Background

Single domain antibodies are antibody fragments consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With beneficial pharmacologic and pharmacokinetic properties, they are ideally suited to targeting cellular antigens for molecular imaging or therapeutic purposes. However, because of their camelid or nonhuman origin, the possible immunogenicity of single domain antibodies when used in the clinic is a concern.

Fig. 1 Schematic overview of the single domain antibody humanization strategy. (Cécile Vincke, 2009)

Human or Humanized SdAb Production Services

Creative Biolabs has extensive experience in generating human single domain antibodies and humanizing single domain antibodies. We have developed synthetic human single domain antibody phage display libraries using humanized single domain antibody scaffolds. Human single domain antibodies against all targets can be rapidly isolated from these libraries. In addition, by immunizing a unique strain of transgenic mice available from a partner that harbors human single domain antibody gene repertoires, Creative Biolabs is able to produce immune human single domain antibody libraries that can produce high affinity human single domain antibodies against the immunogens.

Single domain antibody humanization services are also available at Creative Biolabs. In the first straightforward approach that employs soluble, stable, well expressed universal humanized single domain antibody scaffolds, CDR regions of a parental single domain antibody can be directly grafted with the antigen-specificity and affinity of the parental antibody transferred. Regular CDR grafting plus the back-mutation method is also frequently used to humanize single domain antibodies at Creative Biolabs.

Published Data

• Humanized CD7 sdAb-based Immunotoxins Development

Fig. 2 In vivo evaluation of dhuVHH6-PE38 in CEM xenograft tumor model. (Yuan Yu, 2017)

The researchers humanized previously produced monovalent and bivalent CD7 single-domain antibody-based immunotoxins that effectively trigger apoptosis in CD7-positive malignant cells, and further truncated PE38 toxins derived from Pseudomonas exotoxin A (PE). Three new immunotoxins, dhuVHH6-PE38, dVHH6-PE-LR, and dhuVHH6-PE-LR, were successfully constructed. The results showed that all immunotoxins maintained the ability to specifically bind to CD7 positive T lymphocytes but not to CD7 negative control cells. These proteins can be endocytosed into the cytoplasm after binding to CD7-positive cells, but this phenomenon was not observed in CD7-negative cells. The WST-8 assay showed that all immunotoxins retained efficient and specific growth inhibitory activity against CD7 positive cell lines and primary T-cell acute lymphoblastic leukemia (T-ALL) cells. In vivo animal model experiments showed that humanized dhuVHH6-PE38 immunotoxin could tolerate higher doses and prolong the survival time of mice transplanted with CEM cells.

• Anti-CD72 Humanized SdAb CAR-T Cells Development

Fig. 3 In vitro 24-hour cytotoxicity assays comparing H24, NbD4, and CD19 CAR-T cells against SEM and JeKo-1. (Ying Fu, 2022)

CD72 is described as a promising target for B-cell malignant tumors. The scientists humanized the single-domain antibody framework region derived from the llama to produce a series of humanized anti-CD72 sdAbs. These sdAbs binders were inserted into second-generation CD72 CAR-T cells and evaluated for preclinical models of B-cell acute lymphoblastic leukemia and B-cell non-Hodgkin's lymphoma in vitro and in vivo. Subsequently, the researchers found a clone (H24) that could enhance the efficacy of B-cell tumors. Compared with the full llama framework, H24 has a slightly higher binding affinity for CD72, which may be the basis for improving effectiveness. After treatment with H24 nanoCARs, recurrence in vivo was accompanied by a down-regulation of CD72 antigen, which was partially reversible. The H24 single-domain antibody clone was found to have no off-target binding, so it was designated as a real clinical candidate.

FAQ

1. What is a humanized single domain antibody?

   A humanized single domain antibody is an engineering version of sdAb, modified to improve its compatibility with the human immune system. Humanization involves modifying the amino acid sequences of sdAb to make them more similar to human antibody sequences, thereby reducing their immunogenicity. This process is important for therapeutic applications because it minimizes the risk of adverse immune reactions, such as the production of anti-drug antibodies (ADAs), that neutralize therapeutic effects or cause other complications.

2. What is the therapeutic application of human and humanized single domain antibodies?

   Oncology: sdAbs is small in size and can penetrate tumors more effectively, thus accurately targeting cancer cells or the tumor microenvironment.
   Infectious diseases: sdAbs can bind to conserved regions of viruses or bacteria, making them excellent candidates for neutralizing sources of infection.
   Autoimmune and inflammatory diseases: humanized sdAb can be designed to block cytokines or cell surface receptors involved in autoimmune and inflammatory processes.
   Neurological diseases: sdAbs has the potential to cross the blood-brain barrier and can be used to target pathological proteins related to diseases such as Alzheimer's or Parkinson's disease.

3. How are human or humanized single domain antibodies developed?

   Selection of targets and initial antibody production.
   Sequencing and humanization.
   Synthesis and expression of humanized antibodies.
   Functional and immunogenicity tests.

Resources

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