Camels' Biological Fluids Contained Nanobodies: Promising Avenue in Cancer Therapy

Al‑Numair et al.
Cancer Cell International 2022, 22(1):279

https://doi.org/10.1186/s12935-022-02696-7 Cancer Cell International
REVIEW Open Access
Camels’ biological fluids contained

nanobodies: promising avenue in cancer
therapy
Nouf S. Al‑Numair1,2, Abdulrahman Theyab3,4, Faisal Alzahrani5,6, Anwar M. Shams7, Ibrahim O. Al‑Anazi8,
Atif Abdulwahab A. Oyouni9,10, Osama M. Al‑Amer11, Charalampos Mavromatis12, Islam M. Saadeldin13,
Wed A. Abdali9 and Yousef M. Hawsawi2,14*
Abstract
Cancer is a major health concern and accounts for one of the main causes of death worldwide. Innovative strategies
are needed to aid in the diagnosis and treatment of different types of cancers. Recently, there has been an evolving
interest in utilizing nanobodies of camel origin as therapeutic tools against cancer. Nanotechnology uses nanobodies
an emerging attractive field that provides promises to researchers in advancing different scientific sectors including
medicine and oncology. Nanobodies are characteristically small-sized biologics featured with the ability for deep
tissue penetration and dissemination and harbour high stability at high pH and temperatures. The current review
highlights the potential use of nanobodies that are naturally secreted in camels’ biological fluids, both milk and urine,
in the development of nanotechnology-based therapy for treating different typesQuery of cancers and other dis‑
eases. Moreover, the role of nano proteomics in the invention of novel therapeutic agents specifically used for cancer
intervention is also illustrated.
Keywords: Nanobodies, Arabian camelid, Biological fluids, Cancers, Therapeutic agents, Diagnosis, And nano
proteomics
Cancer and nanobodies at a glance to early detection and prompt implementation of the
Cancer denotes one of the leading causes of death glob- treatment plan which often involves surgery, radiation,
ally albeit with the rapid innovations in the molecular chemotherapy, targeted therapy, and hormone therapy
biology [1]. According to statistics produced by the GLO- [3]. Following decades of research, researchers have now
BOCAN series of the International Agency for Research begun to explore nanotechnology-based therapy using
on Cancer (IARC), over 14.1 million cancer cases nanobodies and evaluating their potential uses as novel
and more than 8.2 million cancer-related deaths were cancer therapeutic modalities. Nanobodies can cross the
reported in 2012 [2]. Approximately, 21.7 million can- blood–brain barrier and invade large solid tumours more
cer cases and over 13 million cancer-related deaths are competently than other conventional antibodies. This
predicted to occur by 2030 [1]. Successful interventions encourages experts to propose nanobodies as ideal thera-
of cancer progression in affected patients are attributed peutic candidates for the solid metastatic cancers [4].
*Correspondence: [email protected]
2
College of Medicine, Alfaisal University, P.O. Box 50927, Riyadh 11533, Saudi
Arabia
Full list of author information is available at the end of the article
© The Author(s) 2022, corrected publication 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0
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Al‑Numair et al. Cancer Cell International 2022, 22(1):279 Page 2 of 18
Distinguished features of the nanobodies

and nanoparticles
Small molecules, or nanoparticles, have a diameter of
roughly 200 nm. Many pharmaceutical companies have
aspired to create efficient drug delivery methods, and as
a result, a variety of nanoparticle-based delivery modali-
ties, including magnetic, polymeric, and inorganic nano-
particles, have been developed [5]. An attachment of the
targeting moieties to the drug cargo has been devised
to improve the transport, permeability, and penetration
of the drug-conjugated nanoparticles into the target tis-
sues. Poly-ethylene glycol (PEG) molecules and nanobod-
ies are examples of these targeted molecules that protect
the nanoparticles [6]. Decorating nanoparticles with
nanobodies enhanced the accumulation of the nanopar-
ticles containing the drug’s cargo into the diseased tis-
sues [7]. By monomerizing the dimeric variable domains
of human or mouse conventional antibodies, nanobod-
ies can be bioengineered. Nanobodies, with a molecular
mass of roughly 15 kDa, are thought to be the smallest
antibody components capable of antigen detection [8].
Alternatively, nanobodies can also be retrieved and iso-
lated from the immunized camel blood and character-
ized as a single variable domain on a heavy chain (VHH) Fig. 1 Nanobodies applications
antibodies/nanobodies [6, 8]. VHH, or Heavy chain anti-
bodies (HCAbs), that are derived from camel blood have
lower lipophilicity and are a single domain consisting of were also reported to be effectively targeted by nanobod-
one amino acid chain. These characteristics confer cer- ies [16]. Other remarkable nanobodies-based therapies
tain advantages to VHH over conventional antibodies include ALX-0081 and viral-based cancer therapeutics
which generally consist of two amino acid chains and are packaged with nanobodies. In a clinical trial, ALX-0081
10 × larger in size (approximately 150 kDa). was successfully used as a target for the Von Willebrand
Nanobodies exemplify unique structural and functional factor to prevent the risk of thrombosis in patients with
features credited to their small size (15–74 kDa) and acute coronary syndrome [4]. Nanobodies have also
large surface area, high stability and solubility, high bind- been used in photothermal therapy due to their ability
ing affinity and detection of different epitopes, fast tissue to bind tumour antigens, like HER2, such as cleft-gold
internalization, ease of production and manipulation, and nanoparticles that absorb light energy to generate heat
low immunogenic reactions [21]. Consequently, as shown that destroys cancer cells. Thus, malignant cells can be
in Fig. 1, nanobodies were engaged in numerous scien- destroyed photothermally after exposure to a laser beam
tific and medical sectors encompassing environment and in an experimental environment [25]. Moreover, due to
agriculture, electronics and energy, transportation and the advent of viral-based cancer therapies that can be
automobile, common consumer products, microscopy integrated with most cells whilst only replicating within
and scientific appliances, engineering and construction, cancer cells, packaging the viral vectors with nanobodies
and medicine and drugs discovery [22]. The implication would ensure tumour-specific targeting. This would be a
of bio-engineered nanobodies as novel therapeutic tools valid tool in targeting metastatic cancer effectively [26].
for many diseases has been substantiated in several stud-
ies. For instance, nanobodies directed against hemag- Nanobodies and nanoparticle‑based cancer
glutinin influenza A H5N1 have been shown to suppress therapy
virus replication in infected mice and reduce both mor- Targeted delivery is a major milestone in cancer ther-
bidity and mortality [23]. Nanobodies target the binding apy as it mitigates the damage caused by non-selective
area of virulence factors, such as toxin A and toxin B of drugs. Nanoparticles allow a controlled release of drugs
Clostridium difficile and neutralize cytopathic effects in inside the cancer cells using multi-functional nanocarri-
fibroblasts in vitro [24]. Gastrointestinal tract syndromes, ers. Consequently, the therapeutic potential of nanobod-
including colon cancer and inflammatory bowel disease, ies as tools to deliver drug-loaded nanoparticles toward
tumours has recently attracted the attention of many or two-humped camel, lives in the cold desert of Asia.
researchers [27, 28]. The nanoparticle-based approach The C. dromedarius exhibits various unique traits, for
concentrates the drug within target cancerous cells and instance, it can survive and reproduce in the extreme
protects the surrounding healthy cells and tissues from heat and drought conditions of the desert. In 2010,
cytotoxic agents. Several researchers have developed there were more than 830,000 different dromedary
modified nanoparticles for use as a potential target for breeds in SA. This number significantly increased to
anticancer drugs with success [29]. For example, nano- 3,113,628 in 2015. Additionally, camels in SA are classi-
particles have been successfully conjugated with nano- fied into seven different C. dromedarius breeds accord-
bodies that express high specificity for the cancer marker ing to their coat colour: Majaheem, Maghateer, Homor,
Mucin-1 [30]. Mucin-1 overexpression is related to breast Shaele, Zargeh, Shageh, and Sofor, as demonstrated in
and colon cancers [31]. Nanoparticles made from poly- Fig. 2 [32, 33].
mers have been designed to carry a “killer gene”, which The genome of the C. dromedarius camel has been
causes cell death when expressed in the target cells. completely sequenced as a result of the joint efforts of
The expression of the killer gene was controlled by the researchers from the Beijing Genomics Institute (BGI),
Mucin-1 promotor gene [30]. This specification allows China and the National Center for Genomic Technol-
the killing of only cancerous cells and shields healthy tis- ogy (NCGT) at King Abdulaziz City for Science and
sue from the toxicities associated with cancer treatment. Technology (KACST), Saudi Arabia [34]. Sequencing
the complete genome is considered a breakthrough
Arabian camel’s genome and Nanobodies and a vital achievement in understanding the distinc-
emergence tive traits of this mammal. Availability of the genomic
There are two surviving species of camels: Camelus data is particularly essential to gaining novel insights
dromedarius and Camelus bactrianus with each hav- into the molecular mechanisms underlying the produc-
ing distinguishing features. Camelus dromedarius (C. tion and secretion of nanobodies in the biological fluids
dromedarius) also called the dromedary, one-humped [35]. Nanobodies were reported in camels in the early
camel, or Arabian camel mainly lives in the hot deserts 1980s, the presence of which makes camels unique.
of North African and Middle Eastern countries, includ- Importantly, camel milk contains nanobodies that
ing Saudi Arabia (SA) [32, 33]. Whereas the Camelus potentially can be used as a promising tool for diagnos-
bactrianus (C. bactrianus), also known as the Bactrian, ing and targeting metastatic cancers [36–39].
Fig. 2 The diversity and distribution of Camels in Saudi Arabia

Camel‑based nanobodies [44]. Similarly, camelid VHH presented robust effects as

The fact that all camelids naturally produce different a talented tool that can be potentially implanted in sev-
antibodies that circulate in their blood should be kept in eral biotechnological and medical fields. For instance,
mind. Two identical heavy chains and two identical light cAbBCII10 is a specific VHH framework that has the
chains joined by disulphide bonds through non-covalent ability for transferring the antigen specificity from the
interactions make up the usual structure for conventional donor VHH onto its scaffolds following the grafting com-
antibodies [40]. On the contrary, as implied by their plementarity determining region (CDR) technique [45].
name, the heavy chain antibodies (HCAbs) are distin- Development of a therapeutic tool targeting selectively
guished molecules that display unique features by hav- the tumour cells and leaving the normal cells unaffected
ing a heavy-chain homodimer while lacking light chains is a challenging aim. Cancer cells are featured by overex-
[16]. In contrast to traditional antibodies, these HCAbs pression of specific receptors or biomarkers that can act
are simple to isolate and clone from the serum of an as an attractive spot to increase the delivery and the accu-
immunized camelid and show lesser immunogenic reac- mulation of the drug into the affected tissues [25]. Fol-
tivity [8]. In C. dromedarius (dromedary) the HCAbs are lowing a ligand-receptor or antibody-antigen-based
devoid of the standard first constant domain (CH1) and approach, conventional monoclonal antibodies have been
instead possess a single antigen binding site for which widely used in biomedical research and clinical medicine.
they called the variable domain of heavy chain of HCAbs Accordingly, numerous vehicles for carrying drugs to
(VHH) or nanobodies [35, 41]. The protein-engineer- their therapeutic destination for treating various malig-
ing strategy has used a single-domain unit approach to nancies have been constructed [6, 46]. These involve
enhance the efficacy of the large size conventional anti- arcitumomab (anti-carcinoembryonic antigen (CEA)),
bodies. Besides several biological and economic proper- capromab (anti-prostate-specific membrane antigen
ties, the single-domain binding-based antibodies showed (PSMA)), trastuzumab (anti-human epidermal growth
an endogenous agonistic or antagonistic effect on their factor receptor 2 (HER2)), bevacizumab (anti-vascular
target cells. In comparison to conventional antibod- endothelial growth factor (VEGF)), and cetuximab (anti-
ies, nanobodies, with single-domain moiety, provided epidermal growth factor receptor (EGFR)) antibodies
a unique therapeutic avenue by exhibiting smaller size, [6, 47, 48]. Although the use of traditional nanoparticle
are costless and easier to make, and higher solubility or murine monoclonal antibody therapy in clinics has
and heat stability [17, 18]. Likewise, single-domain anti- produced positive outcomes, there are still a number of
bodies are more resistant to gastric acid due to having drawbacks. [9–12]. These include their fragile and com-
a smaller number of cleavage sites and are more resist- plex structure that requires higher expenses during the
ant to proteolytic enzymes than conventional antibodies production [46]. Additionally, it was shown that murine
[19]. This resistance can be increased by further optimiz- antibodies induced an immunogenic response by gener-
ing the structure of single-domain antibodies to make ating human anti-mouse antibodies, which led to their
them more permeable to the gastrointestinal tract and neutralization and loss of potency. Additionally, typical
thus suitable for local per-oral applications [17]. Further- antibodies with a large molecular weight (approximately
more, as their molecular mass is below the renal thresh- 150 kDa) have limited tissue penetration, extravasation,
old, single-domain antibodies have a very short plasma and permeability, which results in a heterogeneous distri-
retention time and can be excreted via the kidneys into bution of the medication within the intended tissue [13].
the urine [20]. They also show less cytotoxicity due to the Likewise, conventional nanoparticles exhibited a slower
absence of the FC fragment, which revealed a role in the diffusion pattern and pose a significant hindrance in the
activation of the complement system. Additionally, high drug release to the tumours which is associated with the
specificity and affinity to the targeted antigen in combi- and suboptimal therapeutic effects [14, 15]. Accordingly,
nation with rapid cellular internalization allow the nano- tumour cells don’t expose to similar concentrations of
bodies to work faster and exhibit a superior efficacy over the drugs and further this concentration doesn’t reach
the conventional antibodies [19, 42]. Interestingly, the the therapeutic level that ensued cellular death resulting
single-domain antigen-binding property exhibited by in reducing treatment efficacy [11–13]. As a result, many
camelids VHH was utilized to generate various antibody cancer patients exhibited resistance to antibody-based
constructs (bivalent, biparatopic or bispecific constructs) approach, which was linked to therapy failure and poor
to enhance their specificity and affinity to their target results. It is advised that the size of these nanoparticles
antigens, or to bind two individual antigen particles [43]. should be between 5 and 200 nm in order to increase the
This approach was applied to produce vastly stable anti- effectiveness of the antibody-based therapy. In fact, nan-
body constructs recognizing clinically significant human oparticles less than 5 nm can be removed from the blood-
prostate-specific antigen (hPSA) within only 15 min stream with ease, whereas those larger than 200 nm will
get caught in organs with reticuloendothelial systems, as phosphatidyl-ethanolamine (PE), phosphatidyl-choline

like the liver and spleen. Consequently, it was recom- (PC), lysophosphatidylcholine (LPC), and phosphati-
mended that the ideal size of the utilized nanoparticles dylinositol (PI) [53].
be less than 100 nm and have a hydrophilic capacity [6, The highest milk production is found in the Majaheem
49]. The HCAbs camelid antibodies revealed a molecu- breed (of C. dromedarius species) however there is a sig-
lar mass of around 95 kDa and the VHH domain showed nificant difference in the milk components among the
about 12–14 kDa mass thus they are considered as the C. dromedarius dairy camel breeds. For example, milk
minute intact antigen-binding entities that are naturally produced by the Sofor camel is higher in protein, fat,
occurring for that named nanobodies [8]. Therefore the and lactose than that produced by other camel breeds in
current trend in the establishment of drug delivery has SA [54]. On the other hand, the milk from the Magha-
switched to the use of smaller molecules with high spe- teer breed contains a high quantity of potassium and cal-
cificities, such as camelid nanobodies, to overcome the cium [54]. An analytical study conducted by Salmen et al.
existing obstacles confronted in the current nanothera- compared the camel milk casein components profile of
peutic modality [46]. different camel breeds including Majaheim, Wadah, and
The employment of the camelid-derived nanobod- Safrah in Saudi Arabia [55]. Among these breeds, Saf-
ies in diverse areas of nanomedicine and nano therapy rah camel milk contained the highest concentration of
has contributed to the advancement of disease screen- casein proteins (67.54%) followed by Majaheim (66.26%),
ing and prevention, diagnosis, treatment, and follow-up. and Wadah (64.07%). Furthermore, the highest level of
Indeed, many commercial companies such as Ablynx, the casein ash content of the camel milk was found to
Camel-IDS, and Hybrigenics were reported to generate be expressed in the Wadah breed (4.1%) while the Saf-
nanobodies [6]. In addition to their small size, camelid- rah breed revealed the lowest casein ash level of 2.95% of
based nanobodies offer exceptional properties including the total contents. On the other hand, the Wadah breed
high sensitivity and specificity spectrum, higher safety showed the lowest casein moisture content (6.07%) and
level, water solubility and biostability, and are originally the Safrah breed had the highest casein moisture com-
produced. These features allow nanobodies to participate ponent of 9.33% of the total content [55]. The Majaheim
significantly in the improvement of drug manufactur- breed had the intermediate value among the other breeds
ers and the treatment of various disorders. The tumour’s and showed a 3.30% and 8.89% for casein ash and mois-
microenvironment presented a good example to demon- ture contents, respectively. The amino acid components
strate the efficient engagement of nanobodies in deliver- both essential and non-essential were relatively dem-
ing the drug cargo to the targeted tissues. Smaller size onstrated similar concentrations among the investi-
and higher penetration capacity that portray nanobodies gated breeds [55]. A subsequent comparative study that
allow them to overcome the limitations harnessed with described the protein and fat composition differences
conventional antibody-based therapy in the treatment of among various Indian camel breeds including Bikaneri,
solid tumours. Numerous studies including experimen- Jasialmeri, and Kachchhi has been conducted [56]. In
tal preclinical investigations and clinical trials have been this study, the highest protein amount was detected in
conducted to examine the effect of camelid-based nano- the camel milk of the Kachchhi breed (4.22%), followed
bodies in the management of different diseases such as by Bikaneri (3.61%) and Jasialmeri (3.37%). Addition-
breast and lung cancers, infectious diseases, and inflam- ally, the fat content also displayed different values among
matory conditions [46]. the three breeds and thus ascribed to the level of intake
nourishment, lactation status, breed type, age, and sea-
Involvement of camels’ biological secretions in remedying son. The Jasialmeri camels showed a 2.99% fat compo-
diverse health conditions nent while the Bikaneriand and the Kachchhi possess
Camels milk components and biological proprieties 2.47% and 1.95% fat content, respectively [56].
Camels’ milk has a high nutritional value [50] as it con- The milk proteins, particularly lactoferrin, deliver
sists of numerous proteins, fats, oligosaccharides (lac- potent probiotics, antioxidant, anti-microbial along
tose), nucleotides, essential amino acids, vitamins, and with anti-inflammatory effects [57] as shown by Table1.
minerals [51]. Certainly, camel milk is enriched with Anti-microbial and anti-inflammatory roles were pro-
amply endowed components including vitamin C and vided by lysozyme and immunoglobulins contents of
E, lactic acid bacteria (LAB), caseins (α, β and κ iso- the camel milk [37]. While antioxidant’s function was
forms), and plenty of the whey acidic proteins such as attributed to the structural proprieties of the milk pro-
lysozyme, alpha-lactalbumin, immunoglobulin, lacto- teins specifically β-caseins and to their greater compo-
ferrin, and lactoperoxidase [52]. Additionally, different sition of antioxidants amino acids [58]. Importantly,
camel milk phospholipids have been also detected such these milk proteins were found to alleviate the burden
Table 1 Experimental studies on camel milk (CM) and urine (CU) and their active molecules
CM/CU/Ab/Active molecule Studied models Status References
HCAb/VHH BW5147 T-cell lymphoma in vitro [71]

Camel antibodies/nanobodies Lewis lung carcinoma
(Bivalent or univalent)
CM (lactoferrin) Huh 7.5 cells in vitro [180]
HCT-116
Camel milk (α-lactalbumin) HepG2, HeLa, in vitro [57]
Camel milk Human prostate cancer cells in vitro [60]
Camel milk (lyophilized) Breast cancer MCF7 cell in vitro [70]
Camel milk (Casein) Breast Cancer BT-474 in vitro
HepG2, HeLa
Camel urine (PM 701) Healthy humans Clinical trial [85]
L1210 cell in vitro/in vivo
A549 cell in vitro
Healthy mice in vivo
Camel urine (PMF) Healthy mice in vivo [181]
HEPG2, HCT116, U251, A549, MCF-7, leukemia in vitro [86]
Rodent lung cancer in vivo
Camel urine PMF 701 nanoparticles A549 cells (human alveolar basal epithelial cell carcinoma) in vitro [121]
(Tyrosine, glycine, cysteine, arginine, hippuric and benzoic
acids, ZnO, Ag, Y, Cs, and Rb)
Nanobodies labelled with 18F Breast cancer HER2 overexpression in vitro/in vivo [112, 113]
Anti-HER2-specific 5F7GGC Nb nanobodies radioiodinated Breast cancer HER2 overexpression in vitro/in vivo [114]
with131I IB-Mal-D-GEEEK
Anti-HGF nanobodies (1E2-Alb8 and 6E10-Alb8) labelled Brain glioblastoma in vivo [116]
with positron emitter zirconium-89
Anti-EGFR nanobodies combination modality Brain glioblastoma in vitro/in vivo [123]
Nanobodies conjugated to PEG-liposomes multivalent EGFR expressing tumors in vitro/in vivo [119]
system
Nanobody-based targeting module (Nb-based TM) EGFR expressing tumors in vitro/in vivo [120]
Nb6 and Nb17 nanobodies Pulmonary disease in vivo [122]
of oxidative stress and the production of the damag- intracellular vacuoles formation, elevated LC3-II/LC3-I
ing free oxygen radicals that harnessed with cancer’s ratio, and formation of the autophagosomes [61].
microenvironment thus consequently suppressing The therapeutic ability of camel milk can be delivered
cancer cells’ proliferation [59]. As well, Korashy et al. by its gifted biological components such as vitamin E
suggested that camel milk exhibits effective apoptotic and C [64], casein, lactoferrins, lactoperoxidase, and fatty
ability against liver and breast cancer cells [57]. Fur- acids, several ions and metals, and immunoglobulins
thermore, the active camel whey fraction (TR35) was [65]. It was postulated that lactoferrin delivers the major-
found to display anti-tumorigenic effects in non-small ity of antitumorigenic effects of the camel milk [59, 61,
lung cancer. This was accomplished through promoting 62, 66]. Lactoferrin, the principal iron-binding glycopro-
in vitro cancer cells apoptosis and inhibiting tumour tein of camel milk, exerts an antioxidant influence and
growth in vivo model via activation of JNK phospho- was found to induce in vitro anti-proliferative capacity
rylation and suppression of P38 and STAT3 phospho- against colon cancer cells (HCT-116) and prevent DNA
rylation [39]. The tumours battling power provided by damage [59]. Additionally, lactoferrin produces an inhibi-
the camel milk were validated by numerous in vitro and tory effect of cytochrome P4501A1 causing murine mela-
in vivo investigations [59–63]. Exposure of different noma cancer cells (B160F10) death [62]. Furthermore,
cancer cell lines such as breast cancer (BT-474), laryn- camel milk shows a high concentration level of vitamin
geal (HE-p2), and human hepatoma (HepG2) cells to C that revealed protection against mutagenesis and clas-
lyophilized camel milk blocked the growth and prolif- togenic influences [67]. Selenium and zinc elements [68],
eration of these cells [61]. Moreover, treated the breast and casein [69] play a pivotal role in eliminating certain
cancer cells MCF-7 and the colorectal cancer cells HCT genotoxic effects of the toxic compounds and ensuring
116 with the commercial camel milk induced cancer the correct synthesis of DNA and RNA thus preventing
cell autophagy manifested by cell membrane deformity, cancer development [68]. Indeed, camel milk endowed
with casein containing α-lactalbumin induces cellular virus infection (HCV), peptic ulcers, inflammatory and
death and activates the apoptosis pathway in HepG2 and infectious conditions, and different types of cancers [60,
HeLa cells [70]. Nonetheless, the exact active compo- 74–76].
nents of the camel milk that specifically exert the tumour To complete the picture, we investigated the correla-
suppression effects and their downstream molecular tion between the expression of the camel milk protein
pathways required further investigations. components combined and the overall survival (OS) of
Moreover, camel milk expressed distinctive immuno- cancer patients with different tumour types using the
globulins characterized as VHH antibodies or nanobod- online KM plotter software (http://kmplot.com/analysis/)
ies [20]. These immunoglobulins are featured by their [77]. This tool analyses the effect of 54,675 genes on the
small size, unlike human IgGs, giving them the ability survival outcome of cancer patients using 10,293 cancer
for tissue penetration and intracellular localization and samples of different types. The genes expression signa-
functioning [53]. Additionally, VHH nanobodies with ture (consists of beta-caseins (CSN2), lysozyme (LYZ),
their biophysical proprieties can easily target the tumour alpha-lactalbumin (LALBA), lactoferrin (LTF), and lac-
tissues and the metastatic loci [71–73]. Cortes-Reta- toperoxidase (LPO)) was obtained using the multigene
mozo et al.’s investigated nanobodies in camel milk and classifier and the mean expression levels of all selected
were able to provide insight into the structural arrange- genes, auto select best cut off value, and ten years follow
ments of the secreted nanobodies. They demonstrated up period with HR of 95% confidence. As displayed in
that nanobodies are naturally occurring single-domain Fig. 3, high expression of the genes signature is associated
antigen-binding units and are less hydrophilic than the with a significant prolongation of OS of patients with
camel variable domain of HCAbs (VHH) domains. Addi- breast, cervical, head & neck, renal, sarcoma, and lung
tionally, they possess nonimmunogenic fragments and cancers confirming favourable patient outcomes. We also
show potent specificity for targeting the solid tumours showed a marginally significant correlation between ele-
[71]. These features assist in the production of functional vated expression of the genes signature and patients’ sur-
and stable antibody constructs with excellent target vival with rectal, ovarian, uterine, and bladder cancers.
specificity against cancer cells and have been suggested
as therapeutic or diagnostic tools [71]. Individual stud- Camels’ urine components and their biological proprieties
ies have reported the promising use of naturally secreted Despite being a waste product, camel urine has been
nanobodies in camels’ biological fluids, including urine utilized as a foundation for several therapeutic media-
and milk, as therapeutic agents for the treatment of vari- tors. In alignment with the previous reports, a study
ous disorders such as chronic hepatitis and hepatitis C conducted by Abdul Qader et al. showed that camel
Fig. 3 The effect of camel’s secretion on different diseases

urine consists of more than 32 proteins including alpha- cell types [88]. Yet the optimal dose with fewer side
1B-glycoprotein, alpha-1-acid glycoprotein, serotrans- effects needs to be defined experimentally and preclini-
ferrin, vitamin D-binding protein, serum albumin, and cally before camel urine could be proposed for cancer
thyroxine-binding globulin. These proteins could explain therapy [89]. Indeed, the age, sex, and breeds of the cam-
the anti-inflammatory and anti-infectious effects deliv- els play an essential role in modulating the camel urine’s
ered by camel urine [78]. In vitro studies suggested that anti-proliferative properties. The proliferation capac-
camel milk and urine can inhibit mutagenesis and the ity of lung cancer cells (A549) was measured after being
proliferation of mutant cells and can induce apoptosis in treated with camel urine extracted from Magateer and
cancerous cells [79]. Romli et al. analyzed the effects of Majaheem camels of different age and sex groups. The
nanobodies-containing camel urine on breast cancer cells utmost cancer cellular death was observed upon admin-
(4T1) in vitro and in vivo. They found that exposure to istration of the male young and adult Magateer urine as
purified camel urine in vitro inhibited the growth of 4T1 compared to other breeds. Nonetheless, the biological
cancer cells and restricted the metastatic process of these ingredients with potential contributions to persuade this
cells. In a double-blind assessment, mice inoculated with result required further investigations [82].
4T1 cells treated with camel urine showed that the size of Likewise, mitigation of tumour angiogenesis that is
the tumour was significantly reduced in the treated group delivered by the camel urine was recently confirmed by
compared to the control group [80]. Likewise, two inde- Gader and Alhaider [76]. Additionally, Alhaider et al.
pendent studies by Evers et al. [81] and Alebie et al. [82] reported the inhibition of inflammatory angiogenesis by
showed the therapeutic potential of camel milk and urine adding camel urine and milk into murine sponge cells.
as anti-neoplastic agents [83, 84]. This was achieved by reducing the key components of
Moreover, camels’ secretions, including milk and urine, fibrovascular tissue, VEGF expression, macrophage
revealed favourable results as a therapeutic modality in recruitment, TGF-beta, and several cytokines [63]. The
the hepatoma [70], breast cancer [57], and lungs cancer anticancer role of camel urine was further evidenced in
[85] (Fig. 4). In 2006, Khorshid and Moshref carried out a report by Cyplal in which camel urine was found to
in vitro experiments that demonstrated the ability of lyo- cause a substantial transcriptional suppression of the
philized camel urine to inhibit the growth of tumour cells gene that encodes a carcinogen-activating enzyme [88].
in different cancer cell lines: hepatocellular carcinoma Moreover, camel urine was reported to induce antitu-
(HEPG2), colon cancer, lung cancer, and leukemia [85]. morigenic effects by reducing the expression of differ-
Khorshid and Moshref hypothesized that the anticancer ent cytokines promoting tumour growth such as IL-4,
action of camel urine occurred via both direct cell cyto- IL6, and IL-10 [88, 89]. Interestingly, upregulation of
toxicity and anti-angiogenic action (reduction of blood the chemoprotective gene expression such as Nqo1 and
flow to tumour cells) [86]. The cytotoxic effect of the Gsta1 was another suggested mechanism by which camel
camel urine was further evaluated by individual reports urine promoted tumour cytotoxicity while protecting the
that demonstrated the proliferative inhibitory effects normal cells from reactive oxygen radicles produced by
and cellular cytotoxicity following camel urine stimula- chemotherapeutic agents [82]. Collectively, these findings
tion of different cancer cells [87, 88]. Ten types of cancer authenticated the strong therapeutic potential of camel
cells were subjected to lyophilized camel urine stimula- biological secretions in both urine and milk against can-
tion that was divided at the end of the treatment into two cer cell progression.
groups. The first group of cells including MDA-MB-231, The precise function of the anticancer components in
DAOY, MED-4, and MED-13 showed over 50% cellular camel urine and milk remains poorly understood. The
death, especially in the breast cancer cells MDA-MB- tumorigenic suppression capacity depicted by the camel
231where the cellular apoptosis effect was triggered by urine was attributed mostly to the abundance of iron-
80%. While the other group of cells, MCF 10A, HFSN- binding lactoferrin component [62, 90–92]. Lactofer-
1, U2OS, MCF-7, MED-8, LoVo and HCT-116, revealed rin was reported to induce cancer inhibitory effect both
insensitivity or marginal response to the camel urine in vitro and in vivo studies by inactivation of CYP1A1 in
treatment [88]. The antiproliferative effects of camel hamster buccal pouch carcinoma causing tumour regres-
urine on the MDA-MB-231 resulted from induction of sion [93]. Different mechanisms have been suggested for
cyclin-dependent kinase inhibitor p21 causing cellu- lactoferrin proliferative suppression effects these include
lar senescence [87], activation of the apoptosis intrinsic activation of Fas signalling pathway upon interaction
pathway by reducing Bcl-2 expression, and suppression with polysaccharides ligands ensued tumour growth inhi-
of several cancer-associated proteins including survivin, bition [94]. Moreover, lactoferrin induces cell cycle arrest
β-catenin, and cyclin D1 [88]. Thus indicating the selec- at the G1 phase and stimulates the expression of cyclin-
tivity of the cytotoxic effect of camel urine on different dependent kinase (CDK) inhibitor p21 cip1 protein and
Fig. 4 The impact of Camel’s secretion on overall survival in several diseases
p27 while reducing the expression of pAkt, cyclin E, and which is a regulator of PI3K/Akt pathway [97]. A study
pRb protein levels [95]. Indeed, lactoferrin by its inter- using GC–MS and ICP–MS analysis of the camel urine
action with insulin-like growth factor-binding protein-3 was conducted to identify the elemental component’s
[96] modulates the stability of insulin-like growth factor, profile of the camel urine [98]. Numerous metabolites
with a potential contribution to the biological effects of area. Nanobodies with their small size and short half-
the camel urine were detected these include benzene life in circulation presented a precise imaging tool with
propanoic acid derivatives, fatty acid derivatives, amino higher tumour targeting and retention and a less noisy
acid derivatives, sugars, prostaglandins, erythritol, meli- background [110, 111]. Furthermore, nanobodies bound
biose, and canavanine [98]. Canavanine is an anti-metab- with a diagnostic isotope such as 18F were successfully
olite of L-arginine derivatives that possess a toxic feature. used in the detection of HER2 expression in breast can-
Canavanine accounts for 2% of total camel urine compo- cer cases using PET/CT imaging technique [112, 113].
nents and is found to deliver tumour suppression effects Trastuzumab is a monoclonal anti-HER2 antibody that
[99]. Consequently, camel urine provides a potentially showed favourable results in breast cancer patients over-
promising therapeutic tool in combatting different types expressing HER2 receptors. While breast cancer cases
of malignancies [89]. with a low or heterogenous expression of HER2 revealed
Furthermore, several forms of camel urine have existed partial response to trastuzumab. Anti-HER2-specific
such as Prophet Medicine Fraction (PMF) and PM701 5F7GGC Nb nanobodies that are radioiodinated w ith131I
found to be enriched in crystals, nanorods, nanoparti- IB-Mal-D-GEEEK were produced for determining HER2
cles, and different organic and inorganic compounds. expression in breast cancer patients before trastuzumab
PMF crystals constitute numerous elements such as administration. This conjugated nanobody revealed
calcium oxalate, cystine, uric acid crystals, ammonium encouraging results in targeting HER2 tumours promot-
urate, calcium phosphate, benzoic acids, glycine, alanine, ing receptor internalization and subsequent signals inac-
and arginine. Additionally, PMF is highly concentrated tivation both in vitro and in mice models, (Table. 1) [114].
in ions including Cs, Rb, K, Ca, Cd, Y, Eu, Th, and Zn Besides HER2 detection, nanobodies against other
[82]. Importantly, the PMF compositions play an essen- growth factor receptors, that are overexpressed in vari-
tial role in producing cytotoxic impact against different ous malignancies, such as EGFR1, VEGFR2, c-Met, HGF,
cancer cell types by increasing the permeability of can- and CXCR7 have been generated [115–118]. In aggres-
cer cells’ membranes allowing their lysis and destruction sive types of cancer where HGF and its receptors c-Met
[82]. An important element is Zn which is present in the were highly expressed, anti-HGF nanobodies (1E2-Alb8
camel urine as ZnO this when bound to the nanoparti- and 6E10-Alb8) labelled with positron emitter zirco-
cle produces a distinctive metal oxide nanomaterial fea- nium-89 was developed to be used for in vivo detection
tured with cytotoxic effects [100]. This biological effect of HGF expression. Additionally, these designated nano-
is achieved by the generation of cellular oxidative stress bodies showed therapeutic benefits demonstrated by
[101, 102] accompany by the deformation and rupture hindrance the tumour growth in the treated mice as com-
of cancer cells’ membrane [103]. Other PMF elements pared to the control group [116]. A system composed of
such, as Cs and Rb can effectively target cancer cells and nanobodies conjugated to PEG-liposomes was examined
stem cells by elevating their pH level and leaving normal both in vitro and in vivo for the ability to recognize the
cells unaffected [82, 104]. Camel urine PMF was found EGFR. This system was found to exhibit an antagonistic
to modulate the growth functions of both normal and effect on EGFR expression resulting in receptor expres-
cancer cells via its amino acid components [105]. Tyros- sion internalization and downregulation and subsequent
ine increases PMF targeting to the cancer cells while gly- inhibition of the tumour cells proliferation [119]. Nano-
cine and cysteine improve PMF antioxidant capacity thus body-based targeting module (Nb-based TM) is another
strengthening the immune system [106]. Furthermore, example of a generated nanobody that targets the expres-
arginine plays important role in the modulation of the sion of EGFR. This effect is achieved by inducing the
immune response and recruitment of T-cells and natural recruitment of the T cells selectively to EGFR overex-
killer cells against cancer cells via activation of different pression cancer cells followed by destroying tumour cells
cytokines including IL-12 [107], IL-23, and IFN generat- both in vitro and in vivo [120].
ing antitumor immune response [108]. Additionally, Gehan et al. analyzed the antican-
cer effects of PMF extracted from dried camel urine
Contribution of camels derived Nanobodies (PM701). They studied the consequence of adding PMF
in nano‑oncology nanoparticles to a lung cancer cells line (A549) using var-
Nano-oncology is a field of science that combines medi- ious microscopic and spectroscopic techniques, includ-
cine, biochemistry, and engineering and helps in estab- ing scanning electron microscopy. A549 cells treated with
lishing tumour screening, diagnosis, and therapeutic plan PMF showed that the PMF exhibited two modes of action
[109]. To achieve an accurate cancer diagnosis, especially against the target cells. First, these nanobodies attacked
in the early stages of the disease, the diagnostic or imag- the membrane of A549 cells and changed the packing
ing agent needs to localize selectively into the affected and polarity of the membrane. Second, these nanobodies
selectively targeted cancer cells and transferred the PMF Thrombotic thrombocytopenic purpura (TTP). TTP is
into the nucleus and other organelles of abnormal cells an autoimmune hematological disorder manifested by
through endocytosis. In this regard, this nanobody-based the presence of autoantibodies that target the von Wille-
drug delivery system (DDS) achieved the purpose of the brand factor causing platelet clumps and microvascular
smart drug treatment [121]. Similarly, Nanobodies were thrombosis and blockage. The standard management for
also used in animal studies to target the pulmonary sur- TTP is daily plasma exchange to eliminate the autoanti-
factant protein A (SPA) associated with airway diseases. bodies from the circulation, in addition to immunosup-
Nb6 and Nb17 nanobodies were developed and demon- pressive therapy. ALX-0681 in combination with the
strated rapid accumulation in the pulmonary tissues as standard remedy for TTP displayed significant mitigation
shown by the imaging technique. This effect was accom- of platelet aggregation and a satisfactory safety profile,
panied by fast clearance from the circulation with a mini- low disease burden and recurrence, and prolonged over-
mum build-up in the liver and spleen [122]. Moreover, all survival [129].
the involvement of nanobodies as therapeutic agents for
different brain tumours such as glioma and glioblastoma Effects of camel milk on modulation of various signalling
was also investigated [123]. In vitro anti-EGFR nano- pathways
bodies suppressed tumour cell proliferation and growth As previously described the camels’ biological secre-
[124]. Also, a combination modality consisting of an anti- tions have shown to exert therapeutic benefits in treat-
EGFR nanobody, pro-apoptotic EGFR-specific nanobody, ing numerous health conditions. However, the precise
and an immune conjugate targeting tumour necrosis fac- subcellular molecular mechanisms that are regulated by
tor-related apoptosis-inducing ligand (TRAIL) displayed these secretions necessitate further investigations. Camel
a potent inhibitory effect on tumour growth and aggres- milk was reported to induce a hypoglycemic effect in
siveness both in vitro and in vivo [123]. both human and animal models and was suggested as a
The insinuation of nanobodies in medical determina- complementary therapy in the type 1 diabetes [130–132].
tions has immensely progressed as numerous nanobod- To understand by which mechanism can camel milk
ies have been tested in the preclinical setting, while many regulate insulin signalling to produce its glycemic effect,
of them are being examined in clinical trials as demon- the human embryonic kidney 293 (HEK293) cells, that
strated in Table 2 [111, 125, 126]. The respiratory dis- expressed transiently human insulin receptors (hIR),
ease mostly benefits from the aerosol or inhaler route of were exposed to camel milk stimulation. Biolumines-
administration to ensure the accumulation of the drug cence resonance energy transfer (BERT) assay was used
into the lungs and thus avoiding systemic side effects to examine the activation of insulin signalling by meas-
[127]. Nanobodies with their unique biological features uring the intensity of the physical interaction between
provide an excellent vehicle for drug pulmonary delivery. hIR and insulin receptor signalling proteins (IRS1) and
ALX-0171 is a 42 kDa trivalent nanobody inhaler that is the growth factor receptor-bound protein 2 (Grb2). Only
currently being tested in clinical trials (Table 2) to fight simultaneous treatment with insulin and camel milk was
against the human respiratory syncytial virus (RSV) and found to significantly enhanced the intensity of BERT sig-
shorten the duration of the infection [128]. A phase I nals between hIR and Grb2 but not IRS1. Furthermore,
clinical trial demonstrated the safety and tolerability of camel milk was found to potentiate ERK1/2 but not Akt
ALX-0171 in the examined individuals [127]. Caplaci- activation downstream insulin signalling [133, 134].
zumab is the first EMA and FDA-approved 28 kDa biva- The anti-inflammatory and antioxidant effects exhib-
lent nanobody (ALX-0681) for treating patients with ited by camel milk components were also validated
Table 2 Examples of nanobodies that are currently being tested in clinical trials
Nanobodies compound Clinical use Target Stage References
Caplacizumab Thrombotic Ultra large von Willebrand factor Clinical trial phase III [129]
ALX-0681 thrombocytopenic
ALX-0081 purpura
ALX-0171 Lower respiratory RSV (respiratory syncytial virus) Clinical trial phase I-II [128]
tract infection
Bispecific nanobody-derived CAR-T cells Lymphoma CD19 Clinical trial phase I [182]
CD20
[131I]-SGMIB anti-HER2 VHH1 Breast cancer HER2 Ongoing [114]
in a rats model with acute respiratory distress syn- protecting remedy against renal damage triggered by
drome (ARDS). ARDS is defined as the formation of sig- cytotoxic drugs [140].
nificant inflammatory background causing damagethe Camel urine, the other camel’s biological secretion,
to alveolar epithelial and endothelial barriers ending in was also reported to exert a tumour suppression effect
respiratory failure [135, 136]. The animal with ARDS and augment the doxorubicin treatment for breast can-
treated with camel milk displayed a notable reduction cer cells. This anticancer impact was obtained by induc-
in the pulmonary wet: dry weight ratio, neutrophil infil- ing cancer cells apoptosis and DNA damage, reversing
tration, alveolar and interstitial edema, lung damage, the EMT markers, and retrieving the expression of the
and improvement in pulmonary functions. Addition- E-cadherin epithelial marker. The author suggested the
ally, camel milk administration alleviated the pulmo- potential involvement of the camel urine component in
nary recruitment of numerous cytokines (TNF, IL-10, attenuation of NF-κB-Snail signalling and its downstream
and IL-1b) and oxidative stress factors. Interestingly, the inflammatory mediators thus abrogating breast cancer
observed camel milk therapeutic effects were postulated cells’ aggressive phenotype [141].
to happen through suppression of the MAPK signalling
cascade [137]. Likewise, the camel milk’s anti-inflamma- Nano proteomics
tory power was further authenticated in another animal The camel genome sequencing project was completed
model presented with arthritis and air pouch edema, in 2003, and the data it produced gave researchers the
mirroring human rheumatoid arthritis (RA) disease. chance to use proteomics to analyze and gain insight into
RA is an autoimmune disease characterized by chronic all of the cellular processes that could not be explained
inflammatory status causing destructive lesions in the by genomics alone. A proteomics study offers help-
bones and cartilages of the small joints particularly the ful information on the post-translational modifications,
hands [138]. In this study, the administration of camel localizations, structures, and interactions of all proteins
milk resulted in the improvement of paw edema and the expressed by an organism in addition to merely studying
inflammatory features associated with arthritis. Inter- the entire proteome [142]. Also, proteomics has different
estingly, camel milk inhibits the phosphorylation of p38 clinical applications which include helping researchers
resulting in the downregulation of MAPK, ERK1/2, and evaluate the safety and efficiency of therapeutic inter-
JNK1/2 cascade activities. Additionally, in comparison to ventions [143, 144]. Examining the processes underly-
the control group the treated animal demonstrated lower ing drug responses, discovering new therapeutic targets,
levels of lipid peroxidase and nitric oxide and elevated understanding the etiology of disease, finding new bio-
glutathione levels and antioxidants factors. Thus sug- markers for disease early detection [145, 146]. Function-
gesting the potent anti-inflammatory power delivered by ally, biomarkers are classified into three major categories:
camel milk contents in attenuating the arthritis process biomarkers for early disease detection, prognostic bio-
[139]. markers that provide information about the potential for
Cyclosporin is an immune suppressant agent that is malignancy, and predictive biomarkers that distinguish
widely used for several autoimmune diseases. The most between various cancers to provide an appropriate ther-
harmful side effects of this drug are kidney injury that can apy [147].
progress to renal failure. Camel milk, as an anti-inflam- The immense challenge of finding minute quanti-
matory/antioxidant mediator, was proposed as a natural ties of biomarkers from natural fluids such as urine,
agent to ameliorate the side effects associated with cyclo- saliva and blood, compared to the simpler detection of
sporine administration. In vivo Administration of camel high-abundance proteins, has led to the emergence of
milk for 3 weeks reduced dramatically the levels of the a new field called nano proteomics, which integrates
biomarkers related to renal damage such as serum creati- proteomics and nano techniques [148–150]. Nano pro-
nine, BUN, and KIM-1. Furthermore, camel milk treat- teomics has been utilized in the discovery of biomark-
ment is associated with a marked lowering in numerous ers in two main areas: nanostructured surfaces and
inflammatory cytokines and degradation signals such nanoporous materials. Several nanomaterials including
as MCP-1, IL-8, TNF, MMP-2, and MMP-9. Besides the gold nanoparticles, quantum dots, nanowires and car-
ability of the camel milk to inhibit p38/ERK/JNK MAPK bon nanotubes have been developed to help researchers
signalling, camel milk blocked NF-κB pathway activation overcome the challenges of detecting low-abundance
by suppressing the expression of NF-κBp65, p-NF-κBp65, proteins [151, 152]. Nano proteomics provides a sensi-
and p-IκBα proteins. Curtailing of pathways together tive and robust analytical platform for screening low-
with enhancement of glutathione and antioxidant activi- abundance biomarkers in a high-throughput manner
ties resulted in alleviating of the oxidative stress on the [150]. Of note, nano proteomics has been used to detect
kidneys. Therefore, camel milk can be used as a natural biomarkers associated with various human diseases,
including autoimmune diseases [153] and cardiovas- to cure solid tumours [124]. Similarly, nanobodies tar-
cular disorders [154]. Therefore, the main concern of geting the tumour necrosis factor-α (TNF) could be
cancer researchers is being able to use nano proteom- applied to cure malignant tumours [170].
ics to facilitate prompt and safe cancer diagnosis. Con-
sequently, various analyses have been conducted on Bioinformatics and nanobodies
select biomarkers in distinctive types of cancer, includ- Advancements in computational molecular biology, such
ing breast and prostate cancers [155, 156]. However, the as the formation of large genomic and proteomic data-
compatibility and toxicity of nano techniques remain a bases and the development of various tools and bioin-
health concern and further research needs to be imple- formatic software, facilitated the achievement of various
mented to guarantee their safety in biological applica- benchmarks that can be used for therapeutic purposes.
tions [157]. Bioinformatics is playing a vital role in understanding
The exceptional pharmacokinetic and physicochemi- complex metabolic pathways and discovering various
cal properties of camel nanobodies are consistent with vital components of living systems [171]. Several bioin-
those needed for cancer therapy and provide added formatic platforms such as MATLAB Symbiology and
benefits over conventional antibody technology in drug Pathway Studio, amongst others, have proven useful in
delivery, immunotherapy, and diagnostics. The affini- studying and identifying various nanobodies. These bio-
ties of camel-heavy antibodies are higher than those of informatic tools have been used in an integrated method
conventional antibodies and can reach up to 100 pM for graph-based visualization of the components of com-
affinity constants [158, 159]. This facilitates protein- plex networks. Such bioinformatics systems use literature
protein interactions and can act as a strong basis for and microarray databases of nanobodies to identify novel
identifying cancer biomarkers and intracellular signal- nanobodies that are involved in genetic regulatory path-
ling. Heavy-chain antibodies can also be bonded with ways. This strategy has revealed novel interactions among
fluorescent dyes to generate chrome bodies that can nanoparticles and genes, providing valuable details that
be used in single-molecule localization with super- contribute to the current understanding of the various
resolution imaging techniques [160–162]. The pres- nanoparticles that are involved in complex cellular path-
ence of cysteines at amino acid positions 54 and 78 ways. Also, the approach presents an advanced research
makes a more stable disulphide bond at the hydro- platform to find nanoparticles that can be used in DDS
phobic region [163, 164]. Additionally, nanobodies are for therapeutic purposes and to advance the bio- and
very robust as their melting points are in the range of chemo-informatics analyses of molecular pharmacology.
67 °C–78 °C, with the ability to refold after thermal Research has also been carried out to utilize and
unfolding has been detected and with functional activ- explore the combined potential of nanoparticles and
ity up to 90 °C [18]. The robustness of nanobodies bioinformatic tools for the treatment of cancer [172].
(heavy-chain antibodies) makes them very suitable for In 2012, Arvizo and colleagues reported that integrated
the antibody engineering [165, 166], tumour targeting bioinformatics, proteomics, and nanotechnology strat-
[8], and pharmaceutical formulations [167]. Moreover, egy could be used to discover unique therapeutic targets
nanobodies can distinguish recessed antigenic domain, for cancer by proposing that protein corona are formed
a property that has been attributed to their small size when proteins bind to nanoparticles [172]. Using corona
and the capacity of the prolonged CDR3 loop to rap- with engineered surface-functionalized gold nanopar-
idly infiltrate the epitopes [159, 168]. Thus, nanobod- ticles (AuNPs) revealed proteins that provided insight
ies can be successfully applied to target some enzymes into the development and stages of ovarian cancer. They
or transmembrane proteins or even signalling path- concluded that protein corona modulation around nano-
ways in certain tumour cells. This was demonstrated by particles is a promising therapeutic approach for various
Cortez-Retamozo et al. in 2004 who targeted tumour diseases, including ovarian cancer. Given the advance-
cells through the nanobodies fused to the β-lactamase ment in animal biotechnologies, including camel clon-
enzyme to identify the carcinoembryonic antigen ing and transgenesis, the design of targeted nanobodies
[169]. The enzyme converts an injected non-toxic pro- secreted from genetically engineered camels would be
drug into a toxic drug with an elevated concentration specific tools to treat tumours. Recent reports have
in the targeted tumour cells. Such like nanobody con- revealed the possibility of delivering anti-EGFR thera-
jugates hold much promise for cancer immunotherapy. pies to brain tumours through the invention of stem cell-
It has been well established that cell-surface protein delivered anti-EGFR nanobodies. These inhibited tumour
conjugates used to target the epidermal growth factor cells when combined with cytotoxic molecules, consider-
receptor can hinder the epidermal growth factor by ably enhancing therapeutic outcome [123]. More so, the
binding to its receptor, this technique has been used isolation of camel stem cells was recently achieved by our
group [173] which could further improve the efficiency of biophysical and biochemical proprieties besides the abil-
camel cloning and transgenesis and thus provide a versa- ity to cross the biological membranes such as the blood-
tile model for generating targeted nanobodies for treating brain barrier. Furthermore, they show the capacity to
cancer cells [123]. enter smoothly into solid organs including the brain,
lymph nodes, lungs, and liver. Because nanobodies pre-
Exosomes sented flexible manufacturing and assembly they have
Exosomes are nano-sized extracellular membrane vesi- been incorporating into multipotent constructs or con-
cles that range in size from 50 to 200 nm and are unique jugating with chemotherapeutic drugs to produce highly
nanoparticle drug carriers with important nanoparti- specific and efficient compounds. These features make
cle features [174, 175]. Exosome biomimetic nanopar- nanobody-based therapies useful and powerful tools to
ticles are advantageous in their ability to incorporate deliver the drug into the targeted tissues exclusively. Nev-
both synthetic and natural materials to create a more ertheless, nanobodies still displayed some pitfalls due to
efficient drug delivery method. Yet they still have limita- their minute sizes including rapid renal clearance and a
tions due to the method of synthesis, which is reflected possibility of renal toxicity. This drawback can be modi-
in the protein integrity on the exosome surfaces, which fied by coupling nanobodies with the serum albumin to
compromises their functioning [176]. Natural sources of increase their retention time in the circulation, yet such
exosomes from biological fluids, such as milk, are there- an approach will inexorably reduce their diffusion and
fore important, especially if we are aiming for production penetration benefits. Further investigation is pivotal to
in a large-scale [74, 177]. elucidate their exact molecular mechanisms in modu-
Preliminary studies have shown that camel milk lating oncogenic signalling pathways and to assess their
exosomes could play an important role in inhibiting the safety profile in preclinical experimental studies before
growth of breast cancer cells [178]. They could also mod- the implication in any clinical trials.
ulate cyclophosphamide-induced oxidative stress and
immunotoxicity in mammals. These effects stem from Abbreviations
the importance of biomolecules carried by exosomes, A549: Lung cancer cell line; BGI: Beijing genomics institute; C. bactrianus:
such as bioactive lipids, a specialized functional pro- Camelus bactrianus; C. dromedaries: Camelus dromedarius; CM: Camel milk;
DDS: Drug delivery systems; HCV: Hepatitis C virus; IARC: International agency
teome, nucleic acids (including DNA, microRNA and for research on cancer; KACST: King Abdulaziz city for science and technol‑
ncRNA), metabolites, and signalling molecules that can ogy; N/A: Not applicable; NCGT: National center for genomic technology; PMF:
be transported over distance within the protection of a Prophet medicine fraction; PRP: Peptidoglycan recognition protein; SA: Saudi
Arabia; TNF: Tumor necrosis factor-Α; VHH: Variable domain of HC antibodies.
lipid bilayer–enclosed structure [179].
Furthermore, EL-Kattawy et al. found that camel Acknowledgements
colostrum-derived exosomes, that are enriched in milk Y.M.H and N.S.N would like to thank the Genetics Department at King Faisal
Specialist Hospital & Research Centre (Gen. Org).
protein contents, showed remarkable apoptotic effects
on liver cancer cells exclusively sparing the normal cells Author contributions
unaffected. These carcinogenic suppression effects are Conceptualization and writing the original draft preparation: N.S.A and A.T.
Formal analysis, writing, review, and editing: F.A.Z, I.O.A, A.A.O, A.M.S, O.M.A,
attained by elevation in the expression level of both and C.H.M. Resources and visualization: I.M.S and W.A.A. Writing, editing, gen‑
Bax and caspase3 and reduction in Bcl2 level. This was erating figures and bioinformatics KM plotter data analysis, and final revisions
accompanied by a reduced expression level of the inflam- correction: A.M.S. Supervision and project administration: Y.M.H. All authors
have read and agreed to the published version of the manuscript. All authors
matory mediators such as TNFα, NFkB, TGFβ1, and read and approved the final manuscript.
Cox2 as well as the angiogenic-related factors VEGF [36].
Funding
There is no fund for this article.
Concluding remarks and perspectives
In this review, we discussed the therapeutic value of the Availability of data and materials
Data sharing is not applicable.
camelid-derived nanobodies along with the therapeutic
effects provided by the ingredients of camels’ biological
Declarations
secretion both milk and urine in combatting different
human diseases mostly cancers. Many research areas Ethics approval and consent to participate
such as medical, biotechnology, engineering, and eco- Not applicable.
nomics have been cultivated after the discovery of the Consent for publication
naturally occurring camelids nanobodies. Nanobod- Not applicable.
ies have recently emerged as attractive robust strategies
Competing interests
for diagnostic and therapeutic aims, particularly in the The authors declare that they have no competing interests.
cancer field. Providentially, nanobodies exhibit several
Author details 17. Harmsen MM, et al. Selection and optimization of proteolytically stable
1
Center of Genomic Medicine, King Faisal Specialist Hospital & Research llama single-domain antibody fragments for oral immunotherapy. Appl
Center, Riyadh, Saudi Arabia. 2 College of Medicine, Alfaisal University, P.O. Microbiol Biotechnol. 2006;72(3):544–51.
Box 50927, Riyadh 11533, Saudi Arabia. 3 Department of Laboratory & Blood 18. van der Linden RH, et al. Comparison of physical chemical properties
Bank, Security Forces Hospital, P.O. Box 14799, Mecca 21955, Saudi Arabia. of llama VHH antibody fragments and mouse monoclonal antibodies.
4
College of Medicine, Al-Faisal University, P.O. Box 50927, Riyadh 11533, Saudi Biochim Biophys Acta. 1999;1431(1):37–46.
Arabia. 5 Department of Biochemistry, Faculty of Science, Embryonic Stem 19. Harmsen MM, et al. Escherichia coli F4 fimbriae specific llama
Cells Unit, King Fahad Medical Center, King Abdulaziz University, Jeddah, Saudi single-domain antibody fragments effectively inhibit bacterial
Arabia. 6 Centre of Artificial Intelligence in Precision Medicines (CAIPM), King adhesion in vitro but poorly protect against diarrhoea. Vet Microbiol.
Abdulaziz University, Jeddah, Saudi Arabia. 7 Department of Pharmacology, 2005;111(1–2):89–98.
College of Medicine, Taif University, P.O. BOX 11099, Taif 21944, Saudi Arabia. 20. Harmsen MM, De Haard HJ. Properties, production, and applications of
8
The National Center for Genomic Technology, King Abdulaziz City for Sci‑ camelid single-domain antibody fragments. Appl Microbiol Biotechnol.
ence and Technology, P.O Box 6086, Riyadh 11442, Saudi Arabia. 9 Depart‑ 2007;77(1):13–22.
ment of Biology, Faculty of Sciences, University of Tabuk, Tabuk, Saudi Arabia. 21. Salvador JP, Vilaplana L, Marco MP. Nanobody: outstanding features
10
Genome and Biotechnology Unit, Faculty of Sciences, University of Tabuk, for diagnostic and therapeutic applications. Anal Bioanal Chem.
Tabuk, Saudi Arabia. 11 Department of Medical Laboratory Technology, 2019;411(9):1703–13.
Faculty of Applied Medical Sciences, University of Tabuk, Tabuk, Saudi Arabia. 22. Singer A, et al. Nanobiotechnology medical applications: Over‑
12
Department of Biological Sciences, Faculty of Science and Arts (Rabigh coming challenges through innovation. The EuroBiotech Journal.
Campus), King Abdulaziz University, Jeddah, Saudi Arabia. 13 Research Institute 2018;2(3):146–60.
of Veterinary Medicine, Chungam National University, Daejeon 34134, Korea. 23. Ibanez LI, et al. Nanobodies with in vitro neutralizing activity
14
Research Center, King Faisal Specialist Hospital and Research Center, MBC- protect mice against H5N1 influenza virus infection. J Infect Dis.
J04, PO Box 40047, Jeddah 21499, Saudi Arabia. 2011;203(8):1063–72.
24. Hussack G, et al. Neutralization of Clostridium difficile toxin A with
Received: 13 May 2022 Accepted: 30 August 2022 single-domain antibodies targeting the cell receptor binding domain. J
Published: 7 September 2022 Biol Chem. 2011;286(11):8961–76.
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branched gold nanoparticles for photothermal therapy. ACS Nano.
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Al‑Numair et al.

Cancer Cell International 2022, 22(1):279

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