked ImmunoSorbent Assay (ELISA) tests, depending on the toxin to be tested [121][122][123] .
In the livestock sector, the main Nbs application is on early disease detection 124,125 to avoid zoonoses 3,126,127 . Nbs can also provide passive immunity in animals, especially those sensitive to diseases after weaning, such as piglets 128 . This is very important to avoid using antibiotics that could generate resistant bacterial strains 89,129 .
Nanobodies used in water and soil allow the quick and accurate identification and detection of toxins and contaminants to the nanogram levels so, becoming a handy tool for these purposes [130][131][132] .
The ability of nanobodies to bind to specific compounds makes them good alternatives in high-affinity chromatography 133,134 . They can also help to determine the protein structure 135 with dynamic properties in case of proteins with various conformations and shapes which are difficult to solubilize 64,136 . Another application is identifying protein functions and protein-protein interactions by mass spectrometry that could replace conventional antibodies by Nbs 137 .
The use of intrabodies that consist of nucleic acid sequences with the genetic coding information of an antibody or Nb 138,139 that can be expressed inside a cell to modify cellular activity 140,141 . This biological application could be used to alter cellular functions 142 , blocking or reducing the activity of endogenous proteins 143 .

Future Perspectives and Conclusions
This mini-review presented applications of nanobodies that could promote the use of Andean camelids in Ecuador as new sources of products of high social, economic and public health interest.
The SARS-CoV-2 virus generated a global health crisis. Therefore, several methods for the diagnosis of COVID-19 were rapidly developed 144 . Nonetheless, there is still no defined treatment 100,110 . Becker's study in 2020 145 e mentions that a great deal of research is being carried out, mainly focused on blocking the replication and translation of the virus RNA; moreover, some treatments seek to block the binding of the virus and its ACE2 receptors [146][147][148] so, Nbs could be used as therapeutic molecules against COVID-19.
Ecuador has the greatest number of reptile species per unit area; one of the groups of greatest interest are snakes.
Among them, about 35 species are reported to be venomous and are distributed along the Ecuadorian coast and Amazon 149 . On the other hand, scorpions, of which 40 species are known to be venomous, have been little studied 150 .
In the past, serums to treat poisonings were produced by the "Instituto Nacional de Investigación en Salud Pública" INSPI, but production was suspended in 2014 so only antivenoms were imported 151 . In 2022, INSPI produced a batch of 300 experimental antivenom sera 152 . The Nbs could be used to act as high-affinity antivenom. The incidence of cancer in Ecuador is 61% in women while 41% in men 153 ; the types of cancer most reported in male patients are prostate, lymphoma and stomach 154 , while for female patients, they are breast cancer, cervix and lymphoid leukemia 153 . Nanobodies could be an interesting alternative for treating these types of cancer in Ecuador. However, the fact that they are rapidly filtered by the kidney makes them less attractive as a therapeutic alternative 58 .
In conclusion, the most common application of Nbs after analyzing more than 50 articles was the treatment and diagnosis of infectious diseases in cancer, so the future development of nanobodies should be linked to the treatment and diagnosis of these diseases. It is suggested that Nbs that achieve a balance between tissue permeability and a longer half-life in serum be developed for treating diseases such as cancer. However, other exciting applications in other fields are also not ruled out for the use of nanobodies.