Creative Biolabs has established a world-leading platform for single domain antibody discovery against membrane protein targets using our proprietary DNA immunization strategy. With over 10 years of experience in providing professional antibody phage display and antibody engineering services, we have become a well recognized leader in the fields of single domain antibody discovery and single domain antibody library. Our scientists are experienced in generating high affinity single domain antibodies against any targets, particularly integral membrane proteins in their native conformation.

Background

Camelid single domain antibodies (also known as sdAb and VHH) from camels, llamas and alpacas have been proven to be an excellent antibody format in cancer drug research. The small size and unusually long CDR3s have given them potential as diagnostic tools and for therapeutic applications. During the past years, therapeutic single domain antibodies against cancer-specific drug targets such as EGFR, HER2, and VEGFA have successfully been developed. There are also numerous reports on the isolation and characterization of VHH biomarkers specific to cancer, biochemical and blood diseases, neuroinflammation, and infectious pathogens. At present, a number of therapeutic single-domain antibodies are in clinical trials or are about to enter clinical trials. Nevertheless, it is still extremely difficult to obtain single domain antibodies against membrane protein targets, especially for multiple-spanning membrane embedded proteins that present the most important group of principal therapeutic targets, such as GPCRs, transporters, and ion channels.

Fig. 1 The single domain antibodies take effects in several ways against bacterial infection. (Qian Qin, 2022)

SdAbs Discovery Targeting Membrane Proteins

Creative Biolabs has developed a robust and highly effective strategy for generating single domain antibodies targeting membrane proteins. With rich successful experience in conducting DNA immunization on various species, our staff scientists are pleased to design the best fit immunization procedure for your interest membrane target to ensure maximum immune response, which incorporates an optimized boost schedule and electroporation; whole cell immunization as well as other unique methods will be adopted to enhance the immunization efficiency. Followed by high-throughput screening on cell lines expressing target membrane proteins in native conformation and subsequent analysis by FACS, we have successfully selected a great many potent single domain antibodies at the nanomolar or sub-nanomolar level, and several of them have inhibitory or antagonist activities. Besides developing single domain antibodies against membrane proteins, Creative Biolabs also provides a full range of single domain antibody services. We are especially experienced in single domain antibody affinity maturation and human or humanized single domain antibody production projects. In addition, we provide large scale production of recombinant single domain antibodies at the most competitive price.

Published Data

• Generate SdAbs against Membrane Proteins by cDNA Immunization Strategy

Fig. 2 Schematic diagram of the genetic immunization strategy to generate sdAbs against membrane proteins. (Thomas Eden, 2017)

Single domain antibodies have a great prospect as new therapeutic biological agents, and membrane protein is one of the most interesting targets for therapeutic sdAbs. Membrane proteins usually contain one or more hydrophobic transmembrane domains, so it is very difficult to obtain sdAbs targeting membrane proteins with natural conformations. Here, scientists describe a genetic immune strategy. That is, cDNA expression plasmids encoding one or more membrane proteins of interest and other costimulatory proteins were used to transfect skin cells, and the extracellular domain of membrane proteins was exposed on the surface of transfected cells. Repeated immunization drives the somatic hypermutation and affinity maturation of target-specific heavy chain antibodies. The VHH/sdAb coding region was amplified by PCR from B cells obtained from peripheral blood or lymph node biopsies. Specific sdAb was selected by phage display screening of sdAb-based heavy chain antibodies expressed as secretory proteins in transfected HEK cells. Using this strategy, the researchers successfully generated agonistic and antagonistic sdAbs for several extracellular enzymes and ligand gated ion channels on the cell surface.

• CD38-Specific sdAbs-Based Constructs

Fig. 3 Schematic illustration of the mode of action of the CD38-specific sdAb-based hcAbs, BiKEs, CARs and sdAb-displaying AAVs. (Julia Hambach, 2022)

Single-domain antibodies are very suitable for the construction of biological agents because of their high solubility. Scientists have generated single-domain antibodies against CD38 (a type II transmembrane glycoprotein whose extracellular domain is an enzyme that hydrolyzes NAD⁺). Then, based on these CD38-specific single domain antibodies, the researchers constructed heavy chain antibodies, bispecific killer cell engagers (BiKEs), chimeric antigen receptor (CAR)-NK cells, and sdAb display AAV vectors. These constructs are effective in specifically and effectively targeting myeloma cells expressing CD38. Further studies can evaluate the potential of these CD38-specific sdAbs-based constructs in the treatment of multiple myeloma.

FAQ

1. How do single domain antibodies interact with membrane proteins?

   The small size of single-domain antibodies enables them to come into contact with epitopes that traditional antibodies do not normally have access to, which makes them particularly useful for targeting membrane proteins. Membrane proteins embedded in the lipid bilayer of cells play an important role in various biological processes and are important targets for drug discovery. Single-domain antibodies can bind specific parts of these proteins with high affinity and specificity, thusmaking it possible to inhibit or alter the function of proteins in a targeted manner.

2. What are the advantages of using single-domain antibodies for membrane protein research and treatment?

   Size: Single-domain antibodies are smaller, can better penetrate dense tissue, and are closer to the target.
   Stability: single-domain antibodies are usually more stable than traditional antibodies, which makes it easier to operate.
   Specificity: they can be designed to show high specificity to unique epitopes on the target protein, thereby minimizing miss effects.
   Production: it can be produced in a microbial system, which is cheaper and faster than the mammalian cell culture needed for traditional antibodies.
   Versatility: they can easily connect to other molecules, such as cytotoxins or imaging reagents, to create efficient treatment or diagnostic tools.

3. What are the specific examples of the application of single-domain antibodies to target membrane proteins?

   In the study of cardiovascular diseases, single-domain antibodies are used to target and inhibit hyperactive cardiac ion channels, which play an important role in diseases such as arrhythmias. In cancer treatment, sdAbs can be used to accurately target specific proteins on the surface of tumor cells, such as overexpressed growth factor receptors, which helps to effectively deliver chemotherapeutic drugs or radioactive elements, thus improving the therapeutic effect and reducing the effect on normal cells. In addition, sdAbs is also used to study neurodegenerative diseases, such as Alzheimer's disease, to help researchers better understand the development of these diseases by targeting the accumulation of abnormal proteins in the brain.

4. How is the role of single domain antibodies enhanced in immunotherapy?

   The small size and high customization of single domain antibodies make them an ideal tool for enhancing immune responses. SdAbs can be designed to target specific immune cell surface molecules, thereby activating or inhibiting the specific function of immune cells. For example, some sdAbs are used to target and block the inhibitory PD-1/PD-L1 pathway, a common immune checkpoint that enhances the ability of T cells to attack cancer cells. In addition, sdAbs can also be used to construct multifunctional immune complexes, which can target multiple immune regulatory molecules at the same time, thus improving the effectiveness and scope of treatment.

Resources

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