Recombinant B8R protein (Carrier-free)

InvitroFit - Carrier-free - Cell-culture tested

Inhibition of IFN-γ signaling by B8R

The recombinant viral B8R is a potent inhibitor of interferon-gamma (IFN-γ) signaling. The secreted protein B8R is a viral immunomodulatory protein encoded by the vaccinia virus. It functions as a decoy receptor that binds and neutralizes IFN-γ, enabling the virus to evade host immune responses [1].

 More details

B8R effectively inhibits IFN-γ signaling across multiple species—including humans, cows, rats, rabbits, and chickens—but not mice [2]. In research, B8R is used to study viral immune evasion by blocking IFN-γ signaling [1], to develop safer live attenuated vaccines through gene deletion [3], and more recently, as an immunogenic target to redirect antiviral T cells toward tumors in cancer immunotherapy [4]. In addition, B8R could potentially be used to reduce IFN-γ-mediated toxicities in CAR-T cell therapy — similar to the effects of IFN-γ-neutralizing antibodies [5] — while preserving antitumor efficacy.

Key features

• Each lot is functionally tested and validated.
• The absence of endotoxins is determined using the EndotoxDetect™ assay.
• InvitroFit™ grade: each lot is highly pure (≥95%) and functionally tested.
   

InvivoGen's recombinant B8R protein is produced in Chinese hamster ovary (CHO) cells, which ensures protein glycosylation and bona fide 3D structure. Of note, glycosylation stabilizes proteins against physicochemical instabilities. It specifically blocks type II IFN-γ-mediated signaling, as verified in HEK-Blue™ IFN-γ cells stimulated with recombinant human IFN-γ (see figure). B8R is unable to block type I or type III IFNs. Also, it does not inhibit mouse IFN-γ signaling (data not shown). 

References:

1.  Mossman K, et al., 1995. Species specificity of ectromelia virus and vaccinia virus interferon-gamma binding proteins. Virology. 208(2):762-9.
2. Puehler F, et al., 1998. Vaccinia virus-encoded cytokine receptor binds and neutralizes chicken interferon-gamma. Virology. 248(2):231-40.
3. Yakubitskiy SN, et al., 2015. Attenuation of Vaccinia Virus. Acta Naturae. 7(4):113-21.
4. Cao D, et al., 2022. Redirecting anti-Vaccinia virus T cell immunity for cancer treatment by AAV-mediated delivery of the VV B8R gene. Mol Ther Oncolytics. 25:264-275.
5. Manni S, et al., 2023. Neutralizing IFNγ improves safety without compromising efficacy of CAR-T cell therapy in B-cell malignancies. Nat Commun. 2023 Jun 9;14(1):3423.

Figures

Dose-dependent neutralization of IFN-γ signaling by B8R. Increasing concentrations (0.001 - 100 ng/ml)  of recombinant B8R were incubated with recombinant human IFN-γ (2 ng/ml) for 30 min prior to the addition of HEK-Blue™ IFN-γ cells. After overnight incubation, SEAP activity in the cell culture supernatant was assessed using QUANTI-Blue™ Solution.  Data are shown as the percentage of activity (mean ± SEM).

Specifications

Applications: Interferon-gamma inhibition, signaling pathway studies, CAR-T cell research

Species:  Vaccinia Virus

Source: Chinese hamster ovary (CHO) cells

Tag: Hexahistidine (His6) N-terminal

Solubility: 100 µg/ml

Working concentration: 0.1 - 100 ng/ml

Purity: ≥95% (SDS-PAGE)

Molecular weight: ~30.15 kDa, due to glycosylation

Quality control: Each lot is functionally tested and validated

Contents

Recombinant B8R protein is provided lyophilized, carrier-free, and provided in two quantities:

• inh-b8r: 25 μg
• inh-b8r-5: 5 x 25 μg

The product is shipped at room temperature.

 Upon receipt, store at -20 °C. 

 The resuspended product is stable for 1 month when properly stored.

 Avoid repeated freeze-thaw cycles.

Details

IFN-γ background

IFN-γ, also known as Type II IFN or immune interferon, is predominantly produced by innate immune cells, such as Natural Killer (NK) cells and innate lymphoid type 1 cells (ILC1), and activated adaptive immune cells, such as Th1 CD4+ T cells and cytotoxic CD8+ T cells [1]. This cytokine is produced as a secreted homodimeric molecule in response to infections and growing tumors [1, 2]. IFN-γ engages a receptor composed of two IFN-γR1 chains and two IFNγ-R2a, thus forming a hexameric complex [2]. While IFN-γR1 is constitutively expressed on all nucleated cells, the expression of IFNγ-R2 is tightly regulated. The binding of IFN-γ to its receptors triggers a JAK1/JAK2 signal transduction leading to the activation of STAT1. Activated STAT1 forms homodimers that are translocated to the nucleus where they bind interferon-gamma-activated sites (GAS) in the promoter of interferon-stimulated genes (ISGs). ISGs encode many products with direct effector or regulatory immune functions [1]. Thus IFN-γ plays a versatile role in immune responses and tissue homeostasis [1].

Relevance for therapeutics development

IFN-γ contributes to the pathogenesis of inflammatory diseases, such as Crohn's disease, psoriasis, haemophagocytic lymphohistiocytosis (HLH), and macrophage activation syndrome (MAS) [3-5].
Fontolizumab (aka HuZAF™) is a therapeutic, humanized monoclonal antibody (mAb) that targets IFN-γ. By binding to IFN-γ, Fontolizumab prevents it from interacting with its receptor (IFN-γR) on the surface of immune cells, thus inhibiting downstream inflammatory response. Fontolizumab was used as an immunosuppressive drug to treat inflammatory diseases, including Crohn's disease and psoriasis. While the mode of action was promising, clinical trials did not demonstrate sufficient clinical benefit [4, 5].
Emapalumab is a fully human monoclonal antibody (mAb) that targets both free and receptor-bound IFN-γ, preventing downstream signaling. This therapeutic antibody was FDA-approved in 2018 for treating pediatric and adult patients with HLH [3]. 

The administration of recombinant IFN-γ is a strategy for enhancing anti-infectious and anti-tumoral innate and adaptive immune responses.
Interferon gamma-1b (Actimmune®) is a form of recombinant human IFN-γ approved by the FDA to treat infections associated with chronic granulomatous disease and to slow the progression of severe malignant osteopetrosis [6]. IFN-γ monotherapy to treat cancer has been of limited success. This is partly explained by IFN-γ's short half-life and dual anti- and pro-tumor activities [7, 8]. Multiple clinical trials are ongoing to explore the combination of IFN-γ with other cancer therapeutics [8].
Another strategy relies on the engineering of IFN-γ partial agonists to tune IFN-γ receptor signaling output. Interestingly, such recombinant IFN-γ variants can exhibit biased gene-expression profiles, such as the retention of upregulation of class I molecules and impaired induction of inhibitory checkpoint molecules by cancer cells [2]. These results demonstrate that the two opposing functions of IFN-γ in the tumor microenvironment can be decoupled, offering a route for therapeutic applications.

References:

1. Ivashkiv L.B., 2018. IFNγ: signalling, epigenetics and roles in immunity, metabolism, disease and cancer immunotherapy. Nat Rev Immunol. 18(9):545-558
2. Mendoza, J.L., et al., 2019. Structure of the IFNγ receptor complex guides design of biased agonists. Nature. 567(7746):56-60.
3. Vallurupalli M. & Berliner N., 2019. Emapalumab for the treatment of relapsed/refractory hemophagocytic lymphohistiocytosis. Blood. 134(21):1783-1786.
4. Reinisch W et al., 2009. Fontolizumab in moderate to severe Crohn's disease: A phase 2, randomized, double-blind, placebo-controlled, multiple-dose study. Infl Bowel Dis., 16(2):233-242.
5. Harden J.L., et al., 2015. Humanized anti-IFNg (HuZAF) in the treatment of psoriasis. J. Aller Clin Immunol. 135(2):553.
6. Silva, A.C. & Lobo, J.M. Sousa., 2020. Cytokines and growth factors. Current Applications of Pharmaceutical Biotechnology. 87-113.
7. Castro, F., et al., 2018. Interferon-Gamma at the Crossroads of Tumor Immune Surveillance or Evasion. Front Immunol. 9:847.
8. Yi, M., et al., 2024. Targeting cytokine and chemokine signaling pathways for cancer therapy. Signal Transduction and Targeted Therapy. 9(1):176.