https://doi.org/10.5812/tms.114888

The Potential Applicability of Single-Domain Antibodies (VHH): From Checkpoint Blockade to Infectious Disease Therapy

authors:

avatar Pouya Safarzadeh Kozani ORCID 1 , 2 , avatar Pooria Safarzadeh Kozani ORCID 3 , avatar Fatemeh Rahbarizadeh ORCID 3 , 4 , *

Department of Medical Biotechnology, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran
Student Research Committee, Medical Biotechnology Research Center, School of Nursing, Midwifery, and Paramedicine, Guilan University of Medical Sciences, Rasht, Iran
Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Research and Development Center of Biotechnology, Tarbiat Modares University, Tehran, Iran
Trends in Medical Sciences: Vol.1, issue 2; e114888
published online: May 9, 2021
article type: Review Article
received: March 30, 2021
revised: April 27, 2021
accepted: April 29, 2021

how to cite: Safarzadeh Kozani P , Safarzadeh Kozani P, Rahbarizadeh F. The Potential Applicability of Single-Domain Antibodies (VHH): From Checkpoint Blockade to Infectious Disease Therapy. J Adv Immunopharmacol. 2021;1(2):e114888. https://doi.org/10.5812/tms.114888.

Abstract

Monoclonal antibodies (mAbs) have demonstrated promising clinical efficacy in various therapeutic fields over the past decades. However, their size has emerged as the most restricting factor limiting their application in antibody-drug conjugate platforms or antibody-redirected delivery systems. This limitation was mainly due to the inability of these platforms to penetrate the tumor site and reach their intended destination. Single-domain antibodies, also known as VHHs or nanobodies®, are known as promising alternatives capable of addressing the drawbacks of conventional full-sized antibodies. Alongside a great level of affinity and selectivity towards their target, the small size of nanobodies, which is about 15 kDa, their capability for tissue penetrance, the significant level of stability, their simple and cost-friendly manufacturing process, and fleeting half-life in the circulation system, are all among the reasons that have made them suitable candidates for the development of various types of therapies. In this review, we briefly discuss the application of nanobodies in checkpoint blockade therapy and nanobody-drug conjugate platforms. We also discuss nanobody-assisted redirection of various types of delivery systems carrying different types of cargoes. In the last section, we underline the promising and effective application of nanobodies in the fight against infectious diseases.

1. Context

More than three decades after the first attempt to use monoclonal antibodies (mAbs) for cancer therapy, today, mAb-based cancer therapies are rapidly developing (1-4). Currently, numerous mAbs have been approved by the US Food and Drug Administration (FDA) for medical use in various fields (5). These commercially available mAbs exert their functional effects through binding to cell surface receptors or targeting ligands that are soluble (6, 7). Upon such interactions, a unique downstream signaling pathway will be triggered, which usually disrupts a molecular signaling pathway, consequently leading to the blockage of the proliferation signaling of malignant tumor cells (8, 9). Therefore, tumor cells can be eliminated in a very selective manner in comparison with the commonly available cancer treatment methods such as chemotherapy. Moreover, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) are also other mechanisms by which mAbs mediate the elimination of their target cells (10). In these mechanisms, mAbs can activate the complement or engage the effector cells of the immune systems to destroy and eliminate tumor cells towards whose cell surface antigens the mAbs have binding affinity (10). mAbs have also been utilized for redirection purposes. In this regard, they have been used for the surface decoration of nanoparticles, which can be loaded with various types of cargoes, including cytotoxic chemotherapeutic drugs, toxins, nucleotides with therapeutic value, photosensitizers, and radiosensitizers (11-13). Despite the advantages of these platforms, there are several hindrances as well, such as the large size of mAbs (even without linking them to nanoparticles), which overshadows their tumor site penetration and trafficking capability (14-16).

Typically, in mammals, plasma cells, which are the immunoglobulin (Ig)-secreting type of B cells, secrete antibodies that are composed of two heavy chains and two light chains. Such antibodies are known as full-length antibodies. Each chain of these full-length antibodies harbors variable and constant regions. However, there are natural or synthetic types of antibodies that are made of only a part of full-length antibodies (17). These smaller structures include antigen-binding fragment (Fab), variable fragment (Fv), or single-chain variable fragment (scFv). These small antibody fragments offer great advantages over the conventional full-sized ones; however, they also suffer from several drawbacks. In comparison to the full-length antibodies, Fabs, Fvs, and scFvs tend to have diminished stability, generally lower affinity, and production difficulties (18).

The discovery of an additional IgG isotype, made of only a homodimer of heavy chains, has been known as a revolution in the field of antibody investigations (19, 20). Since then, heavy-chain antibodies (HCAbs) have been recognized as potential targeting tools in various developmental and clinical investigations. The outstanding part is that even though these antibodies do not harbor light chains, they are completely functional and they tend to have the same level of affinity to their specific antigen, as compared with that of conventional murine or human antibodies. These heavy-chain antibodies are mostly attained from the Camelidae family members, including camels and alpacas. It is believed that the simple process of immunizing these animals is the main reason behind this choice, considering the fact that other animals such as sharks also harbor HCAbs (19). Ever since the discovery of HCAbs, researchers all over the world have exploited the variable domain of the heavy chain (also known as a VHH or a nanobody®) of such HCAbs for therapeutic and diagnostic purposes, and they have reported encouraging outcomes (4, 21, 22). Figure 1 is a simplified representation of a conventional antibody, an HCAb, and a VHH. Nanobodies also tend to have other favorable characteristics such as their high solubility and stability rates (23, 24), their simple production process in bacteria (allowing for large-scale productions) (25), their outstanding tissue trafficking capability (resulting from their small size) (26), and their rapid clearance from the blood circulation (27). However, the rapid elimination of antibodies from the circulation tends to be an unfavorable factor in therapeutic applications, where long-term persistence is required for efficient responses. Moreover, due to their origination from camelid species, they tend to be immunogenic in humans. In this regard, humanization may be helpful for minimizing the immunogenicity of nanobodies (28). So far, various nanobodies have been developed against numerous targets involved in different oncological and immunological indications, some of which have been summarized in Table 1. In this review, we briefly discuss the use of VHHs in various fields, including direct cytotoxic drug conjugation, signaling blockade purposes, VHH-facilitated redirection of delivery systems, and cancer immunotherapy.

A simplified representation of a conventional immunoglobulin molecule, a heavy chain antibody, and a single domain antibody (also known as a nanobody or VHH). The size (in nanometer) and molecular weights (in kDa) are also featured.
A simplified representation of a conventional immunoglobulin molecule, a heavy chain antibody, and a single domain antibody (also known as a nanobody or VHH). The size (in nanometer) and molecular weights (in kDa) are also featured.
Figure 1.

A simplified representation of a conventional immunoglobulin molecule, a heavy chain antibody, and a single domain antibody (also known as a nanobody or VHH). The size (in nanometer) and molecular weights (in kDa) are also featured.

Table 1.

A Summary of Different Nanobodies That Target Distinct Antigen Molecules Involved in Numerous Oncological and Immunological Indications

Nanobody DesignationTarget AntigenInvolved IndicationAnimal SourceReference
H11CTLA-4Various oncological indicationsalpaca(29)
Nb_7, Nb_21, and Nb_22CD33AMLLlama glama(30)
11A4HER2Breast cancerLlama glama(31)
SRB-37 and SRB-85CD19Hematologic malignanciesCamelus bactrianus(32)
RR-B7 and RR-D40MUC1Advanced solid tumorsCamelus dromedarius and Camelus bactrianus(33, 34)
NB5CXCR7Neck cancerLlama glama(35)
Anti-TNF-VHHTNFαSolid tumors-(36)
VHH4MHC-IIGvHDVicugna pacos(37)
C3, E2PD-L1Advanced solid tumorsCamelus dromedarius(38)
K2PD-L1Advanced solid tumorsalpaca(39)
cAb-CEA5CEABreast, colorectal, ovarian, pancreatic, and cancerCamelus dromedarius(40)
JJB-B2CEABreast, colorectal, ovarian, pancreatic, and cancerVicugna pacos(41)
HuNb1-IgG4CD47NHL, colorectal cancer, ovarian cancer, and AMLCamelus bactrianus(42)
C3PSMAProstate cancerCamelus bactrianus(43)
N14, C24, C9PSMAProstate cancerCamelus dromedarius(44)
MU738, JK2, WF211, MU523, and MU1067CD38Multiple myelomaLlama glama(45)
127D1 and 163E3CXCR2Solid tumors and immunological indicationsLlama glama(46)
VHH6CD7LeukemiasLlama glama(47)
V36, 81, 51, and B10CD11bImmunological indicationsLlama glama(48)
K24CAIXBreast cancerCamelus dromedarius(49)

Abbreviations: AML, acute myeloid leukemia; NHL, non-Hodgkin’s lymphoma; GvHD, graft versus host disease.

2. Nanobodies in Therapy

2.1. Nanobodies Application in Infectious Diseases

The neutralizing effects of nanobodies have been promising in various studies paving the way for their investigation in various kinds of virus- or bacteria-induced conditions. In this regard, researchers have investigated the protective effects of llama-derived hemagglutinin-targeting nanobodies in preclinical models of influenza A virus subtype H5N1 (50). They demonstrated that the intranasal administration of these nanobodies successfully suppresses influenza A virus replication (50). They also added that using bivalent nanobodies may lead to a more efficient virus replication suppression than using monovalent nanobodies, in this case (50). Moreover, Hussack et al. investigated the neutralization of Clostridium difficile toxin A using nanobodies targeting its cell receptor-binding domain (51). To date, Clostridium difficile has been a significant challenge for healthcare professionals in hospitals (51). The authors of the mentioned study concluded that these nanobodies show effective toxin neutralization alongside exhibiting a satisfactory level of stability (51).

Researchers have reported that high oral doses of the recombinant nanobody clone K609, which is directed against Escherichia coli F4 fimbriae, and decrease diarrhea induced by E. coli in piglets (52). The same research group further investigated the stability of a more stable clone of these nanobodies named K922 (52). They demonstrated that K922 exhibits significant binding affinity to F4 fimbriae, which is higher than that of K609 (52). They also added that K922 inhibits fimbrial adhesion at lower doses in comparison with that of K609 (52). Ultimately, they concluded that K922 may be a favorable choice for the prevention of diarrhea in piglets (52).

Malassezia furfur is a yeast species known as the reason for various dermatological diseases (including dandruff) triggered by fungal infections (53). Dolk et al. have demonstrated that llama-originated nanobodies isolated using the phage display method can effectively inhibit the growth of organisms associated with dandruff (53). This study indicated that detergent-resistant species of nanobodies targeting the surface protein of Malassezia furfur can be generated for application in anti-dandruff shampoos (53).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently emerged virus that causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 viral particles harbor an envelope protein called spike (S) which interacts with the cell surface receptors (known as ACE-2) of their host cells mediating virus entry into cells (54). Considering the vital role of the spike protein in the functionality of these viruses, it has become a popular target for blocking the entry of the virus into the host cells and preventing the replication of the virus (54). In 2020, Wrapp et al. isolated VHHs from a llama immunized with coronavirus spikes (54). They demonstrated that these nanobodies were capable of binding to the ribosome binding domain (RBD) of the spike protein and neutralizing coronaviruses such as SARS-CoV-2 (54).

2.2. Nanobody-Drug Conjugates

Nanobody-drug conjugation, powered by the selective targeting ability of nanobodies, is another platform that has shown promising results in targeted cancer therapy investigations. The main advantage of this approach is that it is capable of alleviating the off-target side effects of the unconjugated therapeutic agents. Nanobodies have attracted much more attention towards themselves since they tend to have advantages over full-length antibodies. The main advantage of nanobody-drug conjugates in comparison with full-length antibody-drug conjugates is that they can penetrate the tumor sites more easily due to their small size.

Wu et al. conjugated Pt(IV) prodrugs to anti-EGFR nanobodies to selectively deliver them to tumor cells (55). They showed that this platform is capable of inducing antitumor effects both in vitro and in vivo (55). They added that clustered nanobody-drug conjugates exhibit superior antitumor effects in vivo as compared to those of monomeric nanobody-drug conjugates (55). Such pronounced antitumor effects could be a result of the prolonged circulation time and lower kidney clearance rate of clustered nanobody-drug conjugates in comparison with monomeric nanobody-drug conjugates, which are smaller in size.

Our investigational team has conjugated the PE38 toxin (a truncated form of Pseudomonas exotoxin A) to nanobodies and has shown that this nanobody-toxin conjugate can exhibit selective tumor cell targeting ability and cytotoxicity towards tumor cells that express the target antigen (data under publication). Moreover, our experience demonstrates that the nanobody-toxin conjugate platform might serve as a flexible and potential treatment modality against various hematologic malignancies and solid tumors.

2.3. Their Application in Checkpoint Blockade Therapies

Checkpoint blockade therapy is a type of immunotherapy that uses a wide range of agents, from chemical drugs to mAbs, to block the signaling pathway of immune checkpoints or other critical regulators of the immune system (56-60). PD-1 and CTLA-4 are two well-known T cell exhaustion markers whose blocking using mAbs has been effective in creating potent antitumor responses in various types of cancers (56-60).

KN035 is a PD-L1-targeting nanobody capable of mediating robust T cell antitumor activity and suppressing tumor proliferation in preclinical in vivo models (61). Furthermore, a CTLA-4-targeting nanobody named H11 has shown promising results in suppressing tumor growth in preclinical mouse models of melanoma (29).

Ma et al. have generated multivalent bispecific antibody (BsAb) co-targeting PD-L1 and TIGIT (also called T cell immunoreceptor with Ig and ITIM domains) (62). TIGIT is a recently known immune checkpoint molecule that has mediated encouraging results in immune checkpoint blockade therapies (62). The BsAbs are made of a tetravalent anti-PD-L1 Fc-fusion nanobody and a tetravalent anti-TIGIT nanobody (62). The results of this study indicated that the anti-PD-L1 nanobodies harbor significant levels of specificity and affinity against PD-L1, and they can improve T cell functionality and antitumor activity both in vitro and in vivo (62). The researchers also added that similar results were also obtained for the anti-TIGIT nanobodies as these nanobodies had significant levels of specificity and affinity towards TIGIT (62). Furthermore, Ma et al. also reported that the BsAb construct was very effective in disrupting both the PD-1/PD-L1 and the TIGIT/CD155 axis in vitro (62).

2.4. Nanobody-Facilitated Redirection of Delivery Systems

As delivery systems are becoming increasingly popular, various redirection methods have been investigated for the targeting of these platforms to the tissues and cells of interest (63, 64). Delivery platforms, including liposomes, nanoparticles, micelles, etc. have been investigated for the delivery of various cargoes such as therapeutic drugs (e.g., doxorubicin, paclitaxel, and cisplatin), miRNAs, siRNAs, toxins, etc., since they can mediate the controlled release of their cargoes in the intended tumor sites they are targeted towards (63, 64). These delivery platforms have differences in their mechanism of action and structures, but the common feature among them is the presence of a targeting moiety in their constructs (63, 64). The targeting moieties of these delivery platforms are usually decorated on their surface, allowing their easy interaction with the target tumor cell surface antigens (63, 64). So far, various targeting moieties have been utilized for the redirection of these delivery platforms, all of which have their advantages and disadvantages (63, 64). Besides mAbs, other targeting moieties such as aptamers have also been investigated in this regard (63, 64). However, in comparison to aptamers, mAbs have various advantages. The process of generating a specific mAb against a cell surface of interest is more feasible than that of aptamers. Furthermore, the affinity and specificity of mAb tends to be more selective towards cell surface antigens of interest expressed by tumor cells in comparison with those of aptamers.

As a contribution to this topic, researchers have generated extracellular vesicles surface-decorated with anti-EGFR nanobodies and have demonstrated that these vesicles are specifically redirected towards EGFR-expressing tumor cells (65). Moreover, Liu et al. investigated the applicability of EGFR nanobody surface-decorated polymeric micelles loaded with photosensitizers (66). These researchers loaded the polymeric micelles with the photosensitizer temoporfin (mTHPC). This platform proved efficient in redirecting polymeric micelles towards EGRF-positive cancer cells in vivo (66). The researchers also added that these polymeric micelles tend to have enhanced circulation and diminished the level of clearance as compared with those of naked mTHPC (66). Other researchers have developed tumor-targeting nanobullets which are made of liposomes surface-decorated with anti-EGFR nanobodies (67). These liposomes were loaded with the anti-IGF-1R kinase inhibitor AG538 (67). The researchers of this study indicated that the mentioned nanobullets were capable of inducing robust tumor cell growth suppression in a very efficient and selective manner (67).

3. Conclusions

The favorable characteristics of nanobodies make them exclusive and irreplaceable therapeutics for various types of treatments. For instance, caplacizumab (also known as Cablivi) achieved FDA approval for the treatment of adult patients with acquired thrombotic thrombocytopenic purpura (aTTP) in 2019 (68). Moreover, as discussed throughout this article, the big size of the conventional full-length antibodies creates limitations in tissue penetration itself. The mentioned size limitation leads to more hurdles when these antibodies are bound to delivery vehicles such as nanoparticles for the aim of redirection. In this regard, nanobodies can be alternatives to full-length antibodies for the redirection of delivery vehicles such as nanoparticles with great levels of specificity and affinity. In the case of nanobody-drug conjugates, the same story applies. Therefore, utilizing nanobodies as the targeting moiety of these nanoparticles or nanobody-drug conjugates enables creating more effective platforms. Furthermore, nanobodies have also proven efficient in checkpoint blockade therapies.

Herein, we discussed some of the studies investigating the suitability of nanobodies for fighting against infectious diseases. However, there is still room for improving the functionality of nanobodies. Approaches such as bivalent nanobodies can be used to enhance the affinity of monovalent nanobodies (69). Moreover, there are methods used for elevating the in vivo half-life of nanobodies (22, 69). The fusion of nanobodies to human serum albumin is a great example used for elevating the in vivo half-life of nanobodies (22). Bivalent nanobodies also tend to have superior half-life in the circulation system in comparison with that of monovalent nanobodies (70). However, such innovative nanobody engineering methods require careful in vivo assessments for determining their safety and applicability in various types of therapies. Moreover, nanobodies developed by such engineering techniques should also be meticulously examined in terms of their immunogenicity, stability, and affinity. It is also speculated that these modifications may have negative impacts on various characteristics of nanobodies, such as their affinity, tissue penetration, stability, and solubility. Once the outcomes of in-depth assessments or clinical trials come out, the ambiguities remaining in this field can be cleared out. Therefore, it can be safely concluded that nanobodies might be beneficial in achieving more efficient treatment modalities.

References

  • 1.

    Oldham RK, Dillman RO. Monoclonal antibodies in cancer therapy: 25 years of progress. J Clin Oncol. 2008;26(11):1774-7. [PubMed ID: 18398141]. https://doi.org/10.1200/JCO.2007.15.7438.

  • 2.

    Safarzadeh Kozani P, Safarzadeh Kozani P, Rahbarizadeh F. Novel antigens of CAR T cell therapy: New roads; old destination. Transl Oncol. 2021;14(7):101079. [PubMed ID: 33862524]. [PubMed Central ID: PMC8065293]. https://doi.org/10.1016/j.tranon.2021.101079.

  • 3.

    Safarzadeh Kozani P, Safarzadeh Kozani P, Rahbarizadeh F, Khoshtinat Nikkhoi S. Strategies for dodging the obstacles in CAR T cell therapy. Front Oncol. 2021;11:627549. [PubMed ID: 33869011]. [PubMed Central ID: PMC8047470]. https://doi.org/10.3389/fonc.2021.627549.

  • 4.

    Hashem Boroojerdi M, Rahbarizadeh F, Safarzadeh Kozani P, Kamali E, Safarzadeh Kozani P. Strategies for having a more effective and less toxic CAR T-cell therapy for acute lymphoblastic leukemia. Med Oncol. 2020;37(11):100. [PubMed ID: 33047234]. [PubMed Central ID: PMC7549730]. https://doi.org/10.1007/s12032-020-01416-3.

  • 5.

    Oliveira S, Heukers R, Sornkom J, Kok RJ, van Bergen En Henegouwen PM. Targeting tumors with nanobodies for cancer imaging and therapy. J Control Release. 2013;172(3):607-17. [PubMed ID: 24035975]. https://doi.org/10.1016/j.jconrel.2013.08.298.

  • 6.

    Oldfield V, Dhillon S, Plosker GL. Tocilizumab: A review of its use in the management of rheumatoid arthritis. Drugs. 2009;69(5):609-32. [PubMed ID: 19368420]. https://doi.org/10.2165/00003495-200969050-00007.

  • 7.

    Shepard HM, Phillips GL, D. Thanos C, Feldmann M. Developments in therapy with monoclonal antibodies and related proteins. Clin Med. 2017;17(3):220-32. [PubMed ID: 28572223]. [PubMed Central ID: PMC6297577]. https://doi.org/10.7861/clinmedicine.17-3-220.

  • 8.

    Marhelava K, Pilch Z, Bajor M, Graczyk-Jarzynka A, Zagozdzon R. Targeting negative and positive immune checkpoints with monoclonal antibodies in therapy of cancer. Cancers. 2019;11(11). [PubMed ID: 31717326]. [PubMed Central ID: PMC6895894]. https://doi.org/10.3390/cancers11111756.

  • 9.

    Koustas E, Karamouzis MV, Mihailidou C, Schizas D, Papavassiliou AG. Co-targeting of EGFR and autophagy signaling is an emerging treatment strategy in metastatic colorectal cancer. Cancer Lett. 2017;396:94-102. [PubMed ID: 28323034]. https://doi.org/10.1016/j.canlet.2017.03.023.

  • 10.

    Holz E, Raab R, Riethmuller G. Antibody-based immunotherapeutic strategies in colorectal cancer. Recent Results Cancer Res. 1996;142:381-400. [PubMed ID: 8893351]. https://doi.org/10.1007/978-3-642-80035-1_21.

  • 11.

    Senter PD. Potent antibody drug conjugates for cancer therapy. Curr Opin Chem Biol. 2009;13(3):235-44. [PubMed ID: 19414278]. https://doi.org/10.1016/j.cbpa.2009.03.023.

  • 12.

    Allahyari H, Heidari S, Ghamgosha M, Saffarian P, Amani J. Immunotoxin: A new tool for cancer therapy. Tumour Biol. 2017;39(2):1010428317692230. [PubMed ID: 28218037]. https://doi.org/10.1177/1010428317692226.

  • 13.

    Darwish WM, Bayoumi NA, El-Shershaby HM, Allahloubi NM. Targeted photoimmunotherapy based on photosensitizer-antibody conjugates for multiple myeloma treatment. J Photochem Photobiol B. 2020;203:111777. [PubMed ID: 31931387]. https://doi.org/10.1016/j.jphotobiol.2020.111777.

  • 14.

    Siontorou CG. Nanobodies as novel agents for disease diagnosis and therapy. Int J Nanomedicine. 2013;8:4215-27. [PubMed ID: 24204148]. [PubMed Central ID: PMC3818023]. https://doi.org/10.2147/IJN.S39428.

  • 15.

    Baker JH, Lindquist KE, Huxham LA, Kyle AH, Sy JT, Minchinton AI. Direct visualization of heterogeneous extravascular distribution of trastuzumab in human epidermal growth factor receptor type 2 overexpressing xenografts. Clin Cancer Res. 2008;14(7):2171-9. [PubMed ID: 18381959]. https://doi.org/10.1158/1078-0432.CCR-07-4465.

  • 16.

    Rudnick SI, Adams GP. Affinity and avidity in antibody-based tumor targeting. Cancer Biother Radiopharm. 2009;24(2):155-61. [PubMed ID: 19409036]. [PubMed Central ID: PMC2902227]. https://doi.org/10.1089/cbr.2009.0627.

  • 17.

    Chiu ML, Goulet DR, Teplyakov A, Gilliland GL. Antibody structure and function: The basis for engineering therapeutics. Antibodies. 2019;8(4). [PubMed ID: 31816964]. [PubMed Central ID: PMC6963682]. https://doi.org/10.3390/antib8040055.

  • 18.

    Bell A, Wang ZJ, Arbabi-Ghahroudi M, Chang TA, Durocher Y, Trojahn U, et al. Differential tumor-targeting abilities of three single-domain antibody formats. Cancer Lett. 2010;289(1):81-90. [PubMed ID: 19716651]. https://doi.org/10.1016/j.canlet.2009.08.003.

  • 19.

    Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, et al. Naturally occurring antibodies devoid of light chains. Nature. 1993;363(6428):446-8. [PubMed ID: 8502296]. https://doi.org/10.1038/363446a0.

  • 20.

    Padlan EA. Anatomy of the antibody molecule. Mol Immunol. 1994;31(3):169-217. [PubMed ID: 8114766]. https://doi.org/10.1016/0161-5890(94)90001-9.

  • 21.

    Jovcevska I, Muyldermans S. The therapeutic potential of nanobodies. BioDrugs. 2020;34(1):11-26. [PubMed ID: 31686399]. [PubMed Central ID: PMC6985073]. https://doi.org/10.1007/s40259-019-00392-z.

  • 22.

    Bannas P, Hambach J, Koch-Nolte F. Nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics. Front Immunol. 2017;8:1603. [PubMed ID: 29213270]. [PubMed Central ID: PMC5702627]. https://doi.org/10.3389/fimmu.2017.01603.

  • 23.

    Tanha J, Xu P, Chen Z, Ni F, Kaplan H, Narang SA, et al. Optimal design features of camelized human single-domain antibody libraries. J Biol Chem. 2001;276(27):24774-80. [PubMed ID: 11335716]. https://doi.org/10.1074/jbc.M100770200.

  • 24.

    van der Linden RH, Frenken LG, de Geus B, Harmsen MM, Ruuls RC, Stok W, et al. Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies. Biochim Biophys Acta. 1999;1431(1):37-46. [PubMed ID: 10209277]. https://doi.org/10.1016/s0167-4838(99)00030-8.

  • 25.

    Kijanka M, Dorresteijn B, Oliveira S, van Bergen en Henegouwen PM. Nanobody-based cancer therapy of solid tumors. Nanomedicine. 2015;10(1):161-74. [PubMed ID: 25597775]. https://doi.org/10.2217/nnm.14.178.

  • 26.

    Fang T, Lu X, Berger D, Gmeiner C, Cho J, Schalek R, et al. Nanobody immunostaining for correlated light and electron microscopy with preservation of ultrastructure. Nat Methods. 2018;15(12):1029-32. [PubMed ID: 30397326]. [PubMed Central ID: PMC6405223]. https://doi.org/10.1038/s41592-018-0177-x.

  • 27.

    Tijink BM, Laeremans T, Budde M, Stigter-van Walsum M, Dreier T, de Haard HJ, et al. Improved tumor targeting of anti-epidermal growth factor receptor Nanobodies through albumin binding: taking advantage of modular Nanobody technology. Mol Cancer Ther. 2008;7(8):2288-97. [PubMed ID: 18723476]. https://doi.org/10.1158/1535-7163.MCT-07-2384.

  • 28.

    Jank L, Pinto-Espinoza C, Duan Y, Koch-Nolte F, Magnus T, Rissiek B. Current approaches and future perspectives for nanobodies in stroke diagnostic and therapy. Antibodies. 2019;8(1). [PubMed ID: 31544811]. [PubMed Central ID: PMC6640704]. https://doi.org/10.3390/antib8010005.

  • 29.

    Ingram JR, Blomberg OS, Rashidian M, Ali L, Garforth S, Fedorov E, et al. Anti-CTLA-4 therapy requires an Fc domain for efficacy. Proc Natl Acad Sci U S A. 2018;115(15):3912-7. [PubMed ID: 29581255]. [PubMed Central ID: PMC5899492]. https://doi.org/10.1073/pnas.1801524115.

  • 30.

    Romao E, Krasniqi A, Maes L, Vandenbrande C, Sterckx YG, Stijlemans B, et al. Identification of nanobodies against the acute myeloid leukemia marker CD33. Int J Mol Sci. 2020;21(1). [PubMed ID: 31906437]. [PubMed Central ID: PMC6981622]. https://doi.org/10.3390/ijms21010310.

  • 31.

    Kijanka M, Warnders FJ, El Khattabi M, Lub-de Hooge M, van Dam GM, Ntziachristos V, et al. Rapid optical imaging of human breast tumour xenografts using anti-HER2 VHHs site-directly conjugated to IRDye 800CW for image-guided surgery. Eur J Nucl Med Mol Imaging. 2013;40(11):1718-29. [PubMed ID: 23778558]. https://doi.org/10.1007/s00259-013-2471-2.

  • 32.

    Banihashemi SR, Hosseini AZ, Rahbarizadeh F, Ahmadvand D. Development of specific nanobodies (VHH) for CD19 immuno-targeting of human B-lymphocytes. Iran J Basic Med Sci. 2018;21(5):455-64. [PubMed ID: 29922424]. [PubMed Central ID: PMC6000210]. https://doi.org/10.22038/IJBMS.2018.26778.6557.

  • 33.

    Rahbarizadeh F, Rasaee MJ, Forouzandeh Moghadam M, Allameh AA, Sadroddiny E. Production of novel recombinant single-domain antibodies against tandem repeat region of MUC1 mucin. Hybrid Hybridomics. 2004;23(3):151-9. [PubMed ID: 15312305]. https://doi.org/10.1089/1536859041224334.

  • 34.

    Rahbarizadeh F, Rasaee MJ, Forouzandeh M, Allameh A, Sarrami R, Nasiry H, et al. The production and characterization of novel heavy-chain antibodies against the tandem repeat region of MUC1 mucin. Immunol Invest. 2005;34(4):431-52. [PubMed ID: 16302687]. https://doi.org/10.1080/08820130500265356.

  • 35.

    Maussang D, Mujic-Delic A, Descamps FJ, Stortelers C, Vanlandschoot P, Stigter-van Walsum M, et al. Llama-derived single variable domains (nanobodies) directed against chemokine receptor CXCR7 reduce head and neck cancer cell growth in vivo. J Biol Chem. 2013;288(41):29562-72. [PubMed ID: 23979133]. [PubMed Central ID: PMC3795254]. https://doi.org/10.1074/jbc.M113.498436.

  • 36.

    Ji X, Peng Z, Li X, Yan Z, Yang Y, Qiao Z, et al. Neutralization of TNFalpha in tumor with a novel nanobody potentiates paclitaxel-therapy and inhibits metastasis in breast cancer. Cancer Lett. 2017;386:24-34. [PubMed ID: 27832973]. https://doi.org/10.1016/j.canlet.2016.10.031.

  • 37.

    Van Elssen C, Rashidian M, Vrbanac V, Wucherpfennig KW, Habre ZE, Sticht J, et al. Noninvasive imaging of human immune responses in a human xenograft model of graft-versus-host disease. J Nucl Med. 2017;58(6):1003-8. [PubMed ID: 28209904]. [PubMed Central ID: PMC5450362]. https://doi.org/10.2967/jnumed.116.186007.

  • 38.

    Broos K, Keyaerts M, Lecocq Q, Renmans D, Nguyen T, Escors D, et al. Non-invasive assessment of murine PD-L1 levels in syngeneic tumor models by nuclear imaging with nanobody tracers. Oncotarget. 2017;8(26):41932-46. [PubMed ID: 28410210]. [PubMed Central ID: PMC5522039]. https://doi.org/10.18632/oncotarget.16708.

  • 39.

    Broos K, Lecocq Q, Xavier C, Bridoux J, Nguyen TT, Corthals J, et al. Evaluating a single domain antibody targeting human PD-L1 as a nuclear imaging and therapeutic agent. Cancers. 2019;11(6). [PubMed ID: 31234464]. [PubMed Central ID: PMC6628009]. https://doi.org/10.3390/cancers11060872.

  • 40.

    Cortez-Retamozo V, Backmann N, Senter PD, Wernery U, De Baetselier P, Muyldermans S, et al. Efficient cancer therapy with a nanobody-based conjugate. Cancer Res. 2004;64(8):2853-7. [PubMed ID: 15087403]. https://doi.org/10.1158/0008-5472.can-03-3935.

  • 41.

    Kaliberov SA, Kaliberova LN, Buggio M, Tremblay JM, Shoemaker CB, Curiel DT. Adenoviral targeting using genetically incorporated camelid single variable domains. Lab Invest. 2014;94(8):893-905. [PubMed ID: 24933423]. [PubMed Central ID: PMC4157633]. https://doi.org/10.1038/labinvest.2014.82.

  • 42.

    Ma L, Zhu M, Gai J, Li G, Chang Q, Qiao P, et al. Preclinical development of a novel CD47 nanobody with less toxicity and enhanced anti-cancer therapeutic potential. J Nanobiotechnology. 2020;18(1):12. [PubMed ID: 31931812]. [PubMed Central ID: PMC6956557]. https://doi.org/10.1186/s12951-020-0571-2.

  • 43.

    Zare H, Rajabibazl M, Rasooli I, Ebrahimizadeh W, Bakherad H, Ardakani LS, et al. Production of nanobodies against prostate-specific membrane antigen (PSMA) recognizing LnCaP cells. Int J Biol Markers. 2014;29(2):e169-79. [PubMed ID: 24425321]. https://doi.org/10.5301/jbm.5000063.

  • 44.

    Saerens D, Kinne J, Bosmans E, Wernery U, Muyldermans S, Conrath K. Single domain antibodies derived from dromedary lymph node and peripheral blood lymphocytes sensing conformational variants of prostate-specific antigen. J Biol Chem. 2004;279(50):51965-72. [PubMed ID: 15459193]. https://doi.org/10.1074/jbc.M409292200.

  • 45.

    Fumey W, Koenigsdorf J, Kunick V, Menzel S, Schutze K, Unger M, et al. Nanobodies effectively modulate the enzymatic activity of CD38 and allow specific imaging of CD38(+) tumors in mouse models in vivo. Sci Rep. 2017;7(1):14289. [PubMed ID: 29084989]. [PubMed Central ID: PMC5662768]. https://doi.org/10.1038/s41598-017-14112-6.

  • 46.

    Bradley ME, Dombrecht B, Manini J, Willis J, Vlerick D, De Taeye S, et al. Potent and efficacious inhibition of CXCR2 signaling by biparatopic nanobodies combining two distinct modes of action. Mol Pharmacol. 2015;87(2):251-62. [PubMed ID: 25468882]. https://doi.org/10.1124/mol.114.094821.

  • 47.

    Tang J, Li J, Zhu X, Yu Y, Chen D, Yuan L, et al. Novel CD7-specific nanobody-based immunotoxins potently enhanced apoptosis of CD7-positive malignant cells. Oncotarget. 2016;7(23):34070-83. [PubMed ID: 27083001]. [PubMed Central ID: PMC5085138]. https://doi.org/10.18632/oncotarget.8710.

  • 48.

    Rossotti M, Tabares S, Alfaya L, Leizagoyen C, Moron G, Gonzalez-Sapienza G. Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells. Biochim Biophys Acta. 2015;1850(7):1397-404. [PubMed ID: 25819371]. [PubMed Central ID: PMC4439928]. https://doi.org/10.1016/j.bbagen.2015.03.009.

  • 49.

    Araste F, Ebrahimizadeh W, Rasooli I, Rajabibazl M, Mousavi Gargari SL. A novel VHH nanobody against the active site (the CA domain) of tumor-associated, carbonic anhydrase isoform IX and its usefulness for cancer diagnosis. Biotechnol Lett. 2014;36(1):21-8. [PubMed ID: 24068505]. https://doi.org/10.1007/s10529-013-1340-1.

  • 50.

    Ibanez LI, De Filette M, Hultberg A, Verrips T, Temperton N, Weiss RA, et al. Nanobodies with in vitro neutralizing activity protect mice against H5N1 influenza virus infection. J Infect Dis. 2011;203(8):1063-72. [PubMed ID: 21450996]. https://doi.org/10.1093/infdis/jiq168.

  • 51.

    Hussack G, Arbabi-Ghahroudi M, van Faassen H, Songer JG, Ng KK, MacKenzie R, et al. Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. J Biol Chem. 2011;286(11):8961-76. [PubMed ID: 21216961]. [PubMed Central ID: PMC3058971]. https://doi.org/10.1074/jbc.M110.198754.

  • 52.

    Harmsen MM, van Solt CB, van Zijderveld-van Bemmel AM, Niewold TA, van Zijderveld FG. Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy. Appl Microbiol Biotechnol. 2006;72(3):544-51. [PubMed ID: 16450109]. https://doi.org/10.1007/s00253-005-0300-7.

  • 53.

    Dolk E, van der Vaart M, Lutje Hulsik D, Vriend G, de Haard H, Spinelli S, et al. Isolation of llama antibody fragments for prevention of dandruff by phage display in shampoo. Appl Environ Microbiol. 2005;71(1):442-50. [PubMed ID: 15640220]. [PubMed Central ID: PMC544197]. https://doi.org/10.1128/AEM.71.1.442-450.2005.

  • 54.

    Wrapp D, De Vlieger D, Corbett KS, Torres GM, Wang N, Van Breedam W, et al. Structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies. Cell. 2020;181(5):1004-1015 e15. [PubMed ID: 32375025]. [PubMed Central ID: PMC7199733]. https://doi.org/10.1016/j.cell.2020.04.031.

  • 55.

    Wu T, Liu M, Huang H, Sheng Y, Xiao H, Liu Y. Clustered nanobody-drug conjugates for targeted cancer therapy. Chem Commun. 2020;56(65):9344-7. [PubMed ID: 32672289]. https://doi.org/10.1039/d0cc03396k.

  • 56.

    Reck M, Rodriguez-Abreu D, Robinson AG, Hui R, Csoszi T, Fulop A, et al. Pembrolizumab versus chemotherapy for PD-L1-positive non-small-cell lung cancer. N Engl J Med. 2016;375(19):1823-33. [PubMed ID: 27718847]. https://doi.org/10.1056/NEJMoa1606774.

  • 57.

    Schmid P, Cortes J, Pusztai L, McArthur H, Kummel S, Bergh J, et al. Pembrolizumab for early triple-negative breast cancer. N Engl J Med. 2020;382(9):810-21. [PubMed ID: 32101663]. https://doi.org/10.1056/NEJMoa1910549.

  • 58.

    Marin-Acevedo JA, Dholaria B, Soyano AE, Knutson KL, Chumsri S, Lou Y. Next generation of immune checkpoint therapy in cancer: New developments and challenges. J Hematol Oncol. 2018;11(1):39. [PubMed ID: 29544515]. [PubMed Central ID: PMC5856308]. https://doi.org/10.1186/s13045-018-0582-8.

  • 59.

    Marin-Acevedo JA, Kimbrough EO, Lou Y. Next generation of immune checkpoint inhibitors and beyond. J Hematol Oncol. 2021;14(1):45. [PubMed ID: 33741032]. [PubMed Central ID: PMC7977302]. https://doi.org/10.1186/s13045-021-01056-8.

  • 60.

    Weber J. Immune checkpoint proteins: A new therapeutic paradigm for cancer--preclinical background: CTLA-4 and PD-1 blockade. Semin Oncol. 2010;37(5):430-9. [PubMed ID: 21074057]. https://doi.org/10.1053/j.seminoncol.2010.09.005.

  • 61.

    Zhang F, Wei H, Wang X, Bai Y, Wang P, Wu J, et al. Structural basis of a novel PD-L1 nanobody for immune checkpoint blockade. Cell Discov. 2017;3:17004. [PubMed ID: 28280600]. [PubMed Central ID: PMC5341541]. https://doi.org/10.1038/celldisc.2017.4.

  • 62.

    Ma L, Gai J, Qiao P, Li Y, Li X, Zhu M, et al. A novel bispecific nanobody with PD-L1/TIGIT dual immune checkpoint blockade. Biochem Biophys Res Commun. 2020;531(2):144-51. [PubMed ID: 32782142]. https://doi.org/10.1016/j.bbrc.2020.07.072.

  • 63.

    He F, Wen N, Xiao D, Yan J, Xiong H, Cai S, et al. Aptamer-based targeted drug delivery systems: Current potential and challenges. Curr Med Chem. 2020;27(13):2189-219. [PubMed ID: 30295183]. https://doi.org/10.2174/0929867325666181008142831.

  • 64.

    Cui Y, Cui P, Chen B, Li S, Guan H. Monoclonal antibodies: Formulations of marketed products and recent advances in novel delivery system. Drug Dev Ind Pharm. 2017;43(4):519-30. [PubMed ID: 28049357]. https://doi.org/10.1080/03639045.2017.1278768.

  • 65.

    Kooijmans SA, Aleza CG, Roffler SR, van Solinge WW, Vader P, Schiffelers RM. Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting. J Extracell Vesicles. 2016;5:31053. [PubMed ID: 26979463]. [PubMed Central ID: PMC4793259]. https://doi.org/10.3402/jev.v5.31053.

  • 66.

    Liu Y, Scrivano L, Peterson JD, Fens M, Hernandez IB, Mesquita B, et al. EGFR-targeted nanobody functionalized polymeric micelles loaded with mTHPC for selective photodynamic therapy. Mol Pharm. 2020;17(4):1276-92. [PubMed ID: 32142290]. [PubMed Central ID: PMC7140040]. https://doi.org/10.1021/acs.molpharmaceut.9b01280.

  • 67.

    van der Meel R, Oliveira S, Altintas I, Haselberg R, van der Veeken J, Roovers RC, et al. Tumor-targeted Nanobullets: Anti-EGFR nanobody-liposomes loaded with anti-IGF-1R kinase inhibitor for cancer treatment. J Control Release. 2012;159(2):281-9. [PubMed ID: 22227023]. https://doi.org/10.1016/j.jconrel.2011.12.027.

  • 68.

    Duggan S. Caplacizumab: First global approval. Drugs. 2018;78(15):1639-42. [PubMed ID: 30298461]. [PubMed Central ID: PMC6280848]. https://doi.org/10.1007/s40265-018-0989-0.

  • 69.

    Sadeghi A, Behdani M, Muyldermans S, Habibi-Anbouhi M, Kazemi-Lomedasht F. Development of a mono-specific anti-VEGF bivalent nanobody with extended plasma half-life for treatment of pathologic neovascularization. Drug Test Anal. 2020;12(1):92-100. [PubMed ID: 31476257]. https://doi.org/10.1002/dta.2693.

  • 70.

    Rahbarizadeh F, Ahmadvand D, Moghimi SM. CAR T-cell bioengineering: Single variable domain of heavy chain antibody targeted CARs. Adv Drug Deliv Rev. 2019;141:41-6. [PubMed ID: 31004624]. https://doi.org/10.1016/j.addr.2019.04.006.