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Your Position: Casa > IgG Fc Proteins——Ideal Isotype Control for IgG Structured Drug Development

IgG Fc Proteins——Ideal Isotype Control for IgG Structured Drug Development

IgG Fc Proteins——Ideal Isotype Control for IgG Structured Drug Development
Background
Product Features
Product List
Verification Data
Related Reading
References

Immunoglobulin G (IgG) antibodies have attracted great attention in the pharmaceutical industry due to its high specificity and superior pharmacokinetics since the late 1990s, especially following the inaugural approval of Ritux*mab, the first anti-tumor antibody drug, in 1997. Nowadays, IgG antibodies have been used in the treatment of cancer and autoimmune diseases with great success. Fc-mediated effector functions (ADCC, ADCP, CDC) play an important role in the therapeutic effects among the mechanisms by which various IgG antibody drugs exert their therapeutic effects.

There are four distinct subclasses of human IgG with decreasing concentrations, namely IgG1, IgG2, IgG3, and IgG4 (approximately 61%，32%，4%，3%). Although all subclasses are more than 90% identical at the amino acid level and have similar spatial structures. They are highly conserved in terms of the length of the hinge regions, the number of disulfide bonds, and the Fc effect function. These regions are involved in binding to both Ig-FC receptors (FcγR) and C1q. As a result, the different subclasses have different effector functions, both in terms of triggering FcγR-expressing cells, resulting in phagocytosis (ADCP) or antibody-dependent cell mediated cytotoxicity (ADCC), and activating complement (CDC).


           Human IgG subclasses

IgG1 is the most potential subclass in tumor immunotherapy. In addition to Human IgG1 can also effectively bind Mouse Fcγs, significant effects can also be observed in vivo models. The half-life of IgG1 in the body is relatively long. Moreover, from an industrial perspective, IgG1 can be highly expressed and can be purified by protein-A, which makes it highly economical and stable. Therefore, IgG1 has become the most commonly used IgG subtype.

IgG2 is mainly used to neutralize the antigen or block the binding of receptor ligands. The ADCC effect is very weak. Although the binding to C1q is relatively weak, but it can still trigger the CDC effect when the antigen or antibody solubility is high. At the same time, IgG2 is the only subtype that can bind to FcγRIIa (CD32a). In the early days, only antibodies targeting EGFR were marketed based on the IgG2 subtype. With the rise of immune checkpoint research, more and more drugs of the IgG2 subtype have entered the clinic and are on the market.

IgG3 has a longer hinge region, a larger molecular weight and a stronger biological function. IgG3 has the strongest binding ability to FcγRs, which can trigger ADCC and ADCP (antibody dependent cellular phagocytosis), and the CDC effect is stronger than that of IgG1. However, the half-life of IgG3 is shorter, because R435 is different from H435 of other IgG molecules, which affects its binding to FcRn. Considering that pharmacokinetics, frequently administration is required, it is rarely used to develop antibody drugs and from an economic point of view. IgG3 antibodies cannot be purified by protein-A, which is commonly used in industrialization, and are easy to form multimers. This will undoubtedly greatly increase the purification cost, which further inhibits the development of antibodies based on this subtype.
IgG4 has a shorter hinge region and weakly binds to FcγRs other than FcγRI (CD64), but may have a stronger affinity than clone-related IgG1, and may compete with IgG1 for binding to tumor antigens. IgG4 binds to inhibitory FcγRIIb with higher affinity than other IgG subtypes. It can bind to FcγRIIb with other antibodies such as IgG1 to inhibit the function mediated by FcγRs. IgG4 can also exchange Fab-arm with other IgG4 to produce monovalent antibodies with bispecific functions or antibodies with increased affinity. IgG4 cannot immobilize complement and bind to activated FcγRs on immune effector cells, which may lead to antibody-mediated CDC, ADCC and ADCP blockade. Different from the previous mechanism that antibodies rely on ADCC activity to kill cells, the mechanism of PD-1 antibody or PD-L1 antibody is to block the PD-1/PD-L1 pathway and release the inhibition of T cells, etc. For example, Opdivo and Keytruda are designed with the IgG4 subtype with weak ADCC activity.
FDA approved tumor-therapeutic antibodies with different IgG subclasses
https://doi.org/10.1186/s13045-020-00876-4

The structure of IgG Fc (shown in green)
In terms of structure, monoclonal antibodies currently used for autoimmune diseases, inflammation and tumors all use IgG backbone. In addition, IgG-like bispecific antibodies，ADC and IgG Fc fusion proteins are also inseparable from the structural framework of IgG. In addition to increasing the serum half-life, the structure of Fc also improves other properties, such as stability and solubility, and at the same time it facilitates the production process and simplifies the downstream purification. In terms of action, as one of the most effective biotherapeutics at present, an important aspect of antibodies is that they can specifically bind to antigens and at the same time stimulate immune response. In addition to specific binding of Fab fragments to target antigen epitopes, antibody-derived Fc fragments can also bind to Fc receptors on target cells and other cells.
Based on scientific rigor, in order to prove that the binding of the antibody to the target antigen, the effect of the target fusion protein is specific. The non-specific binding signal or basic function verification generated by the IgG Fc fragment cannot be ignored, and a suitable isotype control product is indispensable.
To facilitate the development of IgG structured drug development including monoclonal antibodies, IgG-like bispecific antibodies and IgG Fc-fusion proteins, ACROBiosystems can provide you with a series of IgG Fc proteins. Our recombinant protein IgG Fc only contains the hinge region, CH2, CH3 sequence, but not the Fab sequence. It can be used as an ideal isotype control for monoclonal antibodies, IgG-like bispecific antibodies, ADC and IgG Fc- fusion drugs in the process of drug screening, functional verification and so on.
In addition, our IgG Fc proteins can be used as endogenous IgG for competitive validation of FcRn binding to FcRn targeted antibody drugs. (Click here to view: Increase or Decrease: The Two Therapeutic Strategies via FcRn-Mediated Mechanism)
Product Features

Expressed by HEK293 cells: which realize post-translational glycosylation and other modifications and correct protein folding

Various species and subclasses:

Human IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc

Mouse IgG1 Fc, IgG2a Fc, IgG2b Fc

Llama IgG2b Fc，Rabbit IgG Fc

Various tags: Tag Free，AvitagTM，His Tag，gD Tag，Flag Tag，AvitagTM &His tag

Low endotoxin: Less than 1.0 EU/μg by the LAL method

High purity:

more than 95% as verified by SDS-PAGE

more than 90% as verified by SEC-MALS

High bioactivity: verified by ELISA & SPR with free protocols

Product List
MoleculeCat. No.SpeciesProduct DescriptionPreorder/Order
IgG1 FcFCC-H5214HumanHuman IgG1 Fc Protein, Tag Free (MALS verified)

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IgG1 FcIG1-H8213HumanBiotinylated Human IgG1 Fc protein, Avitag™ (MALS verified)

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IgG1 FcIG1-H5225HumanHuman IgG1 Fc Protein, His Tag (MALS verified)

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IgG1 FcIG1-H52C9HumanHuman IgG1 Fc Protein, Flag Tag (MALS verified)

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IgG1 FcIG1-H52G6HumanHuman IgG1 Fc Protein, gD Tag (MALS verified)

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IgG2 FcIG2-H5206HumanHuman IgG2 Fc Protein, Tag Free (MALS & SPR verified)

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IgG3 FcIG3-H5200HumanHuman IgG3 Fc Protein, Tag Free (MALS & SPR verified)

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IgG4 FcIG4-H5205HumanHuman IgG4 Fc Protein, Tag Free (MALS & SPR verified)

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IgG1 FcIG1-M5208MouseMouse IgG1 Fc Protein, Tag Free (HPLC verified)

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IgG1 FcIG1-M8211MouseBiotinylated Mouse IgG1 Fc protein, Avitag™ (MALS verified)

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IgG2a FcIGA-M5207MouseMouse IgG2a Fc Protein, Tag Free (MALS verified)

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IgG2a FcIGA-M8210MouseBiotinylated Mouse IgG2a Fc Protein, Avitag™ (MALS verified)

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IgG2b FcIGB-M5203MouseMouse IgG2b Fc Protein, Tag Free (MALS verified)

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IgG2b FcIGB-L5204LlamaLlama IgG2b Fc Protein, Tag Free (MALS verified)

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IgG FcIGG-R5203RabbitRabbit IgG Fc Protein, Tag Free (MALS verified)

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Verification Data

High purity verified by SDS-PAGE and SEC-MALS

2Human IgG Fc, Tag Free (Cat. No. FCC-H5214) on SDS-PAGE under reducing (R) condition. The purity of the protein is greater than 95%. The purity of Human IgG Fc, Tag Free (Cat. No. FCC-H5214) is more than 95% and the molecular weight of this protein is around 51-65 kDa verified by SEC-MALS.

Human IgG4 Fc, Tag Free (Cat. No. IG4-H5205) on SDS-PAGE under reducing (R) condition. The purity of the protein is greater than 95%. The purity of Human IgG4 Fc, Tag Free (Cat. No. IG4-H5205) is more than 90% and the molecular weight of this protein is around 54-66kDa verified by SEC-MALS.

The Secondary Structure of IgG Fc Verified Using the Microfluidic Modulaton Spectroscopy (MMS) Platform (Collaborated With RedShiftBio)
The Secondary Structure of IgG Fc Verified Using the Microfluidic Modulaton Spectroscopy (MMS) Platform
The Secondary Structure of IgG Fc Verified Using the Microfluidic Modulaton Spectroscopy (MMS) Platform

MMS analysis showed that different IgG Fc subclasses exhibit distinct secondary structures. Specifically, IgG1 and IgG2 exhibit the highest degree of structural similarity, while IgG3 and IgG4 show the greatest structural difference.

Explore the In-depth Secondary Structure Analysis of IgG Fc

High Bioactivity verified by ELISA&SPR

Immobilized Human CD64, His Tag (Cat. No. FCA-H52H1) at 1 μg/mL (100 μL/well) can bind Human IgG Fc, Tag Free (Cat. No. FCC-H5214) with a linear range of 1-16 ng/mL.

Immobilized Recombinant Protein G, His Tag (Cat. No. RPG-S3140) at 2 μg/mL (100 μL/well) can bind Biotinylated Human IgG1 Fc, Avitag (Cat. No. IG1-H8213) with a linear range of 0.013-0.512 μg/mL.

Affinity verified by SPR

Human FCGRT&B2M Heterodimer Protein, His Tag (Cat. No. FCN-H52W7) captured on CM5 Chip via anti-His antibody can bind Human IgG1 Fc, Tag Free (Cat. No. FCC-H5214) with an affinity constant of 0.957 μM as determined in SPR assay (Biacore 8K).

Human FCGRT&B2M Heterodimer Protein, His Tag (Cat. No. FCN-H52W7) captured on CM5 Chip via anti-His antibody can bind Human IgG4 Fc, Tag Free (Cat. No. IG4-H5205) with an affinity constant of 0.715 μM as determined in SPR assay (Biacore 8K).

Application 1: IgG Fc protein is used as isotype control to verify the specific binding of monoclonal antibody to target antigen

Mouse IgG2a Fc protein is used as an isotype control to verify the specific binding of Mouse IgG2a CD3 monoclonal antibody (Clone: OKT3) to Human CD3 protein.

Human CD3 epsilon & CD3 delta Heterodimer Protein, His Tag&Tag Free (MALS verified) (Cat. No. CDD-H52W1) can bind Monoclonal Anti-Human CD3 Antibody, Mouse IgG2a (Clone: OKT3), Ultra-low endotoxin (Cat. No. CDE-M120a) with a linear range of 0.2-3 ng/mL and cannot bind Mouse IgG2a Fc Protein, Tag Free (MALS verified) (Cat. No. IGA-M5207).

Application 2: IgG Fc protein is used as an isotype control to verify the specific binding of bispecific antibody/IgG Fc fusion protein to target antigen

Human IgG1 Fc is used as an isotype control to verify the specific binding of anti-BCMA×CD3 scFv- Human IgG1 Fc Tag to Human CD3 protein.

Human CD3 epsilon & CD3 delta Heterodimer Protein, His Tag&Tag Free (MALS verified) (Cat. No. CDD-H52W1) can bind anti-BCMA×CD3 scFv- Human IgG1 Fc Tag with a linear range of 0.08-3 ng/mL and cannot bind Human IgG1 Fc Protein, Tag Free (MALS verified) (Cat. No. FCC-H5214).

Human IgG1 Fc is used as an isotype control to verify the specific binding of anti-BCMA×CD3 scFv- Human IgG1 Fc Tag to Human BCMA protein.

Human BCMA / TNFRSF17 Protein,His Tag (HPLC-verified) DMF Filed (Cat. No. BCA-H522y) can bind anti-BCMA×CD3 scFv- Human IgG1 Fc Tag with a linear range of 0.08-3 ng/mL and cannot bind Human IgG1 Fc Protein, Tag Free (MALS verified) (Cat. No. FCC-H5214).

Related Reading

＞＞Programmable half-life and anti-tumor effects of Nanobody

＞＞Increase or Decrease: The Two Therapeutic Strategies via FcRn-Mediated Mechanism

References

Fc gamma receptors: glycobiology and therapeutic prospects

How to select IgG subclasses in developing anti-tumor therapeutic antibodies

Fc fusion as a platform technology: potential for modulating immunogenicity

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