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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Using an original subclass combination of mouse IgG2a and rat IgG2b, Lindhofer and collaborators demonstrated preferential species-restricted heavy/light chain pairing, in contrast to random pairing in conventional quadromas, and sequential pH elution on protein A to separate the desired bispecific antibody from parental monoclonal antibody.
“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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“Early efforts to address both challenges exploited the unique pairing preferences of rat and mouse IgG2 heavy and light chains (Figure 8) (128).”
Zeidler, R., Reisbach, G., Wollenberg, B., Lang, S., Chaubal, S., Schmitt, B., & Lindhofer, H. (1999) · The Journal of Immunology · doi:10.4049/jimmunol.163.3.1246 · Cited by 204 · see citing works
Self-citationProgress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies
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“Aside from the strong immunogenicity, which is a major drawback, the use of mouse and rat antibodies has three major technical advantages: (1) Preferential intra-species heavy-light chain pairing resulting in a reduction of mispairing; (2) efficient heterologous heavy chain pairing; and (3) different affinities of the…”
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“Initially, the mispairing problem of heavy/light chains was approached by creating a chimeric rat/mouse-derived quadroma in 1995, which guaranteed the correct pairing of cognate heavy chains.(24) For instance, the clinically approved catumaxomab was made up of a murine IgG2a anti-CD3 hapten paired with a rat IgG2b ant…”
BackgroundKiewe, P. M., Hasmüller, S., Kahlert, S., Heinrigs, M., Rack, B. K., Marmé, A., Korfel, A., Jäger, M., Lindhofer, H., Sommer, H. L., et al. (2006) · Clinical Cancer Research · doi:10.1158/1078-0432.ccr-05-2436 · Cited by 198 · see citing works
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“The BiUII Quadroma was produced as previously described (Lindhofer et al, 1995).”
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“matography (Protein A) and ionic exchange chromatography are always required to obtain homogeneous bispecific antibody preparations (Lindhofer et al., 1995).”
BackgroundEngineering antibodies for clinical applications
Jain, M., Kamal, N., & Batra, S. K. (2007) · Trends in biotechnology · doi:10.1016/j.tibtech.2007.05.001 · Cited by 129 · see citing works
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BackgroundZheng, J., Zhao, S., Yu, X., Huang, S., & Liu, H. Y. (2017) · Theranostics · doi:10.7150/thno.17826 · Cited by 120 · see citing works
“also suffered by high immunogenicity and difficult production involved in complicated technologies such as hybrid-hybridoma [22] and genetic engineering”
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Heiss, M. M., Ströhlein, M. A., Jäger, M., Kimmig, R., Burges, A., Schoberth, A., Jauch, K., Schildberg, F., & Lindhofer, H. (2005) · International Journal of Cancer · doi:10.1002/ijc.21165 · Cited by 117 · see citing works
“The intact antibodies not only activate T cells via CD3, but simultaneously bind and stimulate Fcc receptor type I and III1 accessory cells like e.g., monocytes/macrophages, NK cells and dendritic cells via their special Fc-region.4,6 trAb consist of the 2 potent and evolutionary related subclasses mouse IgG2a and rat…”
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Liu, H.-Y., Saxena, A., Sidhu, S., & Wu, D.-H. (2017) · Frontiers in Immunology · doi:10.3389/fimmu.2017.00038 · Cited by 109 · see citing works
“Two additional bsAbs produced using the quadroma method, HER2 × CD3 (triomab; ertumaxomab) and CD20 × CD3 (triomab; FBTA05), are being evaluated in clinical trials for cancer treatment (25, 26).”
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“The advent of monoclonal Ab (mAb) technology allowed the development of BsAb obtained from quadromas (hybrid-hybridomas) [4,5] or based on the chemical conjugation of Fab fragments obtained after pepsinization of hybridomaderived mAb [6,7].”
Ruf, P., Gires, O., Jäger, M., Fellinger, K., Atz, J., & Lindhofer, H. (2007) · British Journal of Cancer · doi:10.1038/sj.bjc.6603881 · Cited by 102 · see citing works
“Catumaxomab (removabs), the trAb variant of HO-3 with a dual specificity for EpCAM and CD3 and the trifunctional control antibody Bi20 (antiCD20 anti-CD3) were manufactured using the quadroma technology (Lindhofer et al, 1995) at TRION Pharma (Munich, Germany) under GMP conditions.”
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“Over the following decades, many different methods within the constraints of the classical “IgG-like” format were devised to overcome or circumvent the “mispairing” problem of heavy and light chains produced by quadromas or genetic engineering (Figure 1c): rat/mouse quadromas, which took advantage of the species-restr…”
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“Therefore, through the purification by protein A, the H chain mispairing problem is also solved [6].”
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“On the other hand, the fusion of two single-species hybridomas leads to the production of mismatched antibodies that cannot fulfill the expected functions [21].”
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Self-citationStepping forward: T-cell redirecting bispecific antibodies in cancer therapy
Qin, X., Ning, W., Liu, H., Liu, X., Luo, W., & Xia, N. (2024) · Acta Pharmaceutica Sinica B · doi:10.1016/j.apsb.2024.03.027 · Cited by 59 · see citing works
“1 and 2, and Table 1) 11 e 15 .”
Engineering bispecific antibodies with defined chain pairing
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World Bispecific Antibody Summit, September 27–28, 2011, Boston, MA
Dhimolea, E., & Reichert, J. M. (2012) · mAbs · doi:10.4161/mabs.4.1.18821 · Cited by 56 · see citing works
Ertumaxomab: a trifunctional antibody for breast cancer treatment
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Two new trifunctional antibodies for the therapy of human malignant melanoma
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Self-citationTherapeutic bispecific antibody formats: a patent applications review (1994-2017)
Godar, M., de Haard, H., Blanchetot, C., & Rasser, J. (2018) · Expert Opinion on Therapeutic Patents · doi:10.1080/13543776.2018.1428307 · Cited by 52 · see citing works
“The rat/mouse Triomab quadroma technology (Frenesius Biotech, Trion Pharma) for which a patent application was filed in 1994, solved both LC/HC and HC/HC mispairing problems (Figure 3Aa and Table 1) [13,29].”
BackgroundMethodWang, C., Vemulapalli, B., Cao, M., et al. (2018) · mAbs · doi:10.1080/19420862.2018.1511198 · Cited by 53 · see citing works
“One manufacturing process used in past generates a hybrid-hybridoma (quadroma) by fusing two antibody-producing cell lines, which allows the combination of the heavy and light chains of two different antibodies.(11,16) This process was used for the production of catumaxomab (Removab), a bispecific antibody therapeutic…”
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Bispecific Antibodies and Trispecific Immunocytokines for Targeting the Immune System Against Cancer
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“TrAb are artificially engineered immunoglobulins with two different Fab-binding sites and an intact Fc-region [11] and represent a novel antibody concept [12].”
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“Previous to our work, asymmetric Protein A-binding strategies for Hc homodimer removal have not been used in combination with a CH3 HD technology but have been successfully used by other groups as stand-alone techniques (15, 32, 33).”
LaFleur, D. W., Abramyan, D., Kanakaraj, P., Smith, R. G., Shah, R. R., Wang, G., Yao, X.-T., Kankanala, S., Boyd, E., Zaritskaya, L., et al. (2013) · mAbs · doi:10.4161/mabs.23043 · Cited by 48 · see citing works
SITE-SPECIFIC FUNCTIONALIZATION OF PROTEINS AND THEIR APPLICATIONS TO THERAPEUTIC ANTIBODIES
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Test System for Trifunctional Antibodies in 3D MCTS Culture
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Emerging classes of armed antibody therapeutics against cancer
Hess, C., Venetz, D., & Neri, D. (2014) · MedChemComm · doi:10.1039/c3md00360d · Cited by 47 · see citing works
Catumaxomab – trifunctional anti-EpCAM antibody used to treat malignant ascites
Bokemeyer, C. (2010) · Expert Opinion on Biological Therapy · doi:10.1517/14712598.2010.504706 · Cited by 47 · see citing works
“2 Mode of action Catumaxomab was constructed as a quadroma of a heavy and a light chain of a rat IgG2b antibody against human CD3 and a heavy and a light chain of a mouse IgG2a antibody directed to human EpCAM [20].”
Background“NextGen” Biologics: Bispecific Antibodies and Emerging Clinical Results
Thakur, A. B., & Lum, L. G. (2016) · Expert Opinion on Biological Therapy · doi:10.1517/14712598.2016.1150996 · Cited by 46 · see citing works
Krah, S., Schröter, C. J., Eller, C., et al. (2017) · Protein Engineering Design and Selection · doi:10.1093/protein/gzw077 · Cited by 46 · see citing works
“Several methodologies for specific heavy chain heterodimerization have been described so far, including quadroma technology (Lindhofer et al., 1995), knobs into holes (Ridgway et al., 1996), strand-exchanged engineered domains (SEED) (Davis et al., 2010; Muda et al., 2011) and others.”
BackgroundDevelopment of Bispecific Antibody for Cancer Immunotherapy: Focus on T Cell Engaging Antibody
Moon, D., Tae, N., Park, Y., Lee, S., & Kim, D. H. (2022) · Immune Network · doi:10.4110/in.2022.22.e4 · Cited by 46 · see citing works
Timm, V., Gruber, P., Wasiliu, M., Lindhofer, H., & Chelius, D. (2010) · Journal of Chromatography B · doi:10.1016/j.jchromb.2010.01.036 · Cited by 43 · see citing works
Self-citationTrifunctional Bispecific Antibodies Induce Tumor-Specific T Cells and Elicit a Vaccination Effect
Eißler, N., Ruf, P., Mysliwietz, J., Lindhofer, H., & Mocikat, R. (2012) · Cancer Research · doi:10.1158/0008-5472.can-12-0146 · Cited by 41 · see citing works
“Surek was generated by quadroma technology and purified as described previously (22).”
BackgroundMethodInfluentialSelf-citationResearch and Clinical Landscape of Bispecific Antibodies for the Treatment of Solid Malignancies
Antonarelli, G., Giugliano, F., Corti, C., Repetto, M., Tarantino, P., & Curigliano, G. (2021) · Pharmaceuticals · doi:10.3390/ph14090884 · Cited by 44 · see citing works
“, catumaxomab) exploit species-restricted heavy/light chain pairing [28], whereas FAEs (i.”
BackgroundNatsume, A., Wakitani, M., Yamane‐Ohnuki, N., Shoji-Hosaka, E., Niwa, R., Uchida, K., Satoh, M., & Shitara, K. (2006) · The Journal of Biochemistry · doi:10.1093/jb/mvj157 · Cited by 42 · see citing works
Labrijn, A. F., Meesters, J. I., Bunce, M. W., Armstrong, A. A., Somani, S., Nesspor, T., Chiu, M. L., Altıntaş, I., Verploegen, S. A. B. W., Schuurman, J., et al. (2017) · Scientific Reports · doi:10.1038/s41598-017-02823-9 · Cited by 42 · see citing works
The intriguing options of multispecific antibody formats for treatment of cancer.
Weidle, U. H., Tiefenthaler, G., Weiss, E. H., Georges, G. J., & Brinkmann, U. (2013) · PubMed · Cited by 40 · see citing works
Stamova, S., Cartellieri, M., Feldmann, A., Arndt, C., Koristka, S., Bartsch, H., Bippes, C. C., Wehner, R., Schmitz, M., Bonin, M. V., et al. (2011) · Molecular Immunology · doi:10.1016/j.molimm.2011.09.019 · Cited by 39 · see citing works
Morecki, S., Lindhofer, H., Yacovlev, E., Gelfand, Y., Ruf, P., & Slavin, S. (2008) · Experimental Hematology · doi:10.1016/j.exphem.2008.03.005 · Cited by 38 · see citing works
Self-citationTaking up Cancer Immunotherapy Challenges: Bispecific Antibodies, the Path Forward?
del Bano, J., Chames, P., Baty, D., & Kerfélec, B. (2015) · Antibodies · doi:10.3390/antib5010001 · Cited by 38 · see citing works
“has considerably improved the classical quadroma approach for producing bsAbs [60].”
BackgroundInfluentialDevelopment of bispecific antibodies in China: overview and prospects
Zhang, J., Yi, J., & Zhou, P. (2020) · Antibody Therapeutics · doi:10.1093/abt/tbaa011 · Cited by 38 · see citing works
“The advances in protein engineering and bioprocesses based on mammalian cells for over expression and cost-effective production of a typical bsAb have allowed many start-up companies in China to bypass the early developed quadroma technology [14,178,179] or even latterly improved chimeric quadroma technology [180,181]…”
BackgroundRECRUIT-TandAbs ® : Harnessing the Immune System to Kill Cancer Cells
McAleese, F., & Eser, M. (2012) · Future Oncology · doi:10.2217/fon.12.54 · Cited by 36 · see citing works
Kiefer, J. D., & Neri, D. (2016) · Immunological Reviews · doi:10.1111/imr.12391 · Cited by 36 · see citing works
“Catumaxomab is a hybrid between a mouse IgG2a and a rat IgG2a and is produced in mouse–rat quadromas (85).”
BackgroundSchäfer, W. M., Völger, H. R., Lorenz, S. H., Imhof-Jung, S., Regula, J. T., Klein, C., & Mølhøj, M. (2015) · mAbs · doi:10.1080/19420862.2015.1111498 · Cited by 35 · see citing works
“To address this problem, Lindhofer et al.(7) developed the rat/mouse bsAbs (Triomabs) with species-restricted HC and LC chain pairing and thereby reduced the mismatch variants to <10%.”
Efficacy of catumaxomab in tumor spheroid killing is mediated by its trifunctional mode of action
Hirschhaeuser, F., Walenta, S., & Mueller‐Klieser, W. (2010) · Cancer Immunology Immunotherapy · doi:10.1007/s00262-010-0894-1 · Cited by 33 · see citing works
“Catumaxomab (anti-EpCAM 9 anti-CD3) which was produced using the quadroma technology [22] out of the parental antibodies 26/II/6 (rat IgG2b, anti-human CD3) and HO-3 (mouse IgG2a, anti-human EpCAM), BiLu (antihuman EpCAM 9 anti-mouse CD3, mouse IgG2a 9 rat IgG2b) and F(ab0)2 fragment of catumaxomab were kindly supplie…”
MethodImmunotherapeutic progress and application of bispecific antibody in cancer
Kang, J., Sun, T., & Zhang, Y. (2022) · Frontiers in Immunology · doi:10.3389/fimmu.2022.1020003 · Cited by 34 · see citing works
“Catumaxomab consists of mouse IgG2 and rat IgG2b and targets CD3 on T cells and EpCAM on tumor cells (74).”
BackgroundSingle domain antibody-based bispecific antibody induces potent specific anti-tumor activity
Li, J., Zhou, C., Dong, B., Zhong, H., Chen, S., Li, Q., & Wang, Z. (2016) · Cancer Biology & Therapy · doi:10.1080/15384047.2016.1235659 · Cited by 32 · see citing works
Stamova, S., Feldmann, A., Cartellieri, M., Arndt, C., Koristka, S., Apel, F., Wehner, R., Schmitz, M., Bornhäuser, M., Bonin, M. V., et al. (2012) · Analytical Biochemistry · doi:10.1016/j.ab.2011.12.042 · Cited by 30 · see citing works
Azadi, A., Golchini, A., Delazar, S., Kahaki, F. A., Dehnavi, S. M., Payandeh, Z., & Eyvazi, S. (2021) · Biological Procedures Online · doi:10.1186/s12575-021-00147-7 · Cited by 31 · see citing works
“Catumaxomab is the first BsAb that generate and approved with this method [91, 92].”
The manufacturing considerations of bispecific antibodies
Underwood, D. J., Bettencourt, J., & Jawad, Z. (2022) · Expert Opinion on Biological Therapy · doi:10.1080/14712598.2022.2095900 · Cited by 31 · see citing works
“In addition, the quadroma technology does lead to mispairing of the HC and LC and up to nine different species have been detected [13,47].”
BackgroundMethodEngineering Antibodies for Cancer Therapy
Boder, E. T., & Jiang, W. (2011) · Annual Review of Chemical and Biomolecular Engineering · doi:10.1146/annurev-chembioeng-061010-114142 · Cited by 31 · see citing works
“Interestingly, fusion of mouse and rat hybridomas was found to yield predominantly interspecies paired IgG under certain conditions; thus, simple generation of mouse/rat hybrid bsAbs was achieved, and the resulting bsAbs were found to induce surprisingly robust stimulation of human FcγRI and FcγRIII via the murine/rat…”
BackgroundRiechelmann, H., Wiesneth, M., Schauwecker, P., Reinhardt, P., Gronau, S., Schmitt, A., Schroen, C., Atz, J., & Schmitt, M. W. (2007) · Cancer Immunology Immunotherapy · doi:10.1007/s00262-007-0283-6 · Cited by 30 · see citing works
Wang, F., Tsai, J. C., Davis, J. H., et al. (2019) · mAbs · doi:10.1080/19420862.2019.1685350 · Cited by 29 · see citing works
- 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
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › 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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