Immunoglobulins Secreted by a Hybrid-Hybridoma: Analysis of Chain Assemblies

Smith, William K.; Jarrett, A.L.; Beattie, Ruth E.; Corvalan, J. R. F.

doi:10.1089/hyb.1992.11.87

Cited by 16 publications

(6 citation statements)

References 18 publications

(35 reference statements)

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“…Catumaxomab (trade name Removab), which has been approved as a therapeutic drug, is produced on this basis using a sophisticated purification method. 23 Since Milstein and Cuello's original publication, 4 graphical representations of the prediction of 10 species in the quadroma approach based on statistical arguments (4 in the case of KiH) can be found in many publications about bsAbs (e.g., Suresh et al 5, 24 Moldenhauer, 6 Smith et al, 25 Tada et al 26 ). However, to our knowledge, this theoretical prediction has never been experimentally verified.…”

Section: Introductionmentioning

confidence: 99%

Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry

Schaefer

Völger

Lorenz

et al. 2015

mAbs

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(2016) Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry, mAbs, 8:1, 49-55, DOI: 10.1080/19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 Keywords: bispecific antibody, CrossMab, chain pairing, ESI-QTOF mass spectrometry, knobs-into-holes, quadromaAbbreviations: Ang2, angiopoietin-2; bsAbs, bispecific antibodies; ESI, electrospray ionization; GuA HCl, guanidine hydrochloride; HEK, human embryonic kidney; HC, heavy chain; IgG1, immunoglobulin G1; ISCID, ion source collision induced dissociation; KiH, knobs-into-holes; LC, light chain; MS, mass spectrometry; TCEP, tris(2-carboxyethyl)phosphine; UHR, ultrahigh-resolution; VEGF-A, vascular endothelial growth factor A; QTOF, quadrupole time-of-flightThe quadroma antibody represents the first attempt to produce a bispecific heterodimeric IgG antibody by somatic fusion of 2 hybridoma cells each expressing monoclonal antibodies with distinctive specificities. However, because of random heavy and light chain pairing, the desired functional bispecific antibody represents only a small fraction of the protein produced. Subsequently, the knobs-into-holes (KiH) approach was developed to enforce correct heavy chain heterodimerization. Assuming equimolar expression of 4 unmodified chains comprising 2 heavy and 2 light chains, the statistical distribution of all paired combinations can be calculated. With equimolar expression as the goal, we transfected HEK cells with 1:1:1:1 plasmid ratios and analyzed the protein A affinity-purified antibodies from the quadroma and KiH approaches qualitatively and quantitatively with regard to the estimated relative amounts of the products using electrospray quadrupole time-of-flight mass spectrometry. Our results show that all expected species are formed, and that, within the methodological limits, the species distribution in the mixtures corresponds approximately to the statistical distribution.

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Section: Introductionmentioning

confidence: 99%

Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry

Schaefer

Völger

Lorenz

et al. 2015

mAbs

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“…The resulting molecules are a combination of the heavy and light chains of the two antibodies (Figure 1). When hybridomas from two different species are fused, light and heavy chains of the same species are preferentially bound [22]. Such cell lines hardly produce antibodies with chain mismatches.…”

Section: Recombinant Antibodies In the Treatment Of Cancermentioning

confidence: 99%

Biological Therapies in the Treatment of Cancer—Update and New Directions

Papież

Krzyściak

2021

IJMS

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Biological therapies have changed the face of oncology by targeting cancerous cells while reducing the effect on normal tissue. This publication focuses mainly on new therapies that have contributed to the advances in treatment of certain malignancies. Immunotherapy, which has repeatedly proven to be a breakthrough therapy in melanoma, as well as B-ALL therapy with CAR T cells, are of great merit in this progress. These therapies are currently being developed by modifying bispecific antibodies and CAR T cells to improve their efficiency and bioavailability. Work on improving the therapy with oncolytic viruses is also progressing, and efforts are being made to improve the immunogenicity and stability of cancer vaccines. Combining various biological therapies, immunotherapy with oncolytic viruses or cancer vaccines is gaining importance in cancer therapy. New therapeutic targets are intensively sought among neoantigens, which are not immunocompromised, or antigens associated with tumor stroma cells. An example is fibroblast activation protein α (FAPα), the overexpression of which is observed in the case of tumor progression. Universal therapeutic targets are also sought, such as the neurotrophic receptor tyrosine kinase (NTRK) gene fusion, a key genetic driver present in many types of cancer. This review also raises the problem of the tumor microenvironment. Stromal cells can protect tumor cells from chemotherapy and contribute to relapse and progression. This publication also addresses the problem of cancer stem cells resistance to treatment and presents attempts to avoid this phenomenon. This review focuses on the most important strategies used to improve the selectivity of biological therapies.

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“…Homodimer impurities often possess physical properties (size, charge, binding affinity, etc) similar to those of the heterodimeric bispecific product, making them difficult to identify and separate by conventional means. Developing efficient processes for removal of homodimers from final products and designing an appropriate control system strategy are critical for successful development of bispecific molecule therapeutics 28,30–32 .…”

Section: Introductionmentioning

confidence: 99%

Development of a bioassay to detect T-cell-activating impurities for T-cell-dependent bispecific antibodies

Lee¹,

Contreras²,

et al. 2019

Sci Rep

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T-cell-dependent bispecific antibodies (TDBs) are promising cancer immunotherapies that recruit a patient’s T cells to kill cancer cells. There are increasing numbers of TBDs in clinical trials, demonstrating their widely recognized therapeutic potential. Due to the fact that TDBs engage and activate T cells via an anti-CD3 (aCD3) arm, aCD3 homodimer (aCD3 HD) and high-molecular-weight species (HMWS) are product-related impurities that pose a potential safety risk by triggering off-target T-cell activation through bivalent engagement and dimerization of T-cell receptors (TCRs). To monitor and control the level of unspecific T-cell activation, we developed a sensitive and quantitative T-cell-activation assay, which can detect aCD3 HD in TDB drug product by exploiting its ability to activate T cells in the absence of target cells. This assay provides in-vivo -relevant off-target T-cell-activation readout. Furthermore, we have demonstrated that this assay can serve as a platform assay for detecting T-cell-activating impurities across a broad spectrum of aCD3 bispecific molecules. It therefore has the potential to significantly benefit many T-cell-recruiting bispecific programs.

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Immunoglobulins Secreted by a Hybrid-Hybridoma: Analysis of Chain Assemblies

Cited by 16 publications

References 18 publications

Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry

Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry

Biological Therapies in the Treatment of Cancer—Update and New Directions

Development of a bioassay to detect T-cell-activating impurities for T-cell-dependent bispecific antibodies

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