RecombiMAb anti-mouse CD71 (TfR1) (LALA-PG)
(switched from rat IgG2a, kappa)
Product Details
The 8D3-CP078 monoclonal antibody is a recombinant, chimeric version of the original 8D3 antibody. The variable domain sequences are identical to the original 8D3 but the constant region sequences have been switched from rat IgG2a to mouse IgG2a, kappa for use in murine models. Additionally, 8D3-CP078 contains LALA-PG mutations in the heavy chain Fc fragment rendering it unable to bind endogenous murine FcĪ³ receptors or C1q to induce antibody-dependent, cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Species-matched chimeric antibodies result in reduced immunogenicity and formation of anti-drug antibodies (ADAs) than xenogenic antibodies. In vivo studies using a murine chimeric 8D3 antibody showed rapid uptake in the brain at a level comparable to the rat 8D3 Mab. Anti-TfR1 antibodies with eliminated Fc effector function have reduced acute clinical signs and reticulocyte depletion. Furthermore, murine studies using a TfR-targeting bispecific antibody showed that chimeric mouse IgG2a LALA-PG effector silencing demonstrated improved safety profiles. The LALA-PG variant had less effector function, C1q binding and C3 fixation compared to other common Fc silencing mutations such as the LALA and DANG variants.The 8D3-CP078 monoclonal antibody reacts with native, soluble and denatured forms of murine CD71, also known as transferrin receptor protein 1 (TfR1). CD71 is a 170-180 kDa type II homodimeric transmembrane glycoprotein expressed on the surface of proliferating cells, reticulocytes, and erythroid precursors. CD71 plays a role in the control of cellular proliferation and is required for iron import from transferrin into cells by endocytosis. Cells of the vascular endothelium of brain capillaries that compose the blood-brain barrier (BBB) express high levels of TfR1 allowing for receptor-mediated transcytosis of large biomolecules into the brain. Murine chimeric anti-TfR1 8D3 has been used as a BBB transporter in mice and is suitable for studying CD71 expression and iron uptake into different tissues.
Specifications
| Isotype | Mouse IgG2a LALA-PG |
|---|---|
| Recommended Isotype Control(s) | RecombiMAb mouse IgG2a (LALA-PG) isotype control, anti-hen egg lysozyme |
| Recommended Dilution Buffer | InVivoPure pH 7.0 Dilution Buffer |
| Conjugation | This product is unconjugated. Conjugation is available via our Antibody Conjugation Services. |
| Mutations | LALA-PG |
| Immunogen | Mouse transformed endothelioma cell line t.end1 |
| Reported Applications |
Targeted drug delivery to the brain Immunohistochemistry Flow Cytometry Western Blot Transport across the BBB Receptor mediated transcytosis |
| Formulation |
PBS, pH 7.0 Contains no stabilizers or preservatives |
| Endotoxin |
<1EU/mg (<0.001EU/Ī¼g) Determined by LAL gel clotting assay |
| Aggregation |
<5% Determined by SEC |
| Purity |
>95% Determined by SDS-PAGE |
| Sterility | 0.2 Āµm 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 4Ā°C. Do not freeze. |
Additional Formats
Recommended Products
-
Recommended Isotype Control(s)
RecombiMAb mouse IgG2a (LALA-PG) isotype control, anti-hen egg lysozyme
-
Recommended Dilution Buffer
InVivoPure pH 7.0 Dilution Buffer
transport across the BBB
Lo M, Kim HS, Tong RK, Bainbridge TW, Vernes JM, Zhang Y, Lin YL, Chung S, Dennis MS, Zuchero YJ, Watts RJ, Couch JA, Meng YG, Atwal JK, Brezski RJ, Spiess C, Ernst JA. (2017). "Effector-attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in Mice" J Biol Chem 292(9):3900-3908. PubMed
The antibody Fc region regulates antibody cytotoxic activities and serum half-life. In a therapeutic context, however, the cytotoxic effector function of an antibody is often not desirable and can create safety liabilities by activating native host immune defenses against cells expressing the receptor antigens. Several amino acid changes in the Fc region have been reported to silence or reduce the effector function of antibodies. These earlier studies focused primarily on the interaction of human antibodies with human Fc-Ī³ receptors, and it remains largely unknown how such changes to Fc might translate to the context of a murine antibody. We demonstrate that the commonly used N297G (NG) and D265A, N297G (DANG) variants that are efficacious in attenuating effector function in primates retain potent complement activation capacity in mice, leading to safety liabilities in murine studies. In contrast, we found an L234A, L235A, P329G (LALA-PG) variant that eliminates complement binding and fixation as well as Fc-Ī³-dependent, antibody-dependent, cell-mediated cytotoxity in both murine IgG2a and human IgG1. These LALA-PG substitutions allow a more accurate translation of results generated with an "effectorless" antibody between mice and primates. Further, we show that both human and murine antibodies containing the LALA-PG variant have typical pharmacokinetics in rodents and retain thermostability, enabling efficient knobs-into-holes bispecific antibody production and a robust path to generating highly effector-attenuated bispecific antibodies for preclinical studies.
transport across the BBB
Couch JA, Yu YJ, Zhang Y, Tarrant JM, Fuji RN, Meilandt WJ, Solanoy H, Tong RK, Hoyte K, Luk W, Lu Y, Gadkar K, Prabhu S, Ordonia BA, Nguyen Q, Lin Y, Lin Z, Balazs M, Scearce-Levie K, Ernst JA, Dennis MS, Watts RJ. (2013). "Addressing safety liabilities of TfR bispecific antibodies that cross the blood-brain barrier" Sci Transl Med 5(183):183ra57, 1-12. PubMed
Bispecific antibodies using the transferrin receptor (TfR) have shown promise for boosting antibody uptake in brain. Nevertheless, there are limited data on the therapeutic properties including safety liabilities that will enable successful development of TfR-based therapeutics. We evaluate TfR/BACE1 bispecific antibody variants in mouse and show that reducing TfR binding affinity improves not only brain uptake but also peripheral exposure and the safety profile of these antibodies. We identify and seek to address liabilities of targeting TfR with antibodies, namely, acute clinical signs and decreased circulating reticulocytes observed after dosing. By eliminating Fc effector function, we ameliorated the acute clinical signs and partially rescued a reduction in reticulocytes. Furthermore, we show that complement mediates a residual decrease in reticulocytes observed after Fc effector function is eliminated. These data raise important safety concerns and potential mitigation strategies for the development of TfR-based therapies that are designed to cross the blood-brain barrier.
transport across the BBB
Boado RJ, Zhang Y, Wang Y, Pardridge WM. (2009). "Engineering and expression of a chimeric transferrin receptor monoclonal antibody for blood-brain barrier delivery in the mouse" Biotechnol Bioeng 102(4):1251-8. PubMed
Protein therapeutics may be delivered across the blood-brain barrier (BBB) by genetic fusion to a BBB molecular Trojan horse. The latter is an endogenous peptide or a peptidomimetic monoclonal antibody (MAb) against a BBB receptor, such as the insulin receptor or the transferrin receptor (TfR). Fusion proteins have been engineered with the MAb against the human insulin receptor (HIR). However, the HIRMAb is not active against the rodent insulin receptor, and cannot be used for drug delivery across the mouse BBB. The rat 8D3 MAb against the mouse TfR is active as a drug delivery system in the mouse, and the present studies describe the cloning and sequencing of the variable region of the heavy chain (VH) and light chain (VL) of the rat 8D3 TfRMAb. The VH and VL were fused to the constant region of mouse IgG1 heavy chain and mouse kappa light chain, respectively, to produce a new chimeric TfRMAb. The chimeric TfRMAb was expressed in COS cells following dual transfection with the heavy and light chain expression plasmids, and was purified by protein G affinity chromatography. The affinity of the chimeric TfRMAb for the murine TfR was equal to the 8D3 MAb using a radio-receptor assay and mouse fibroblasts. The chimeric TfRMAb was radio-labeled and injected into mice for a pharmacokinetics study of the clearance of the chimeric TfRMAb. The chimeric TfRMAb was rapidly taken up by mouse brain in vivo at a level comparable to the rat 8D3 MAb. In summary, these studies describe the genetic engineering, expression, and validation of a chimeric TfRMAb with high activity for the mouse TfR, which can be used in future engineering of therapeutic fusion proteins for BBB drug delivery in the mouse.