Table 4.

Obstacles to clinical translation.

Consideration	Reason	Potential solution
Clinical trial attrition	Failure rates of therapeutic candidates generally increase with each trial phase due to poor translation from preclinical models.	→ Reduce and refine animal-based models → Enhance in vitro and in silico methods
Administration/Dosing	The short serum half-life of unmodified Nbs requires frequent and concentrated IV delivery for therapeutic applications, which increases the risk of renal toxicity.	→ Extend serum half-life through albumin-tagging, Fc-domain fusion, PEGylation, multimerization → Alternative delivery methods: SCs, viral vectors, programmable bacteria, intracellular delivery, intra-arterial delivery (for BBB)
Immunogenicity	As Nbs come from camelids, they possess a low risk of triggering an immune resposne.	→ Humanization of Nbs → Developing different idiotypes for a specific Nb → Developing “camelized,” fully human HcAbs (substituted hydrophilic residues into hydrophobic regions)
Functionalization	Modification of Nbs to conjugate other molecules or build more complex constructs might compromise their original functionality.	→ Site-selective Nb functionalization
On-Target/On-Tumor cytotoxicity	Excessive targeting could cause adverse effects such as cytokine release syndrome and tumor lysis syndrome.	→ “Safety-switch”/Suicide gene therapy → Separating out initial dosing regimen
On-Target/Off-Tumor cytotoxicity	Tumor antigen could also be expressed on non-malignant cells and cause damage to healthy tissue.	→ “Safety-switch”/Suicide gene therapy → Bispecific activation → Improve imaging of patient's tumor antigen profile to determine toxicity threshold
Reaching clinical-grade efficacy	Success in preclinical models is not necessarily indicative of therapeutic efficacy in human patients	→ Improve Nb affinity maturation → Improve Nb orientation → Enhance ADCC
Quality control	Ensure that Nbs are homogenous to avoid variability in functionality and risk adverse effects	→ Good manufacturing practices for microbial-based Nb production

Nbs, nanobodies; IV, intravenous; SCs, stem cells; BBB, blood brain barrier; Fc, fragment crystallizable; HcAb, heavy-chain antibodies; ADCC, antibody-dependent cellular cytotoxicity.