RecombiMAb anti-mouse CD20
(switched from mouse IgG2c)
Product Details
The MB20-11-CP062 monoclonal antibody is a recombinant, chimeric version of the original MB20-11 antibody. The recombinant MB20-11-CP062 antibody targets murine CD20. CD20 is expressed on the surface of immature and mature B cells and their malignant counterparts. MB20-11 rapidly and effectively depletes B cells via antibody-dependent cellular cytotoxicity (ADCC) through engaging Fcγ receptors on monocytes and other effector cells. Isotype-specific interactions contribute significantly to the effectiveness of CD20 mAbs in vivo, with IgG2a/c mAbs having greater potency than IgG1 or IgG2b. IgG2a and IgG2c isotypes are indistinguishable in their specificities to murine FcγR. The recombinant MB20-11-CP062 variable domain sequences are identical to clone MB20-11, but the constant region has been converted from mouse IgG2c to mouse IgG2a. Mouse strains such as C57Bl/6, C57Bl/10, SJL, and NOD mice possesses the Igh1-b allele resulting in only the expression of IgG2c. However, mouse strains such as BALB/c and Swiss Webster mice possess the Igh1-a allele which results in only the expression of IgG2a. It is important to consider matching the Ig-haplotype of the receiving mice to the isotype of the injected antibody to avoid eliciting an undesired immune response. Additionally, the highly controlled sequence and lack of genetic drift in recombinant antibodies provide more reliable and reproducible results over hybridoma derived antibodies. B cell depleting antibodies are valuable tools for studying B cell biology and developing therapies for autoimmune diseases and malignancies.References:
1: Uchida, Junji et al. āThe innate mononuclear phagocyte network depletes B lymphocytes through Fc receptor-dependent mechanisms during anti-CD20 antibody immunotherapy.ā The Journal of experimental medicine vol. 199,12 (2004): 1659-69. doi:10.1084/jem.20040119
2: Xiu, Yan et al. āB lymphocyte depletion by CD20 monoclonal antibody prevents diabetes in nonobese diabetic mice despite isotype-specific differences in Fc gamma R effector functions.ā Journal of immunology (Baltimore, Md. : 1950) vol. 180,5 (2008): 2863-75. doi:10.4049/jimmunol.180.5.2863
3: Hamaguchi, Yasuhito et al. āAntibody isotype-specific engagement of Fcgamma receptors regulates B lymphocyte depletion during CD20 immunotherapy.ā The Journal of experimental medicine vol. 203,3 (2006): 743-53. doi:10.1084/jem.20052283
4: Zhang, Zhiping et al. āPossible allelic structure of IgG2a and IgG2c in mice.ā Molecular immunology vol. 50,3 (2012): 169-71. doi:10.1016/j.molimm.2011.11.006
Specifications
Isotype | Mouse IgG2a,Ā Īŗ |
---|---|
Recommended Isotype Control(s) | RecombiMAb mouse IgG2a isotype control, unknown specificity |
Recommended Dilution Buffer | InVivoPure pH 7.0 Dilution Buffer |
Conjugation | This product is unconjugated. Conjugation is available via our Antibody Conjugation Services. |
Immunogen | Mouse CD20-GFP transfected 300.19 cells |
Reported Applications |
Western Blot Flow Cytometry In vivo B cell depletion *Reported for the original MB20-11 antibody. For information on in vivo applications, please contact technicalservice@bioxcell.com |
Formulation |
PBS, pH 7.0 Contains no stabilizers or preservatives |
Endotoxin |
<1EU/mg (<0.001EU/μg) Determined by LAL gel clotting assay |
Aggregation |
<5% Determined by SEC |
Purity |
>95% Determined by SDS-PAGE |
Sterility | 0.2 µm filtration |
Production | Purified from HEK293 cell supernatant in an animal-free facility |
Purification | Protein G |
Molecular Weight | 150 kDa |
Murine Pathogen Tests |
Ectromelia/Mousepox Virus: Negative Hantavirus: Negative K Virus: Negative Lactate Dehydrogenase-Elevating Virus: Negative Lymphocytic Choriomeningitis virus: Negative Mouse Adenovirus: Negative Mouse Cytomegalovirus: Negative Mouse Hepatitis Virus: Negative Mouse Minute Virus: Negative Mouse Norovirus: Negative Mouse Parvovirus: Negative Mouse Rotavirus: Negative Mycoplasma Pulmonis: Negative Pneumonia Virus of Mice: Negative Polyoma Virus: Negative Reovirus Screen: Negative Sendai Virus: Negative Theilerās Murine Encephalomyelitis: Negative |
Storage | The antibody solution should be stored at the stock concentration at 4°C. Do not freeze. |
Additional Formats
Recommended Products
in vivo B cell depletion
Haas KM, Watanabe R, Matsushita T, Nakashima H, Ishiura N, Okochi H, Fujimoto M, Tedder TF. (J Immunol). "Protective and pathogenic roles for B cells during systemic autoimmunity in NZB/W F1 mice" J Immunol 184(9):4789-800. PubMed
Delineating the relative contributions of B lymphocytes during the course of autoimmune disease has been difficult. Therefore, the effects of depleting all mature B cells using a potent CD20 mAb, or of depleting circulating and marginal zone B cells using a ligand-blocking CD22 mAb, were compared in NZB/W F(1) mice, a model for human systemic lupus erythematosus. Single low-dose mAb treatments depleted B cells efficiently in both NZB/W F(1) and C57BL/6 mice. Prophylactic B cell depletion by repeated CD20 mAb treatments prolonged survival during pristane-accelerated lupus in NZB/W F(1) mice, whereas CD22 mAb had little effect. Despite effective B cell depletion, neither mAb treatment prevented autoantibody generation. In addition, CD20, CD22, and control mAb-treated NZB/W F(1) mice developed anti-mouse IgG autoantibodies in contrast to parental NZB and NZW strains, which may have reduced the effectiveness of B cell depletion. Despite this, low-dose CD20 mAb treatment initiated in 12-28-wk-old mice, and administered every 4 wk thereafter, significantly delayed spontaneous disease in NZB/W F(1) mice. By contrast, B cell depletion initiated in 4-wk-old mice hastened disease onset, which paralleled depletion of the IL-10-producing regulatory B cell subset called B10 cells. B10 cells were phenotypically similar in NZB/W F(1) and C57BL/6 mice, but were expanded significantly in young NZB/W F(1) mice. Thus, B cell depletion had significant effects on NZB/W F(1) mouse survival that were dependent on the timing of treatment initiation. Therefore, distinct B cell populations can have opposing protective and pathogenic roles during lupus progression.
in vivo B cell depletion
Xiu Y, Wong CP, Bouaziz JD, Hamaguchi Y, Wang Y, Pop SM, Tisch RM, Tedder TF. (2008). "Do you think it is possible that I get support with the validation images from QC. Do you think it would be possible that they send me the final version of the website image." J Immunol 180(5):2863-75. PubMed
NOD mice deficient for B lymphocytes from birth fail to develop autoimmune or type 1 diabetes. To assess whether B cell depletion influences type 1 diabetes in mice with an intact immune system, NOD female mice representing early and late preclinical stages of disease were treated with mouse anti-mouse CD20 mAbs. Short-term CD20 mAb treatment in 5-wk-old NOD female mice reduced B cell numbers by approximately 95%, decreased subsequent insulitis, and prevented diabetes in >60% of littermates. In addition, CD20 mAb treatment of 15-wk-old NOD female mice significantly delayed, but did not prevent, diabetes onset. Protection from diabetes did not result from altered T cell numbers or subset distributions, or regulatory/suppressor T cell generation. Rather, impaired CD4+ and CD8+ T cell activation in the lymph nodes of B cell-depleted NOD mice may delay diabetes onset. B cell depletion was achieved despite reduced sensitivity of NOD mice to CD20 mAbs compared with C57BL/6 mice. Decreased B cell depletion resulted from deficient FcgammaRI binding of IgG2a/c CD20 mAbs and 60% reduced spleen monocyte numbers, which in combination reduced Ab-dependent cellular cytotoxicity. With high-dose CD20 mAb treatment (250 microg) in NOD mice, FcgammaRIII and FcgammaRIV compensated for inadequate FcgammaRI function and mediated B cell depletion. Thereby, NOD mice provide a model for human FcgammaR polymorphisms that reduce therapeutic mAb efficacy in vivo. Moreover, this study defines a new, clinically relevant approach whereby B cell depletion early in the course of disease development may prevent diabetes or delay progression of disease.
in vivo B cell depletion
Hamaguchi Y, Xiu Y, Komura K, Nimmerjahn F, Tedder TF. (2006). "Antibody isotype-specific engagement of Fcgamma receptors regulates B lymphocyte depletion during CD20 immunotherapy" J Exp Med 203(3):743-53. PubMed
CD20 monoclonal antibody (mAb) immunotherapy is effective for lymphoma and autoimmune disease. In a mouse model of immunotherapy using mouse anti-mouse CD20 mAbs, the innate monocyte network depletes B cells through immunoglobulin (Ig)G Fc receptor (FcgammaR)-dependent pathways with a hierarchy of IgG2a/c>IgG1/IgG2b>IgG3. To understand the molecular basis for these CD20 mAb subclass differences, B cell depletion was assessed in mice deficient or blocked for stimulatory FcgammaRI, FcgammaRIII, FcgammaRIV, or FcR common gamma chain, or inhibitory FcgammaRIIB. IgG1 CD20 mAbs induced B cell depletion through preferential, if not exclusive, interactions with low-affinity FcgammaRIII. IgG2b CD20 mAbs interacted preferentially with intermediate affinity FcgammaRIV. The potency of IgG2a/c CD20 mAbs resulted from FcgammaRIV interactions, with potential contributions from high-affinity FcgammaRI. Regardless, FcgammaRIV could mediate IgG2a/b/c CD20 mAb-induced depletion in the absence of FcgammaRI and FcgammaRIII. In contrast, inhibitory FcgammaRIIB deficiency significantly increased CD20 mAb-induced B cell depletion by enhancing monocyte function. Although FcgammaR-dependent pathways regulated B cell depletion from lymphoid tissues, both FcgammaR-dependent and -independent pathways contributed to mature bone marrow and circulating B cell clearance by CD20 mAbs. Thus, isotype-specific mAb interactions with distinct FcgammaRs contribute significantly to the effectiveness of CD20 mAbs in vivo, which may have important clinical implications for CD20 and other mAb-based therapies.
in vivo B cell depletion
Uchida J, Hamaguchi Y, Oliver JA, Ravetch JV, Poe JC, Haas KM, Tedder TF. (2004). "The innate mononuclear phagocyte network depletes B lymphocytes through Fc receptor-dependent mechanisms during anti-CD20 antibody immunotherapy" J Exp Med 199(12):1659-69. PubMed
Anti-CD20 antibody immunotherapy effectively treats non-Hodgkin's lymphoma and autoimmune disease. However, the cellular and molecular pathways for B cell depletion remain undefined because human mechanistic studies are limited. Proposed mechanisms include antibody-, effector cell-, and complement-dependent cytotoxicity, the disruption of CD20 signaling pathways, and the induction of apoptosis. To identify the mechanisms for B cell depletion in vivo, a new mouse model for anti-CD20 immunotherapy was developed using a panel of twelve mouse anti-mouse CD20 monoclonal antibodies representing all four immunoglobulin G isotypes. Anti-CD20 antibodies rapidly depleted the vast majority of circulating and tissue B cells in an isotype-restricted manner that was completely dependent on effector cell Fc receptor expression. B cell depletion used both FcgammaRI- and FcgammaRIII-dependent pathways, whereas B cells were not eliminated in FcR common gamma chain-deficient mice. Monocytes were the dominant effector cells for B cell depletion, with no demonstrable role for T or natural killer cells. Although most anti-CD20 antibodies activated complement in vitro, B cell depletion was completely effective in mice with genetic deficiencies in C3, C4, or C1q complement components. That the innate monocyte network depletes B cells through FcgammaR-dependent pathways during anti-CD20 immunotherapy has important clinical implications for anti-CD20 and other antibody-based therapies.
in vivo B cell depletion
Uchida J, Hamaguchi Y, Oliver JA, Ravetch JV, Poe JC, Haas KM, Tedder TF. (2004). "The innate mononuclear phagocyte network depletes B lymphocytes through Fc receptor-dependent mechanisms during anti-CD20 antibody immunotherapy" J Exp Med 199(12):1659-69. PubMed
Anti-CD20 antibody immunotherapy effectively treats non-Hodgkin's lymphoma and autoimmune disease. However, the cellular and molecular pathways for B cell depletion remain undefined because human mechanistic studies are limited. Proposed mechanisms include antibody-, effector cell-, and complement-dependent cytotoxicity, the disruption of CD20 signaling pathways, and the induction of apoptosis. To identify the mechanisms for B cell depletion in vivo, a new mouse model for anti-CD20 immunotherapy was developed using a panel of twelve mouse anti-mouse CD20 monoclonal antibodies representing all four immunoglobulin G isotypes. Anti-CD20 antibodies rapidly depleted the vast majority of circulating and tissue B cells in an isotype-restricted manner that was completely dependent on effector cell Fc receptor expression. B cell depletion used both FcgammaRI- and FcgammaRIII-dependent pathways, whereas B cells were not eliminated in FcR common gamma chain-deficient mice. Monocytes were the dominant effector cells for B cell depletion, with no demonstrable role for T or natural killer cells. Although most anti-CD20 antibodies activated complement in vitro, B cell depletion was completely effective in mice with genetic deficiencies in C3, C4, or C1q complement components. That the innate monocyte network depletes B cells through FcgammaR-dependent pathways during anti-CD20 immunotherapy has important clinical implications for anti-CD20 and other antibody-based therapies.
Uchida J, Lee Y, Hasegawa M, Liang Y, Bradney A, Oliver JA, Bowen K, Steeber DA, Haas KM, Poe JC, Tedder TF. (2004). "Mouse CD20 expression and function" Int Immunol 16(1):119-29. PubMed
CD20 plays a role in human B cell proliferation and is an effective target for immunotherapy. In this study, mouse CD20 expression and biochemistry were assessed for the first time using a new panel of CD20-specific mAb, with CD20 function assessed using CD20-deficient (CD20(-/-)) mice. CD20 expression was B cell restricted and was initiated during late pre-B cell development. The frequency and density of CD20 expression increased during B cell maturation in the bone marrow, with a subpopulation of transitional IgM(hi) B cells expressing higher CD20 levels than the majority of mature recirculating B cells. Transitional T1 B cells in the spleen also expressed high CD20 levels, providing a useful new marker for this B cell subset. In CD20(-/-) mice, immature and mature B cell IgM expression was approximately 20-30% lower relative to B cells from wild-type littermates. In addition, CD19-induced intracellular calcium responses were significantly reduced in CD20(-/-) B cells, with a less dramatic effect on IgM-induced responses. These results reveal a role for CD20 in transmembrane Ca(2+) movement in mouse primary B cells that complements previous results obtained using human CD20 cDNA-transfected cell lines. Otherwise, B cell development, tissue localization, signal transduction, proliferation, T cell-dependent antibody responses and affinity maturation were normal in CD20(-/-) mice. Thus, mouse and human CD20 share similar patterns of expression and function. These studies thereby provide an animal model for studying CD20 function in vivo and the molecular mechanisms that influence anti-CD20 immunotherapy.