Chemically generated IgG2 bispecific antibodies through disulfide bridging

Patterson, James T.; Gros, Edwige; Zhou, Helin; Atassi, Ghazi; Kerwin, Lisa; Carmody, Lisa; Zhu, Tong; Jones, Benjamin F.; Fu, Yanwen; Kaufmann, Gunnar F.

doi:10.1016/j.bmcl.2017.07.021

Cited by 16 publications

(22 citation statements)

References 27 publications

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“…To assess the importance of proximity of BiCAT molecules (FGFR3‐α2 integrin) for cell proliferation inhibition in cancer cells, several (IgG) 2 antibodies (Figure A), similar products to bispecific antibodies, were treated with LK2 cells. The binding capacity of each (IgG) 2 antibody was examined using fluorescence microscopy.…”

Section: Resultsmentioning

confidence: 99%

“…Considering this concept, a bispecific antibody may be the best tool to discuss whether the proximity (interaction) among BiCAT molecules is important for cancer cell proliferation inhibition. In this study, we used (IgG) 2 antibodies for the assay because they can be generated in less time than bispecific antibodies. The (IgG) 2 antibodies containing FGFR3‐α2 integrin‐(IgG) 2 antibody significantly inhibited cell proliferation in LK2 cells (Figure ), suggesting that the proximity (interaction) among BiCAT molecules is also an important factor.…”

Section: Discussionmentioning

confidence: 99%

“…The final concentration of each reagent was determined based on previous reports and the data from the pilot studies (data not shown). For the (IgG) 2 antibody preparation, 4 types of antibody mix were prepared by simply mixing with cross‐linker antibody as 2follows: Ab mix 1 (anti–FGFR3 antibody [Santa Cruz; sc‐123]: 1 μg/mL and anti–rabbit IgG Fc specific antibody [Jackson ImmunoResearch; 111‐005‐046]: 0.5 μg/mL); Ab mix 2 (anti–α2 integrin antibody [Abcam; ab133557]: 1 μg/mL and anti–rabbit IgG Fc specific antibody: 0.5 μg/mL); Ab mix 3 (anti–α2 integrin antibody: 0.5 μg/mL, anti–α2 integrin antibody: 0.5 μg/mL and anti–rabbit IgG Fc specific antibody: 0.5 μg/mL); Ab mix 4 (anti–α2 integrin antibody: 0.5 μg/mL, anti–α2 integrin antibody: 0.5 μg/mL). These Ab mixes were incubated at room temperature for 30 minutes to form (IgG) 2 antibodies, respectively (Ab mix 3 contains FGFR3‐α2 integrin‐(IgG) 2 antibody).…”

Section: Methodsmentioning

confidence: 99%

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Proximity proteomics identifies cancer cell membrane cis‐molecular complex as a potential cancer target

et al. 2019

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Cancer‐specific antigens expressed in the cell membrane have been used as targets for several molecular targeted strategies in the last 20 years with remarkable success. To develop more effective cancer treatments, novel targets and strategies for targeted therapies are needed. Here, we examined the cancer cell membrane‐resident “cis‐bimolecular complex” as a possible cancer target (cis‐bimolecular cancer target: BiCAT) using proximity proteomics, a technique that has attracted attention in the last 10 years. BiCAT were detected using a previously developed method termed the enzyme‐mediated activation of radical source (EMARS), to label the components proximal to a given cell membrane molecule. EMARS analysis identified some BiCAT, such as close homolog of L1 (CHL1), fibroblast growth factor 3 (FGFR3) and α2 integrin, which are commonly expressed in mouse primary lung cancer cells and human lung squamous cell carcinoma cells. Analysis of cancer specimens from 55 lung cancer patients revealed that CHL1 and α2 integrin were highly co–expressed in almost all cancer tissues compared with normal lung tissues. As an example of BiCAT application, in vitro simulation of effective drug combinations used for multiple drug treatment strategies was performed using reagents targeted to BiCAT molecules. The combination treatment based on BiCAT information moderately suppressed cancer cell proliferation compared with single administration, suggesting that the information about BiCAT in cancer cells is useful for the appropriate selection of the combination among molecular targeted reagents. Thus, BiCAT has the potential to contribute to several molecular targeted strategies in future.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Proximity proteomics identifies cancer cell membrane cis‐molecular complex as a potential cancer target

et al. 2019

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“…Although the cross-linked product had significant anti-tumor activity when coated on activated T-cells, the product itself was very heterogeneous. A better strategy at producing a more homogeneous cross-linked product was described by Patterson et al for two IgGs [18] and their Fab fragments [18]. In this approach chemically reduced hinge region cysteines were derivatized with Click reagents that allowed a more specific crosslinking of two different IgGs together.…”

Section: Discussionmentioning

confidence: 99%

Generation of dual specific bivalent BiTEs (dbBIspecific T-cell Engaging antibodies) for cellular immunotherapy

Kujawski

Bhatacharya

et al. 2019

Preprint

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Bispecific T-cell engaging antibodies (BiTES), comprising dual anti-CD3 and anti-tumor antigen scFv fragments, are important therapeutic agents for the treatment of cancer. The dual scFv construct for BiTES requires proper protein folding while their small molecular size leads to rapid kidney clearance. Here we show that an intact (150 kDa) anti-tumor antigen antibody to CEA was joined in high yield (ca. 30%) to intact (150 kDa) anti-murine and anti-human CD3 antibodies using hinge region specific Click chemistry to form dual-specific, bivalent BiTES (db BiTES, 300 kDa). The interlocked hinge regions are compatible with a structural model that fits the electron micrographs of the 300 kDa particles. Compared to intact anti-CEA antibody, dbBiTES maintain high in vivo tumor targeting as demonstrated by PET imaging, and redirect dbBiTE coated T-cells (1 microgram/10 million cells) to kill CEA + target cells both in vitro, and in vivo in CEA transgenic mice.

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“…A relatively universal method of BsAb preparation is the exchange of IgG HL fragments, which occurs stochastically between natural IgG 4 , 60 IgG 1 with a mutation in the CH3 domain, 105 and IgG 2 through disulfide linkers. 122 According to the literature, the HL-fragment exchange occurs in human blood, milk, and placenta between IgG of all subclasses, resulting in polyreactive BsAbs. 61 – 64 However, the exchange of HL fragments between the therapeutic molecules of bispecific IgG 4 and the patient’s IgG 4 leads to the formation of BsAbs that do not possess the original properties, 123 so this imposes significant limitations on this method.…”

Section: Perspectivesmentioning

confidence: 99%

Bispecific antibodies: design, therapy, perspectives

Sedykh¹,

Prinz²,

Buneva³

et al. 2018

DDDT

251

183

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Antibodies (Abs) containing two different antigen-binding sites in one molecule are called bispecific. Bispecific Abs (BsAbs) were first described in the 1960s, the first monoclonal BsAbs were generated in the 1980s by hybridoma technology, and the first article describing the therapeutic use of BsAbs was published in 1992, but the number of papers devoted to BsAbs has increased significantly in the last 10 years. Particular interest in BsAbs is due to their therapeutic use. In the last decade, two BsAbs – catumaxomab in 2009 and blinatumomab in 2014, were approved for therapeutic use. Papers published in recent years have been devoted to various methods of BsAb generation by genetic engineering and chemical conjugation, and describe preclinical and clinical trials of these drugs in a variety of diseases. This review considers diverse BsAb-production methods, describes features of therapeutic BsAbs approved for medical use, and summarizes the prospects of practical application of promising new BsAbs.

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Chemically generated IgG2 bispecific antibodies through disulfide bridging

Cited by 16 publications

References 27 publications

Proximity proteomics identifies cancer cell membrane cis‐molecular complex as a potential cancer target

Proximity proteomics identifies cancer cell membrane cis‐molecular complex as a potential cancer target

Generation of dual specific bivalent BiTEs (dbBIspecific T-cell Engaging antibodies) for cellular immunotherapy

Bispecific antibodies: design, therapy, perspectives

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