Human Transferrin Receptor Research Articles

Combination Therapy of an Antibody Specific for Transferrin Receptor 1 (ch128.1/IgG1) With Bortezomib or Lenalidomide Results in Increased Survival in an In Vivo Model of Human Multiple Myeloma: A Brief Communication.

Transferrin receptor 1 (TfR1) is a universal cancer marker and a meaningful target for antibody-based immunotherapy. We previously developed a mouse/human chimeric antibody (ch128.1/IgG1) specific for the human TfR1 and reported that treatment of SCID-Beige mice bearing disseminated human multiple myeloma (MM) cells with ch128.1/IgG1 results in significant antitumor activity in early-stage and late-stage disease. Both bortezomib and lenalidomide are Food and Drug Administration (FDA) approved therapeutics used to treat MM in combination with other agents. Since combining treatments with different mechanisms of action is an effective antitumor strategy and given the relevance of bortezomib and lenalidomide in MM therapy, we decided to explore, for the first time, the combination of bortezomib or lenalidomide treatment with ch128.1/IgG1 within the context of late-stage MM disease. We found that treatment with a single dose of ch128.1/IgG1, or multiple doses of bortezomib or lenalidomide, used as single agents, results in significant antitumor activity in SCID-Beige mice bearing late-stage disseminated human MM.1S tumors. However, this antitumor activity is superior when ch128.1/IgG1 is combined with either bortezomib or lenalidomide, showing significantly longer survival compared with any therapy used alone. These novel results suggest that the combinations of ch128.1/IgG1 and bortezomib or lenalidomide are promising strategies against MM.

The AJ072 antibody against the human transferrin receptor labels the surface and early endosomes of HEK cells by immunofluorescence

The AJ072 antibody, against the human transferrin receptor, labels the plasma membrane and early endosomes of HEK cells by surface and total immunofluorescence.

Correction for Ferrero et al., "Antibody-Based Inhibition of Pathogenic New World Hemorrhagic Fever Mammarenaviruses by Steric Occlusion of the Human Transferrin Receptor 1 Apical Domain".

Correction for Ferrero et al., "Antibody-Based Inhibition of Pathogenic New World Hemorrhagic Fever Mammarenaviruses by Steric Occlusion of the Human Transferrin Receptor 1 Apical Domain".

Host receptor-targeted therapeutic approach to counter pathogenic New World mammarenavirus infections

Five New World mammarenaviruses (NWMs) cause life-threatening hemorrhagic fever (HF). Cellular entry by these viruses is mediated by human transferrin receptor 1 (hTfR1). Here, we demonstrate that an antibody (ch128.1/IgG1) which binds the apical domain of hTfR1, potently inhibits infection of attenuated and pathogenic NWMs in vitro. Computational docking of the antibody Fab crystal structure onto the known structure of hTfR1 shows an overlapping receptor-binding region shared by the Fab and the viral envelope glycoprotein GP1 subunit that binds hTfR1, and we demonstrate competitive inhibition of NWM GP1 binding by ch128.1/IgG1 as the principal mechanism of action. Importantly, ch128.1/IgG1 protects hTfR1-expressing transgenic mice against lethal NWM challenge. Additionally, the antibody is well-tolerated and only partially reduces ferritin uptake. Our findings provide the basis for the development of a novel, host receptor-targeted antibody therapeutic broadly applicable to the treatment of HF of NWM etiology.

The AJ072 antibody against the human transferrin receptor labels HeLa cells by surface immunofluorescence

The AJ072 antibody against the human transferrin receptor labels the cell membrane of HeLa cells by surface immunofluorescence; AM236 does not.

Motif-driven protein binder design towards transferrin receptor helical domain.

Human transferrin receptor 1 (TfR) is necessary for the delivery of the iron carrier protein transferrin into cells and can be utilized for targeted delivery across cellular membranes. Binding of transferrin to the receptor is regulated by hereditary hemochromatosis protein (HFE), an iron regulatory protein that partly shares a binding site with transferrin on TfR. Here, we derived essential binding interactions from HFE and computationally grafted these into a library of small protein scaffolds. One of the designed proteins, TB08, was further optimized computationally and experimentally to identify variants with improved binding to TfR. The optimized variant, TB08 S3.1, expressed well in the E. coli expression system and had an affinity to TfR in the low micromolar range, Kd ≈ 1 μm, as determined by surface plasmon resonance. A binding competition assay with transferrin further confirmed the interaction of the evolved variant to TfR at the shared binding surface. Additionally, the GFP-tagged evolved variant of TB08 demonstrated cellular internalization as determined by fluorescent and confocal microscopy in HeLa cells. The designed protein is small, allows for robust cargo tagging, and interacts specifically with TfR, thus making it a valuable tool for the characterization of TfR-mediated cellular transport mechanisms and for the assessment of engineering strategies for cargo delivery across cell membranes.

Investigating receptor-mediated antibody transcytosis using blood\u2013brain barrier organoid arrays

BackgroundThe pathways that control protein transport across the blood–brain barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development.MethodsWe applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis.ResultsBBB organoid arrays allowed the simultaneous growth of more than 3000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis.ConclusionsHuman BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies.

Rescue of a lysosomal storage disorder caused by Grn loss of function with a brain penetrant progranulin biologic

GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.

A DNA Aptameric Ligand of Human Transferrin Receptor Generated by Cell-SELEX.

General cancer-targeted ligands that can deliver drugs to cells have been given considerable attention. In this paper, a high-affinity DNA aptamer (HG1) generally binding to human tumor cells was evolved by cell-SELEX, and was further optimized to have 35 deoxynucleotides (HG1-9). Aptamer HG1-9 could be taken up by live cells, and its target protein on a cell was identified to be human transferrin receptor (TfR). As a man-made ligand of TfR, aptamer HG1-9 was demonstrated to bind at the same site of human TfR as transferrin with comparable binding affinity, and was proved to cross the epithelium barrier through transferrin receptor-mediated transcytosis. These results suggest that aptamer HG1-9 holds potential as a promising ligand to develop general cancer-targeted diagnostics and therapeutics.

New Insights into Red Blood Cell Microcytosis upon mTOR Inhibitor Administration

The aim of this study was to evaluate the effect of everolimus, a mammalian target of rapamycin (mTOR) inhibitor, on red blood cell parameters in the context of iron homeostasis in patients with tuberous sclerosis complex (TSC) and evaluate its effect on cell size in vitro. Everolimus has a significant impact on red blood cell parameters in patients with TSC. The most common alteration was microcytosis. The mean MCV value decreased by 9.2%, 12%, and 11.8% after 3, 6, and 12 months of everolimus treatment. The iron level declined during the first 3 months, and human soluble transferrin receptor concentration increased during 6 months of therapy. The size of K562 cells decreased when cultured in the presence of 5 μM everolimus by approximately 8%. The addition of hemin to the cell culture with 5 μM everolimus did not prevent any decrease in cell size. The stage of erythroid maturation did not affect the response to everolimus. Our results showed that the mTOR inhibitor everolimus caused red blood cell microcytosis in vivo and in vitro. This effect is not clearly related to a deficit of iron and erythroid maturation. This observation confirms that mTOR signaling plays a complex role in the control of cell size.

Antibody-Based Inhibition of Pathogenic New World Hemorrhagic Fever Mammarenaviruses by Steric Occlusion of the Human Transferrin Receptor 1 Apical Domain.

ABSTRACTPathogenic clade B New World mammarenaviruses (NWM) can cause Argentine, Venezuelan, Brazilian, and Bolivian hemorrhagic fevers. Sequence variability among NWM glycoproteins (GP) poses a challenge to the development of broadly neutralizing therapeutics against the entire clade of viruses. However, blockade of their shared binding site on the apical domain of human transferrin receptor 1 (hTfR1/CD71) presents an opportunity for the development of effective and broadly neutralizing therapeutics. Here, we demonstrate that the murine monoclonal antibody OKT9, which targets the apical domain of hTfR1, can sterically block cellular entry by viral particles presenting clade B NWM glycoproteins (GP1-GP2). OKT9 blockade is also effective against viral particles pseudotyped with glycoproteins of a recently identified pathogenic Sabia-like virus. With nanomolar affinity for hTfR1, the OKT9 antigen binding fragment (OKT9-Fab) sterically blocks clade B NWM-GP1s and reduces infectivity of an attenuated strain of Junin virus. Binding of OKT9 to the hTfR1 ectodomain in its soluble, dimeric state produces stable assemblies that are observable by negative-stain electron microscopy. A model of the OKT9-sTfR1 complex, informed by the known crystallographic structure of sTfR1 and a newly determined structure of the OKT9 antigen binding fragment (Fab), suggests that OKT9 and the Machupo virus GP1 share a binding site on the hTfR1 apical domain. The structural basis for this interaction presents a framework for the design and development of high-affinity, broadly acting agents targeting clade B NWMs.IMPORTANCE Pathogenic clade B NWMs cause grave infectious diseases, the South American hemorrhagic fevers. Their etiological agents are Junin (JUNV), Guanarito (GTOV), Sabiá (SABV), Machupo (MACV), Chapare (CHAV), and a new Sabiá-like (SABV-L) virus recently identified in Brazil. These are priority A pathogens due to their high infectivity and mortality, their potential for person-to-person transmission, and the limited availability of effective therapeutics and vaccines to curb their effects. While low homology between surface glycoproteins of NWMs foils efforts to develop broadly neutralizing therapies targeting NWMs, this work provides structural evidence that OKT9, a monoclonal antibody targeting a single NWM glycoprotein binding site on hTfR1, can efficiently prevent their entry into cells.

Design and Synthesis of Piperazine-Based Compounds Conjugated to Humanized Ferritin as Delivery System of siRNA in Cancer Cells.

Gene expression regulation by small interfering RNA (siRNA) holds promise in treating a wide range of diseases through selective gene silencing. However, successful clinical application of nucleic acid-based therapy requires novel delivery options. Herein, to achieve efficient delivery of negatively charged siRNA duplexes, the internal cavity of “humanized” chimeric Archaeal ferritin (HumAfFt) was specifically decorated with novel cationic piperazine-based compounds (PAs). By coupling these rigid-rod-like amines with thiol-reactive reagents, chemoselective conjugation was efficiently afforded on topologically selected cysteine residues properly located inside HumAfFt. The capability of PAs-HumAfFt to host and deliver siRNA molecules through human transferrin receptor (TfR1), overexpressed in many cancer cells, was explored. These systems allowed siRNA delivery into HeLa, HepG2, and MCF-7 cancer cells with improved silencing effect on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression with respect to traditional transfection methodologies and provided a promising TfR1-targeting system for multifunctional siRNA delivery to therapeutic applications.

A comparison of transferrin-receptor and epithelial cellular adhesion molecule targeting for microfluidic separation of cancer cells.

Microfluidic, flow cytometry, and immunomagnetic methods for cancer cell isolation have heavily relied on the Epithelial Cellular Adhesion Molecule (EpCAM) for affinity separation. While EpCAM has been used extensively for circulating tumor cell isolation, it cannot be used to isolate non-epithelial cells. The human transferrin receptor (CD71) can also be used for cancer cell isolation and has the advantage that as an affinity target it can separate virtually any cancer cell type, regardless of disease origin. However, direct comparison of the capture ability of EpCAM and CD71 has not been reported previously. In this work, cell capture with both EpCAM and CD71 were studied using a novel higher-throughput herringbone cell separation microfluidic device. Five separation chip models were designed and the one with the highest capture efficiency (average 90 ± 10%) was chosen to compare antigen targets for cell capture. Multiple cancer cell lines including CCRF-CEM, PC-3 and MDA-MB-231 were tested for cell capture performance using both ligands (anti-CD71 and anti-EpCAM) in the optimized chip design. PC-3 and MDA-MB-231 cells were spiked into blood at concentrations ranging from 0.5%-10%. PC-3 cells were separated by anti-CD71 and anti-EpCAM with 32-37% and 31-50% capture purity respectively, while MDA-MB-231 were separated with 35-53% and 33-56% capture purity using anti-CD71 and anti-EpCAM for all concentrations. The enrichment factor for the lowest concentrations of cells in blood ranged from 66-74X. The resulting enrichment of cancer cells shows that anti-CD71 was found to be statistically similar to anti-EpCAM for epithelial cancer cells, while anti-CD71 can be further used for non-epithelial cells, where anti-EpCAM cannot be used.

Transferrin receptor targeting by de novo sheet extension

The de novo design of polar protein-protein interactions is challenging because of the thermodynamic cost of stripping water away from the polar groups. Here, we describe a general approach for designing proteins which complement exposed polar backbone groups at the edge of beta sheets with geometrically matched beta strands. We used this approach to computationally design small proteins that bind to an exposed beta sheet on the human transferrin receptor (hTfR), which shuttles interacting proteins across the blood-brain barrier (BBB), opening up avenues for drug delivery into the brain. We describe a design which binds hTfR with a 20 nM Kd, is hyperstable, and crosses an in vitro microfluidic organ-on-a-chip model of the human BBB. Our design approach provides a general strategy for creating binders to protein targets with exposed surface beta edge strands.

Two peptides targeting endothelial receptors are internalized into murine brain endothelial cells.

The blood-brain barrier (BBB) is one of the main obstacles for therapies targeting brain diseases. Most macromolecules fail to pass the tight BBB, formed by brain endothelial cells interlinked by tight junctions. A wide range of small, lipid-soluble molecules can enter the brain parenchyma via diffusion, whereas macromolecules have to transcytose via vesicular transport. Vesicular transport can thus be utilized as a strategy to deliver brain therapies. By conjugating BBB targeting antibodies and peptides to therapeutic molecules or nanoparticles, it is possible to increase uptake into the brain. Previously, the synthetic peptide GYR and a peptide derived from melanotransferrin (MTfp) have been suggested as candidates for mediating transcytosis in brain endothelial cells (BECs). Here we study uptake, intracellular trafficking, and translocation of these two peptides in BECs. The peptides were synthesized, and binding studies to purified endocytic receptors were performed using surface plasmon resonance. Furthermore, the peptides were conjugated to a fluorophore allowing for live-cell imaging studies of their uptake into murine brain endothelial cells. Both peptides bound to low-density lipoprotein receptor-related protein 1 (LRP-1) and the human transferrin receptor, while lower affinity was observed against the murine transferrin receptor. The MTfp showed a higher binding affinity to all receptors when compared to the GYR peptide. The peptides were internalized by the bEnd.3 mouse endothelial cells within 30 min of incubation and frequently co-localized with endo-lysosomal vesicles. Moreover, our in vitro Transwell translocation experiments confirmed that GYR was able to cross the murine barrier and indicated the successful translocation of MTfp. Thus, despite binding to endocytic receptors with different affinities, both peptides are able to transcytose across the murine BECs.

Naturally acquired blocking human monoclonal antibodies to Plasmodiumvivax reticulocyte binding protein 2b

Plasmodium vivax preferentially invades reticulocytes and recognition of these cells is mediated by P. vivax Reticulocyte Binding Protein 2b (PvRBP2b) binding to human Transferrin receptor 1 (TfR1) and Transferrin (Tf). Longitudinal cohort studies in Papua New Guinea, Thailand and Brazil show that PvRBP2b antibodies are correlated with protection against P. vivax infection and disease. Here, we isolate and characterize anti-PvRBP2b human monoclonal antibodies from two individuals in Cambodia with natural P. vivax infection. These antibodies bind with high affinities and map to different regions of PvRBP2b. Several human antibodies block PvRBP2b binding to reticulocytes and inhibit complex formation with human TfR1-Tf. We describe different structural mechanisms for functional inhibition, including either steric hindrance with TfR1-Tf or the reticulocyte membrane. These results show that naturally acquired human antibodies against PvRBP2b can inhibit its function which is important for P. vivax invasion.

A novel circulating tamiami mammarenavirus shows potential for zoonotic spillover.

A detailed understanding of the mechanisms underlying the capacity of a virus to break the species barrier is crucial for pathogen surveillance and control. New World (NW) mammarenaviruses constitute a diverse group of rodent-borne pathogens that includes several causative agents of severe viral hemorrhagic fever in humans. The ability of the NW mammarenaviral attachment glycoprotein (GP) to utilize human transferrin receptor 1 (hTfR1) as a primary entry receptor plays a key role in dictating zoonotic potential. The recent isolation of Tacaribe and lymphocytic choriominingitis mammarenaviruses from host-seeking ticks provided evidence for the presence of mammarenaviruses in arthropods, which are established vectors for numerous other viral pathogens. Here, using next generation sequencing to search for other mammarenaviruses in ticks, we identified a novel replication-competent strain of the NW mammarenavirus Tamiami (TAMV-FL), which we found capable of utilizing hTfR1 to enter mammalian cells. During isolation through serial passaging in mammalian immunocompetent cells, the quasispecies of TAMV-FL acquired and enriched mutations leading to the amino acid changes N151K and D156N, within GP. Cell entry studies revealed that both substitutions, N151K and D156N, increased dependence of the virus on hTfR1 and binding to heparan sulfate proteoglycans. Moreover, we show that the substituted residues likely map to the sterically constrained trimeric axis of GP, and facilitate viral fusion at a lower pH, resulting in viral egress from later endosomal compartments. In summary, we identify and characterize a naturally occurring TAMV strain (TAMV-FL) within ticks that is able to utilize hTfR1. The TAMV-FL significantly diverged from previous TAMV isolates, demonstrating that TAMV quasispecies exhibit striking genetic plasticity that may facilitate zoonotic spillover and rapid adaptation to new hosts.

A brain penetrant progranulin biotherapeutic rescues lysosomal and inflammatory phenotypes in the brain of GRN knockout mice

AbstractBackgroundHeterozygous loss of function (LOF) mutations in GRN cause frontotemporal dementia (FTD), a neurodegenerative disorder associated with lysosomal dysfunction, TDP‐43 pathology and inflammation in the CNS. Additionally, homozygous LOF mutations cause neuronal ceroid lipofuscinosis, a lysosomal storage disorder. GRN encodes progranulin (PGRN), a soluble protein that is most abundantly expressed in microglia, where it localizes to lysosomes and can undergo secretion. Although the precise function of PGRN is unknown, growing evidence suggests that it regulates lysosomal function, inflammatory responses and promotes neuronal survival. Accordingly, PGRN LOF models are associated with lysosomal defects, hyperinflammatory responses, and decreased neuronal viability, both in vitro and in vivo. Because GRN‐FTD patients exhibit reduced levels of PGRN in biofluids and tissues, including the brain, a protein replacement therapy analogous to enzyme replacement therapy and capable of crossing the blood brain barrier (BBB) more efficiently may represent a powerful approach to slow or prevent disease progression.MethodHere we describe a novel therapeutic for increasing brain penetrance of PGRN, referred to as Protein Transport Vehicle (PTV):PGRN. PTV:PGRN consists of recombinant human PGRN fused to a modified Fc domain engineered to bind to the human transferrin receptor (huTfR), thus facilitating receptor‐mediated transcytosis across the BBB.ResultWe found that extracellular applications of PTV:PGRN rescue a range of Grn KO cell phenotypes in vitro, including lysosomal proteolysis and alteration in the levels of bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid involved in lysosomal lipid catabolism. Intravenously administered PTV:PGRN showed increased acute brain exposure in huTfR knock‐in (TfRmu/hu) mice relative to a regular Fc:PGRN fusion that does not bind to huTfR. Importantly, low doses of PTV:PGRN, but not Fc:PGRN, fully corrected lysosomal lipid alterations, including BMP deficiency, as well as inflammatory markers in the brain of Grn KO x TfRmu/hu mice.ConclusionOur data suggest that PTV:PGRN may represent a viable therapeutic strategy for the treatment of GRN‐FTD and potentially other disorders associated with PGRN deficiency.

Blood-brain barrier transport using a high affinity, brain-selective VNAR antibody targeting transferrin receptor 1.

Transfer across the blood-brain barrier (BBB) remains a significant hurdle for the development of biopharmaceuticals with therapeutic effects within the central nervous system. We established a functional selection method to identify high affinity single domain antibodies to the transferrin receptor 1 (TfR1) with efficient biotherapeutic delivery across the BBB. A synthetic phage display library based on the variable domain of new antigen receptor (VNAR) was used for in vitro selection against recombinant human TfR1 ectodomain (rh-TfR1-ECD) followed by in vivo selection in mouse for brain parenchyma penetrating antibodies. TXB2 VNAR was identified as a high affinity, species cross-reactive VNAR antibody against TfR1-ECD that does not compete with transferrin or ferritin for receptor binding. IV dosing of TXB2 when fused to human Fc domain (TXB2-hFc) at 25nmol/kg (1.875mg/kg) in mice resulted in rapid binding to brain capillaries with subsequent transport into the brain parenchyma and specific uptake into TfR1-positive neurons. Likewise, IV dosing of TXB2-hFc fused with neurotensin (TXB2-hFc-NT) at 25nmol/kg resulted in a rapid and reversible pharmacological response as measured by body temperature reduction. TXB2-hFc did not elicit any acute adverse reactions, bind, or deplete circulating reticulocytes or reduce BBB-expressed endogenous TfR1 in mice. There was no evidence of target-mediated clearance or accumulation in peripheral organs except lung. In conclusion, TXB2 is a high affinity, species cross-reactive, and brain-selective VNAR antibody to TfR1 that rapidly crosses the BBB and exhibits a favorable pharmacokinetic and safety profile and can be readily adapted to carry a wide variety of biotherapeutics from blood to brain.

Tuning the binding interface between Machupo virus glycoprotein and human transferrin receptor.

Machupo virus, known to cause hemorrhagic fevers, enters human cells via binding with its envelope glycoprotein to transferrin receptor 1 (TfR). Similarly, the receptor interactions have been explored in biotechnological applications as a molecular system to ferry therapeutics across the cellular membranes and through the impenetrable blood–brain barrier that effectively blocks any such delivery into the brain. Study of the experimental structure of Machupo virus glycoprotein 1 (MGP1) in complex with TfR and glycoprotein sequence homology has identified some residues at the interface that influence binding. There are, however, no studies that have attempted to optimize the binding potential between MGP1 and TfR. In pursuits for finding therapeutic solutions for the New World arenaviruses, and to gain a greater understanding of MGP1 interactions with TfR, it is crucial to understand the structure–sequence relationship driving the interface formation. By displaying MGP1 on yeast surface we have examined the contributions of individual residues to the binding of solubilized ectodomain of TfR. We identified MGP1 binding hot spot residues, assessed the importance of posttranslational N‐glycan modifications, and used a selection with random mutagenesis for affinity maturation. We show that the optimized MGP1 variants can bind more strongly to TfR than the native MGP1, and there is an MGP1 sequence that retains binding in the absence of glycosylation, but with the addition of further amino acid substitutions. The engineered variants can be used to probe cellular internalization or the blood‐brain barrier crossing to achieve greater understanding of TfR mediated internalization.