Knockout Mouse Catalog | Cyagen APAC

BRGSF (BALB/c Rag2tm1Fwa Il2rgtm1Cgn SirpaNOD Flk2tm1lrl) mouse is one of the most immunodeficient mice on the current market. The knockout of Rag2 and Il2rg genes leads to the losses of T, B, and NK cells in BRGSF mice, SIRPaNOD inhibits the phagocytosis of human-derived cells by mouse macrophages, and the deficiency of Flk2 gene significantly reduces the myeloid cell components (especially dendritic cells, DCs) in mice (Figure 1). With the highly deficient immune system, BRGSF mice are highly compatible with all kinds of CDX and PDX grafts and grafting efficiency is high, so such mice are extremely suitable for the pharmacological and pharmacodynamic researches of anti-tumor drugs. In addition, unlike N*G mice (NOD genetic background: complement C5-/-), BRGSF mice with BALB/c background have a complete complement system in vivo, which is a powerful tool for the studies of complement-dependent cytotoxicity (CDC). For example, the effectiveness and safety of drugs used for on-demand deletion of CAR-T cells by CDC mechanism can be evaluated.

Fig. 1 Features of BRGSF mice

 

Introduction to the Complement-dependent Cytotoxicity (CDC) Mechanism

As a kind of activated serum protein with enzymatic activity, complement is mainly produced by hepatocytes and macrophages and is involved in the body's inflammatory reaction and anti-microbial immune reaction. Complement-dependent cytotoxicity (CDC) is that after the complement system is activated, membrane attack complex (MAC) forms on the surface of the target cell membrane and perforates the cell membrane, which eventually leads to the cell killing effect for cell lysis. There are three pathways for complement activation, i.e. classical pathway, mannan-binding lectin (MBL) pathway and alternative pathway (Fig. 2). All the three pathways require the participation of complement 5 (C5) to finally form MAC composed of C5b6789 (C5b-9). Mice with NOD genetic background are unable to exert the CDC effectiveness due to C5 gene deletion and incomplete complement system in vivo. On the contrary, BRGSF mice with BALB/c background have a complete and effective complement system, which is a powerful tool to study CDC and can be used to assess the effectiveness and safety of drugs which exert their curative effect by CDC mechanism.

 

Fig. 2 Three pathways of complement activation [1]

 

Introduction to CAR-T Cell Immunotherapy

CAR-T cell immunotherapy is a new type of anti-tumor therapy that uses the body's own immune system to attack tumor cells. After two CAR-T therapies targeting CD19 (Kymriah of Novartis and Yescarta of Kite/Gilead) were approved for marketing by FDA at the end of 2017, CAR-T therapy targeting CD19 from Kite/Gilead was also approved in July 2020. Recently (February 5, 2021), we’ ve got another progress that FDA approved Breyanzi, the fourth CAR-T therapy targeting CD19, developed by Juno Therapeutics, a subsidiary of Bristol-Myers Squibb (BMS), for marketing and for the treatment of relapsed or refractory diffuse large B-cell lymphoma (DLBCL).

At present, CAR-T cell immunotherapy play its role in tumor treatment mainly by modifying the genes in selected T cells so that tumor-specific chimeric antigen receptors (CAR) are expressed on their surfaces, which are then amplified and cultured in vitro and transfused back into patient’s body (Fig. 3). With the high specificity and affinity of antigen and antibody, as well as the killing effect of T cells, this therapy can selectively kill tumor cells. However, several factors, including the common toxic and side effects of CAR-T therapy, such as cytokine release syndrome (CRS) and neurotoxicity, have also hindered the further development of CAR-T therapy. Among them, CRS is a lethal acute systemic inflammatory response syndrome (SIRS), which is caused by a large number of pro-inflammatory cytokines secreted by activated immune cells. Therefore, it can also be considered as a manifestation of the effectiveness of CAR-T therapy.

 

Fig. 3 Flow Diagram of CAR-T Cell Immunotherapy [2]

 

Case Study: On-demand Depletion of CAR-T Cells by CDC Mechanism

To avoid the serious harm of the toxic and side effects of CAR-T therapy to patients without reducing the effectiveness, researchers hope to find a method that can control CAR-T cells. That is to say, CAR-T cells are quickly cleared on demand after exerting their curative effect.

In 2018, scientists from Cellectis, a French biopharmaceutical company, published a paper on on-demand deletion of CAR-T cells by CDC mechanism in Scientific Reports: A Versatile Safeguard for Chimeric Antigen Receptor T-Cell Immunotherapies. In this paper, scientists introduced a CAR-T cell, CubiCAR-T, with the functions of detection, purification and on-demand depletion, for the treatment of multiple myeloma (MM).

The CAR in this CubiCAR-T cell contains a single-chain antibody against B cell maturation antigen (anti-BCMA ScFV). BCMA is an important therapeutic target widely present on the surface of multiple myeloma cells. CD34 epitope, binding to anti-CD34 McAb, can be used for cell detection and purification during CAR-T production process. CD20 mimotope, specifically binding to Rituximab (RTX) against CD20, can be used for on-demand depletion of cells by CDC mechanism (Fig. 4). CD20 plays a role of "safety switch" in cubiCAR-T cells.

 

Fig. 4 CAR Structure Design Scheme [3]

 

In vitro experiments have shown that the deletion kinetics of CubiCAR-T cells depends on the concentration of RTX added (Fig. 5). In the presence of complement and 50 μg/mL RTX, the half-life period of CubiCAR-T cells is about 10 minutes, but this concentration is about 10 times lower than the reported RTX plasma drug peak concentration (Cmax) in patients [4].

 

Fig. 5 Kinetics of deleting CubiCAR-T cells by complement [3]

Addition of RTX (10-100 µg/mL) at different concentrations and supplement in 2×105 CubiCAR-T cells, or the cells are not treated. Relative viability of cells = Cell viability after treatment/Cell viability without treatment × 100

 

To further verify the effectiveness of CubiCAR-T cells and the possibility of being specifically depleted by RTX, researchers selected BRGS mice for in vivo experiments (Fig. 6). Like the "upgraded" BRGSF miceBRGS mice have no T, B and NK cells, macrophages have weak phagocytosis of human-derived cells, and there is a functional complement system. Thus, BRGS mice can be used to evaluate the effectiveness and safety of RTX in the depletion of CAR-T cells by CDC mechanism.


Fig. 6 Flow diagram of in vivo experiment of BRGS mice [3]

 

The results of mouse fluorescence imaging experiment showed that (Fig. 7):

  1. In the Mock group (no CAR-T cellswere injected), tumor cell proliferation was not inhibited, and there was no significant difference between RTX and IgG added.
  2. In the CAR-T group(CAR contained no CD20), tumor cell proliferation was obviously inhibited, and there was no significant difference between RTX and IgG added.
  3. In the CubiCAR-T group, tumor cell proliferation was obviouslyinhibited in the IgG control group, while tumor cell proliferation in the RTX group was not affected.

These results indicate that CubiCAR-T cells, like unmodified CAR-T cells, can effectively inhibit tumor cell proliferation, and CubiCAR-T cells can be quickly and specifically depleted by RTX.

 

Fig. 7 CubiCAR-T cells showed anti-tumor activity in mice and could be specifically depleted by RTX. [3] After inoculation with MM.1S-Luc GFP carrying luciferase and GFP markers, BRGS mice were randomly divided into different groups and injected with CubiCAR-T, CAR-T or T cells (mock-transduced T cells) on day 17, and were treated with or without RTX., and the fluorescence imaging analysis was performed on day 21, day 26, and day 31.

 

 

Summary

CAR T cell immunotherapy has a promising future in tumor treatment, and also faces a number of challenges, including severe drug side effects. Researchers have designed various "safety switches" for on-demand depletion of CAR-T cells, all of which, however, also have some disadvantages, such as large drug size, potential immunogenicity, and reliance on unapproved small molecules as activators. In addition, they are common in one point, i.e. all these "safety switches" exist on the surface of cells separated from CAR, and this design may result in an unbalanced ratio of CAR to safety switch (CAR/safeguard), leading to the emergence of CAR-T cells without carrying "safety switchs", The Cubicar-T designed incorporates the "safety switch" CD20 into CAR and uses RTX, an antibody drug approved by the FDA, for the on-demand depletion of CubiCAR-T cells by CDC mechanism, thus improving the drug safety while ensuring the drug effectiveness. In addition, CubiCAR-T cells incorporate CD34 epitope into CAR, which can also effectively improve the detection and purification efficiency of CAR-T cells in the enterprise production process. In particular, researchers have also tried to replace the ScFV targeting BCMA in CubiCAR with ScFVs targeting CD123, CD22 and CD19. These CubiCAR-T cells have also showed anti-tumor activity in in vitro experiment and could be specifically depleted by RTX. These data indicate that CubiCAR is expected to become an "universal safety switch" that can be applied to different types of tumor.

 

 

Download Free Copy of White Paper!

 

References:

  1. Tegla CA., et al. Membrane attack by complement: the assembly and biology of terminal complement complexes[J]. Immunologic Research51(1):45-60 (2011).
  2. Kenar D. Jhaveri and Mitchell H. Rosner. Chimeric Antigen Receptor T Cell Therapy and the Kidney. Clinical Journal of the American Society of Nephrology13(5)796-798(2018).
  3. Valton J., et al. A Versatile Safeguard for Chimeric Antigen Receptor T-Cell Immunotherapies. Scientific Reports8:8972(2018).
  4. RubensteinJL., et al. Multicenter phase 1 trial of intraventricular immunochemotherapy in recurrent CNS lymphoma. Blood 121:745 - 51(2013). 

 

Superior Severe Combined Immunodeficient - BRGSF Mouse Model

BRGSF mice are one of the most highly immunodeficient mouse models on the current market, they are highly compatible with various sources of cell line-derived xenografts (CDXs) and patient-derived xenografts (PDXs), and can be used for solid tumor and hematoma research. Unlike N*G mice, BRGSF mice with BALB/c background, which has a complete complement cascade in vivo, and also a powerful tool for complement-dependent cytotoxicity (CDC) studies. >> Learn more 

BRGSF-HIS mice are reconstructed from human CD34+ hematopoietic stem cells (HSCs) transplantation, which possesse all of the major human hematopoietic cell subsets, effective differentiation of human myeloid cell lines (HMCs), and long lasting reconstituted human cells - making BRGSF-HIS mouse a suitable model for long-term studies. >> Learn more 

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