Rabbit DimAb Development
Our team has decades of experience on high quality monoclonal antibody development services. DIMA’s DimAbTM development platform is a revolutionary technology platform for monoclonal antibody development. Different from hybridoma fusion platform, we can directly isolate IgG genes from B cells of immunized animals. At present, we have successfully completed a number of custom development projects with high satisfaction (Please see our customer testimonials).
Comparing with other animals, the rabbit has a unique B cell development process and is more distant from human than rodent. Besides these, rabbit B cells utilize a dual affinity maturation mechanism, including gene conversion and SHM (somatic hyper-mutation). At the same time, the rabbit IgG has a unique protein structure different from the mouse IgG, including one subtype of IgG, more noncanonical C-C for light chain, great variation in length and sequence for CDR3, etc. These differences might make rabbit produce antibodies with high affinity and wide diversity. In recent years, rabbit monoclonal antibodies are getting popular in different immunological assay development, especially for immunohistochemistry application. So far, 5 FDA approved companion diagnostic IHC antibodies are rabbit monoclonal antibodies (Download). In the past, rabbit monoclonal development technology required a patent protected rabbit myeloma cell line, which limited its applications in many areas. Right now, DIMA’s new proprietary DimAbTM technology platform provides a great technology breakthrough in this area. Without using myeloma cells, we can directly isolate IgG genes from positive B cell clones. This will not only enable us shorten the development time, but also directly acquire the genetic coding information of IgG without worrying of losing hybridoma cells.
|Comparison Items||Rabbit Monoclonal Antibody||Mouse Monoclonal Antibody|
|B cell antibody spectrum||V (D) J recombination; continued development in Appendix or other gut associated lymphoid tissues, continued to increase the diversity of antibody spectrum and the size of antibody library through very slow gene conversion||V (D) J arrangement|
|The mechanism of antibody maturation||Gene conversion + hypermutation||Hypermutation|
|CD1 Family||Five CD1 isoforms have strong immune responses to antigens such as lipids and glycolipids; recognize polypeptides and small molecules||One CD1d; many small molecular compounds and polypeptides do not cause a good immune response in mice|
|IgG Type||One IgG with more disulfide bonds, more complex CDR region and light chain structure||IgG1, IgG2a, IgG2b, IgG2c, IgG3|
|Species response||Stronger response to human and rodent antigens||Weak response to rodent antigens|
|Epitope recognition||Antibodies can recognize multiple epitopes of antigens and have advantages in post-translational modification (such as phosphorylation, methylation, acetylation, ubiquitination like)||Limited epitope recognition by antibody|
|Affinity||The Kd value (equilibrium dissociation constant) can usually reach the picomore level (Kd = 10-12M)||Kd value is in the range of nanomolar (Kd = 10-9M)|
|Production||The recombinant expressed antibody is stable between batches, and the automatic preparation process can be established||The antibody secreted by hybridoma is unstable, which is easy to cause the loss of antibody gene|
|Store||The antibody gene sequence is obtained directly, and the antibody information is easy to store and transmit||The cost of hybridoma cell preservation is very high, which requires a lot of liquid nitrogen and cell storage equipment|
|Application||WB, ELISA, FC, IP, IHC, ICC (obvious advantages in IHC)||WB, ELISA, FC, IP have no obvious advantages in IHC and ICC|
Application in clinical diagnosis Recent evidences indicated that rabbit monoclonal antibody has clear advantages in immunohistochemistry application. Currently rabbit monoclonal antibodies become the favorite choices for pathologists. At present, FDA has approved 11 rabbit monoclonal antibodies for diagnosis, including companion diagnostic IHC antibodies for anti-PD-1 and anti-PD-L1 immunotherapy drug treatment, 4B5 clone for Herceptin treatment, etc.
|Concomitant diagnostic reagents||Antibody drug||Cancer||Detection platform|
|PATHWAY ANTI-HER-2/NEU (4B5)||HERCEPTIN
|VENTANA ALK (D5F3) CDx Assay||XALKORI
|Non-small cell lung cancer||IHC|
|PD-L1 IHC 28-8||OPDIVO
|Melanoma Non-small cell lung cancer||IHC|
|VENTANA PD-L1 (SP142) Assay||TECENTRIQ
|Bladder cancer Non-small cell lung cancer||IHC|
|VENTANA PD-L1 (SP263) Assay||KEYTRUDA®
Application on therapeutic antibody drug development On October 8th, 2019, the first rabbit monoclonal antibody derived drug Beovu® was approved by FDA for the treatment of wet age-related macular degeneration (AMD). Beovu® is a variable region fragment of humanized rabbit monoclonal antibody scFv targeting VEGF. This is also the 83rd antibody drug approved by FDA. Compared with other antibodies, rabbit recombinant monoclonal antibodies have higher affinity, which can not only reduce the clinical dosage of antibodies, but also reduce the side effects caused by the use of a large dosage.
Application in scientific research With the advantages of high specificity and high affinity, rabbit monoclonal antibodies have been widely used in life science research. At present, rabbit monoclonal antibodies have been widely used as reagents for immunoblotting, immunohistochemistry, flow cytometry and other immunology research. Due to its unique B cell development system, rabbit monoclonal antibodies have exhibited special strength in detecting subtle difference on small epitopes, such as post-translational modified amino acids within a protein.
|Step||Service details||Deliverables||Estimated production cycle (week)|
|Immunogen preparation||Plasmid construction, mammalian cell produced immunogens or supplied by customer||7-10 Weeks|
|Animal immunization||Immunize two rabbits against the same antigen||Crude sera and ELISA data on anti-serum titer test||5-8 Weeks|
|B cell isolation and amplification||PBMC collection and cryopreservation; target positive B cell enrichment and single B cell expansion||100ul B cell supernatant;B cell supernatant ELISA test data||2-3 Weeks|
|DimAbTM cDNA cloning, sequencing and storage||IgG gene cloning, Expression vector construction; small-scale antibody production and validation; select 1-2 positive clones for full-length IgG sequencing and sequence analysis||Sequencing report of one clone; ELISA data||3-4 Weeks|
|DimAbTM production and validation||Antibody production, purification and QC validation||1 mg purified recombinant antibody, antibody QC data||3-4 Weeks|
The whole DimAbTM development procedure took around 3.5 months
(delivered 8 flow working rabbit DimAbTMs to customer (Bioraid, a CAR-T company))