RecombiMAb anti-rat/mouse CD71 (TfR1)

transport across the BBB

Chang HY, Wu S, Li Y, Zhang W, Burrell M, Webster CI, Shah DK. (2021). "Brain pharmacokinetics of anti-transferrin receptor antibody affinity variants in rats determined using microdialysis" MAbs 13(1):1874121. PubMed

Receptor-mediated transcytosis (RMT) is used to enhance the delivery of monoclonal antibodies (mAb) into the central nervous system (CNS). While the binding to endogenous receptors on the brain capillary endothelial cells (BCECs) may facilitate the uptake of mAbs in the brain, a strong affinity for the receptor may hinder the efficiency of transcytosis. To quantitatively investigate the effect of binding affinity on the pharmacokinetics (PK) of anti-transferrin receptor (TfR) mAbs in different regions of the rat brain, we conducted a microdialysis study to directly measure the concentration of free mAbs at different sites of interest. Our results confirmed that bivalent anti-TfR mAb with an optimal dissociation constant (K_D) value (76 nM) among four affinity variants can have up to 10-fold higher transcytosed free mAb exposure in the brain interstitial fluid (bISF) compared to lower and higher affinity mAbs (5 and 174 nM). This bell-shaped relationship between K_D values and the increased brain exposure of mAbs was also visible when using whole-brain PK data. However, we found that mAb concentrations in postvascular brain supernatant (obtained after capillary depletion) were almost always higher than the concentrations measured in bISF using microdialysis. We also observed that the increase in mAb area under the concentration curve in CSF compartments was less significant, which highlights the challenge in using CSF measurement as a surrogate for estimating the efficiency of RMT delivery. Our results also suggest that the determination of mAb concentrations in the brain using microdialysis may be necessary to accurately measure the PK of CNS-targeted antibodies at the site-of-actions in the brain.

Candelaria PV, Leoh LS, Penichet ML, Daniels-Wells TR. (2021). "Antibodies Targeting the Transferrin Receptor 1 (TfR1) as Direct Anti-cancer Agents" Front Immunol . PubMed

The transferrin receptor 1 (TfR1), also known as cluster of differentiation 71 (CD71), is a type II transmembrane glycoprotein that binds transferrin (Tf) and performs a critical role in cellular iron uptake through the interaction with iron-bound Tf. Iron is required for multiple cellular processes and is essential for DNA synthesis and, thus, cellular proliferation. Due to its central role in cancer cell pathology, malignant cells often overexpress TfR1 and this increased expression can be associated with poor prognosis in different types of cancer. The elevated levels of TfR1 expression on malignant cells, together with its extracellular accessibility, ability to internalize, and central role in cancer cell pathology make this receptor an attractive target for antibody-mediated therapy. The TfR1 can be targeted by antibodies for cancer therapy in two distinct ways: (1) indirectly through the use of antibodies conjugated to anti-cancer agents that are internalized by receptor-mediated endocytosis or (2) directly through the use of antibodies that disrupt the function of the receptor and/or induce Fc effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). Although TfR1 has been used extensively as a target for antibody-mediated cancer therapy over the years, interest continues to increase for both targeting the receptor for delivery purposes and for its use as direct anti-cancer agents. This review focuses on the developments in the use of antibodies targeting TfR1 as direct anti-tumor agents.

in vivo anti-tumor activity

Daniels-Wells TR, Candelaria PV, Leoh LS, Nava M, Martínez-Maza O, Penichet ML. (2020). "An IgG1 Version of the Anti-transferrin Receptor 1 Antibody ch128.1 Shows Significant Antitumor Activity Against Different Xenograft Models of Multiple Myeloma: A Brief Communication" J Immunother 43(2):48-52. PubMed

The transferrin receptor 1 (TfR1) is a meaningful target for antibody-based cancer therapy given its overexpression on malignant cells and its central role in cancer pathology. We previously developed a mouse/human chimeric IgG3 targeting human TfR1 (ch128.1), which exhibits significant antitumor activity against multiple myeloma (MM) in xenograft models of SCID-Beige mice bearing disseminated ARH-77 or KMS-11 tumors. This activity is observed in early and late disease stages of disseminated KMS-11 tumors and, in this model, the mechanism of antitumor activity is Fc-mediated, involving macrophages. As human IgG1 is the isotype of choice for therapeutic antibodies targeting malignant cells and has several advantages compared with IgG3, including established manufacturability, we now developed an IgG1 version of ch128.1. A single dose of ch128.1/IgG1 shows significant antitumor activity, not only against early and late stages of disseminated KMS-11 tumors (Asian origin) but also against these stages of disseminated disease following injection of human MM cells MM.1S (African American origin) or its variant that is resistant to dexamethasone MM.1R. Treatment with the Fc mutant version of ch128.1/IgG1 (L234A/L235A/P329S) with impaired effector functions fails to confer protection against MM.1S and MM.1R tumors, indicating a crucial role of the Fc fragment in the antitumor activity, similar to its IgG3 counterpart. In fact, we found that ch128.1/IgG1, but not the mutant, elicits antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated phagocytosis in the presence of murine bone marrow-derived macrophages. Our results suggest that ch128.1/IgG1 is a promising therapeutic against human B-cell malignancies such as MM.

transport across the BBB

Amani H, Habibey R, Shokri F, Hajmiresmail SJ, Akhavan O, Mashaghi A, Pazoki-Toroudi H. (2019). "Selenium nanoparticles for targeted stroke therapy through modulation of inflammatory and metabolic signaling" Sci Rep 9(1):6044. PubMed

Ischemic cerebral stroke is a major cause of death and morbidity. Currently, no neuroprotective agents have been shown to impact the clinical outcomes in cerebral stroke cases. Here, we report therapeutic effects of Se nanoparticles on ischemic stroke in a murine model. Anti-transferrin receptor monoclonal antibody (OX26)-PEGylated Se nanoparticles (OX26-PEG-Se NPs) were designed and synthesized and their neuroprotective effects were measured using in vitro and in vivo approaches. We demonstrate that administration of the biodegradable nanoparticles leads to resolution of brain edema, protection of axons in hippocampus region, and myelination of hippocampal area after cerebral ischemic stroke. Our nanoparticle design ensures efficient targeting and minimal side effects. Hematological and biochemical analyses revealed no undesired NP-induced changes. To gain mechanistic insights into the therapeutic effects of these particles, we characterized the changes to the relevant inflammatory and metabolic signaling pathways. We assessed metabolic regulator mTOR and related signaling pathways such as hippo, Ubiquitin-proteasome system (ERK5), Tsc1/Tsc2 complex, FoxO1, wnt/β-catenine signaling pathway. Moreover, we examined the activity of jak2/stat3 signaling pathways and Adamts1, which are critically involved in inflammation. Together, our study provides a promising treatment strategy for cerebral stroke based on Se NP induced suppression of excessive inflammation and oxidative metabolism.

transport across the BBB

Tang X, Liang Y, Zhu Y, Xie C, Yao A, Chen L, Jiang Q, Liu T, Wang X, Qian Y, Wei J, Ni W, Dai J, Jiang Z, Hou W. (2015). "Anti-transferrin receptor-modified amphotericin B-loaded PLA-PEG nanoparticles cure Candidal meningitis and reduce drug toxicity" Int J Nanomedicine . PubMed

Fatal fungal infections in central nervous system (CNS) can occur through hematogenous spread or direct extension. At present, hydrophobic amphotericin B (AMB) is the most effective antifungal drug in clinical trials. However, AMB is hydrophobic and therefore penetrates poorly into the CNS, and therapeutic levels of AMB are hard to achieve. The transferrin receptor (TfR/CD71) located at the blood-brain barrier mediates transferrin transcytosis. In order to enhance the receptor-mediated delivery of AMB into CNS with therapeutic level, an anti-TfR antibody (OX26)-modified AMB-loaded PLA (poly[lactic acid])-PEG (polyethylene glycol)-based micellar drug delivery system was constructed. The prepared OX26-modified AMB-loaded nanoparticles (OX26-AMB-NPs) showed significant reduction of CNS fungal burden and an increase of mouse survival time. In conclusion, OX26-AMB-NPs represent a promising novel drug delivery system for intracerebral fungal infection.

transport across the BBB

Yue J, Liu S, Wang R, Hu X, Xie Z, Huang Y, Jing X. (2012). "Fluorescence-labeled immunomicelles: preparation, in vivo biodistribution, and ability to cross the blood-brain barrier" Macromol Biosci 12(9):1209-19. PubMed

Multifunctional hybrid micelles are prepared from amphiphilic mal-PEG-b-PLA and mPEG-b-P(LA-co-DHC/RhB) block copolymers. A specific anti-transferrin receptor antibody, OX26, is linked onto the surface of the micelles. ELISA indicates that the conjugated antibody preserves its activity. OX26 conjugation can increase the uptake efficiency of micelles by target cell lines (C6). Pharmacokinetics and in vivo biodistribution experiments are carried out to investigate the ability of OX26-conjugated micelles (immunomicelles) to cross the blood-brain barrier. The data show that the brain uptake of OX26-conjugated micelles is much more than that of OX26-free ones. Therefore, OX26-conjugated micelles will be promising drug carriers to cross the blood-brain barrier.

Immunohistochemistry (frozen)

Fabriek BO, Polfliet MM, Vloet RP, van der Schors RC, Ligtenberg AJ, Weaver LK, Geest C, Matsuno K, Moestrup SK, Dijkstra CD, van den Berg TK. (2007). "The macrophage CD163 surface glycoprotein is an erythroblast adhesion receptor" Blood 109(12):5223-9. PubMed

Erythropoiesis occurs in erythroblastic islands, where developing erythroblasts closely interact with macrophages. The adhesion molecules that govern macrophage-erythroblast contact have only been partially defined. Our previous work has implicated the rat ED2 antigen, which is highly expressed on the surface of macrophages in erythroblastic islands, in erythroblast binding. In particular, the monoclonal antibody ED2 was found to inhibit erythroblast binding to bone marrow macrophages. Here, we identify the ED2 antigen as the rat CD163 surface glycoprotein, a member of the group B scavenger receptor cysteine-rich (SRCR) family that has previously been shown to function as a receptor for hemoglobin-haptoglobin (Hb-Hp) complexes and is believed to contribute to the clearance of free hemoglobin. CD163 transfectants and recombinant protein containing the extracellular domain of CD163 supported the adhesion of erythroblastic cells. Furthermore, we identified a 13-amino acid motif (CD163p2) corresponding to a putative interaction site within the second scavenger receptor domain of CD163 that could mediate erythroblast binding. Finally, CD163p2 promoted erythroid expansion in vitro, suggesting that it enhanced erythroid proliferation and/or survival, but did not affect differentiation. These findings identify CD163 on macrophages as an adhesion receptor for erythroblasts in erythroblastic islands, and suggest a regulatory role for CD163 during erythropoiesis.

Flow Cytometry

Jiménez E, Sacedón R, Vicente A, Hernández-López C, Zapata AG, Varas A. (2002). "Rat peripheral CD4+CD8+ T lymphocytes are partially immunocompetent thymus-derived cells that undergo post-thymic maturation to become functionally mature CD4+ T lymphocytes" J Immunol 168(10):5005-13. PubMed

CD4+CD8+ double-positive (DP) T cells represent a minor subpopulation of T lymphocytes found in the periphery of adult rats. In this study, we show that peripheral DP T cells appear among the first T cells that colonize the peripheral lymphoid organs during fetal life, and represent approximately 40% of peripheral T cells during the perinatal period. Later their proportion decreases to reach the low values seen in adulthood. Most DP T cells are small size lymphocytes that do not exhibit an activated phenotype, and their proliferative rate is similar to that of the other peripheral T cell subpopulations. Only 30-40% of DP T cells expresses CD8beta chain, the remaining cells expressing CD8alphaalpha homodimers. However, both DP T cell subsets have an intrathymic origin since they appear in the recent thymic emigrant population after injection of FITC intrathymically. Functionally, although DP T cells are resistant to undergo apoptosis in response to glucocorticoids, they show poor proliferative responses upon CD3/TCR stimulation due to their inability to produce IL-2. A fraction of DP T cells are not actively synthesizing the CD8 coreceptor, and they gradually differentiate to the CD4 cell lineage in reaggregation cultures. Transfer of DP T lymphocytes into thymectomized SCID mice demonstrates that these cells undergo post-thymic maturation in the peripheral lymphoid organs and that their CD4 cell progeny is fully immunocompetent, as judged by its ability to survive and expand in peripheral lymphoid organs, to proliferate in response to CD3 ligation, and to produce IL-2 upon stimulation.