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Preferential species-restricted heavy/light chain pairing in rat/mouse quadromas. Implications for a single-step purification of bispecific antibodies
Open accessAbstract
Conventional mouse/mouse or rat/rat hybrid-hybridoma supernatants contain up to 10 different IgG molecules consisting of various combinations of heavy and light chains. Hence, the yield of functional bispecific Ab is low, and purification is often complicated, hampering a general preclinical evaluation of, e.g., bispecific Ab-mediated tumor immunotherapy in animal models. In experiments to overcome this drawback we found that fusion of rat with mouse hybridomas opens the possibility of large scale production of bispecific Ab due to the increased incidence of correctly paired Ab and facilitated purification. In essence, rat/mouse quadroma-derived bispecific Ab have the following advantages: 1) enrichment of functional bispecific Ab because of preferential species-restricted heavy/light chain pairing (observed in four of four rat-mouse quadromas) in contrast to the random pairing in conventional mouse/mouse or rat/rat quadromas, and 2) a possible one-step purification of the quadroma supernatant with protein A. This simple chromatography step does not bind unwanted variants with parental rat/rat heavy chain configuration, and the desired rat/mouse bispecific Ab are retained, which can then easily be separated from parental mouse Ab by sequential pH elution.
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“By using an original subclass combination (mouse IgG2a and rat IgG2b), they demonstrated a preferential speciesrestricted heavy/light chain pairing, in contrast to the random pairing in conventional mouse/mouse or rat/rat quadromas, as well as use of sequential pH elution on protein A to easily separate the desired bsAb from the parental mAb.”
From Bispecific antibodies for cancer therapy: The light at the end of the tunnel? (2009) · cites this paper as [21]
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Therapeutic antibodies: successes, limitations and hopes for the future
Chames, P., Van Regenmortel, M., Weiss, E., & Baty, D. (2009) · British Journal of Pharmacology · doi:10.1111/j.1476-5381.2009.00190.x · Cited by 1,382
Bispecific antibodies: a mechanistic review of the pipeline
Labrijn, A. F., Janmaat, M. L., Reichert, J. M., & Parren, P. W. H. I. (2019) · Nature Reviews Drug Discovery · doi:10.1038/s41573-019-0028-1 · Cited by 1,303
The making of bispecific antibodies
Brinkmann, U., & Kontermann, R. E. (2017) · mAbs · doi:10.1080/19420862.2016.1268307 · Cited by 961
“Preferential pairing of light chains with its corresponding heavy chain was described.(141) Importantly, the heteromeric Fc part allows fractionated purification by protein A chromatography”
BackgroundMethodKontermann, R. E., & Brinkmann, U. (2015) · Drug Discovery Today · doi:10.1016/j.drudis.2015.02.008 · Cited by 603
Nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics
Bannas, P., Hambach, J., & Koch-Nolte, F. (2017) · Frontiers in Immunology · doi:10.3389/fimmu.2017.01603 · Cited by 572
“, rat IgG2a and rat lambda on the one side and mouse IgG2b and mouse kappa on the other side, reduces mispairing of the VL and VH domains (50).”
Background
Metadata
- Authors
- Lindhofer, H., Mocikat, R., Steipe, B., & Thierfelder, S.
- Affiliations
- Helmholtz Munich (DE)
- ORCID
- Venue
- The Journal of Immunology
- Publisher
- Oxford University Press (OUP)
- Published
- 1995-07-01
- Volume
- 155
- Issue
- 1
- Pages
- 219-225
- ISSN
- 0022-1767 (ISSN-L)
- 1550-6606
- OpenAlex
- W1921660070
- Cited by
- 213
- References
- 0
- Citations by year
- 2026: 3 · 2025: 2 · 2024: 4 · 2023: 7 · 2022: 12 · 2021: 10 · 2020: 4 · 2019: 14 · 2018: 7 · 2017: 16 · 2016: 11 · 2015: 11 · 2014: 17 · 2013: 18 · 2012: 13
- FWCI
- 2.10
- Type
- journal article
- Language
- en
- Open access
- Locations
- The Journal of Immunology (publishedVersion)
- PubMed (publishedVersion)
- Site cant be reached (submittedVersion, other-oa)
- PuSH - Publication Server of Helmholtz Zentrum München (submittedVersion)
- Topics
- Life Sciences › Immunology and Microbiology › Immunology › Monoclonal and Polyclonal Antibodies Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Health Sciences › Medicine › Hematology › Multiple Myeloma Research and Treatments
- Keywords
- bispecific antibodies, hybridoma fusion, antibody purification, protein A chromatography, cancer immunotherapy, monoclonal antibody production
- MeSH terms
- Animals, Chromatography, Ion Exchange, Enzyme-Linked Immunosorbent Assay, Flow Cytometry, Hybridomas, Immunoglobulin Heavy Chains, Immunoglobulin Light Chains, Mice, Inbred AKR, Nerve Tissue Proteins, Rats, Inbred Strains, Recombinant Fusion Proteins, Species Specificity, Staphylococcal Protein A, Immunoblotting, Rats, Sprague-Dawley, Antibodies, Bispecific, Mice, Rats
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