VHH antibodies and uses thereof

Abstract

The present invention relates to heavy chain-only variable domain (VHH) antibodies that specifically bind to transferrin receptor 1 (TfR1) and their use for transporting therapeutic or imaging agents intracellularly and across the blood-brain barrier.

Description

[Technical Field]

[0001] The present invention relates in particular to heavy chain-only variable domain (VHH) antibodies capable of binding to the transferrin receptor and the use of such VHH antibodies to transport molecules across the blood-brain barrier to relevant targets in the brain. [Background technology]

[0002] Brain exposure of drugs targeting central nervous system (CNS) diseases is inherently challenging because the blood-brain barrier (BBB) ​​protects the brain from unwanted substances present in the peripheral circulation, including antibodies and other proteins that may have therapeutic effects in the brain. Some small molecules are excluded from the brain compartment due to properties such as lipophilicity or lack of substrates for efflux pumps. Typically, compounds smaller than 600 daltons (Da) can cross the BBB unless excluded by other forces. Larger molecules, such as proteins, do not readily enter the brain in significant quantities unless assisted by specific active transport. In the case of therapeutic antibodies and protein-based drugs, it is estimated that less than 0.1% of systemically injected therapeutic antibodies reaches the brain compartment. Several strategies to overcome this narrow barrier are being tested and evaluated.

[0003] The BBB is composed of brain endothelial cells (BECs) as the first barrier to entry into the brain. Other cells of the so-called neurovascular unit (NVU) are also important for transport and interaction with target cells within the brain parenchyma. In the human brain, the cerebral blood vessels total 20 m 2 , representing a large surface area, which may present an opportunity for brain exposure for circulatory therapies.

[0004] Therapeutic antibodies or other protein-based drugs have great potential for treating CNS pathologies. However, the low availability of such therapeutic molecules in brain compartments remains a major problem. Recently, therapeutic monoclonal antibodies targeting the brain, such as amyloid beta protofibrils, have reported clinical efficacy. However, the exposure of these therapeutic molecules in human brain compartments after each administration is not considered optimal.

[0005] Therefore, there is a need to increase brain exposure of therapeutic molecules to improve the safety, dosing, and overall cost of CNS therapeutics.

[0006] Receptor-mediated transcytosis, a natural mechanism using endogenous receptors expressed on the luminal surface of the BBB, has been reported to be successful in increasing brain exposure of therapeutic molecules and to be clinically effective and safe.

[0007] WO2020 / 144233 discloses camelid heavy chain-only variable domain (VHH) molecules that bind to the transferrin receptor (TfR) and their uses for the delivery of molecules of pharmaceutical or diagnostic interest within cells and organs in pathological conditions, including cancer.

[0008] WO2016 / 077840, WO2019 / 089395, WO2020 / 056327, and WO2022 / 103769 disclose TfR-specific binding moieties that can be used to transport biomolecules across membranes, including the BBB and the gastrointestinal tract. These TfR-specific binding moieties comprise single-domain nurse shark variable domains of novel antigen receptor (VNAR) antibodies that bind to TfR.

[0009] WO2016 / 081643 relates to anti-transferrin receptor antibodies and methods of using same.

[0010] There remains a need for effective transporters of therapeutic molecules into the brain. These transporters should be capable of being conjugated or fused to therapeutic or diagnostic molecules in a manner that does not affect the target binding of the transporter or the therapeutic effect of the therapeutic molecule. Summary of the Invention

[0011] It is a general objective to provide VHH molecules that are specific for transferrin receptor 1 and are able to efficiently transport cargo to a desired compartment by receptor-mediated transcytosis.

[0012] These and other objects are achieved by embodiments of the present invention.

[0013] The invention is defined in the independent claims. Further embodiments of the invention are defined in the dependent claims.

[0014] An embodiment of the present invention relates to a heavy chain-only variable domain (VHH) antibody that specifically binds to transferrin receptor 1 (TfR1). The VHH antibody comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence GX1X2FX3X4X5Y, where X1 is T or N, X2 is P, D, or R, X3 is S, T, or G, X4 is N, L, M, or I, and X5 is N or E. The VHH antibody also comprises a CDR2 consisting of the amino acid sequence FTX6X7GX8T as defined in SEQ ID NO: 1, where X6 is S, T, or A, X7 is G, A, H, S, or T, and X8 is S, N, or D. The VHH antibody further comprises a CDR2 consisting of the amino acid sequence X9X 10 LX 11 X 12 wherein X is Y or H, and X 10 is F or Y and X 11 is D or G, and X 12 is V, N or D.

[0015] Another aspect of the present invention relates to a VHH antibody that specifically binds to TfR1. The VHH antibody has the amino acid sequence DSAFX as defined in SEQ ID NO: 38. 28 MNT, wherein X 28 is S or N. The VHH antibody also comprises a CDR2 consisting of the amino acid sequence IVSDDNT as defined in SEQ ID NO: 39. The VHH further comprises a CDR3 consisting of the amino acid sequence KGDVV as defined in SEQ ID NO: 40.

[0016] A further aspect of the present invention relates to a fusion molecule comprising a VHH antibody as described above linked to at least one molecule.

[0017] A related aspect of the present invention defines the above fusion molecules for use as a medicament, wherein at least one molecule is a therapeutic agent or for use in treating a disease or disorder of the central nervous system (CNS), and at least one molecule is a therapeutic agent capable of treating a disease or disorder of the CNS.

[0018] Yet another aspect of the present invention relates to a pharmaceutical composition comprising the above-described fusion molecule and a pharmaceutically acceptable vehicle, wherein at least one molecule is a therapeutic agent.

[0019] Other aspects of the present invention relate to nucleic acid molecules encoding the above-described VHH antibodies or fusion molecules, expression vectors comprising the above-described nucleic acid molecules operably linked to a promoter, and host cells comprising the above-described nucleic acid molecules or the above-described expression vectors.

[0020] The VHH antibodies of the present invention specifically bind to TfR1 without interfering with the binding of transferrin to TfR1. The binding properties of the VHH antibodies are tailored to optimize transcytosis on endothelial cells at the BBB. This allows the VHH antibodies to be used as transporters of various molecules, including therapeutic agents or diagnostic imaging agents, to the brain compartment when administered systemically.

[0021] The embodiments, together with further objects and advantages thereof, may best be understood by reference to the following description taken in conjunction with the accompanying drawings, in which: [Brief explanation of the drawings]

[0022] [Figure 1A]Graphical representation of llama immunization with immunogens. Immunization of llamas (Llama glama) (N=2) was performed at Modiquest Research BV (The Netherlands) according to the ModiPhage™ method. The primary immunization was performed on day 1 with the human transferrin receptor 1 ectodomain (hTfR1) (500 μg protein + complete Freund's adjuvant (CFA), intramuscularly (im)). A boosting dose (500 μg protein + incomplete Freund's adjuvant (IFA), im) was administered on day 21 for mouse TfR1 (mTfR1) and day 42 for hTfR1. Peripheral blood was withdrawn on day 54, and the immune response was tested by enzyme-linked immunosorbent assay (ELISA) to test for the presence of both immunoglobulin G1 (IgG1) and IgG2 / 3 antibodies and their reactivity to mTfR1 and hTfR1. Immunizations were repeated on days 86 (hTfR1 + IFA) and 107 (hTfR1 + IFA). New peripheral blood samples were analyzed on day 117. An additional pre-harvest boost dose with a 1:1 mixture of hTfR1 and mTfR1 (250 μg + 250 μg + IFA) was given on day 120, followed by peripheral blood lymphocyte (PBL) harvest on day 124. [Figure 1B]Strategy for establishing TfR1-binding VHHs. Graphical overview of phage library establishment. PBLs were isolated by density gradient centrifugation using approximately 1.5 x 109 Ficoll-Paque™ PLUS. Ribonucleic acid (RNA) was extracted, followed by reverse transcription into complementary deoxyribonucleic acid (cDNA). The cDNA was then used as a template in polymerase chain reaction (PCR) to amplify the IgG repertoire (IgG2 / 3, variable heavy chain (VH) domain to constant heavy chain 2 (CH2), heavy chain-only antibodies). A first PCR reaction was performed to amplify all antibodies (IgG1 and IgG2 / 3) and a second nested PCR reaction was performed to amplify and isolate the VHH repertoire (described in Pardon et al., A general protocol for the generation of Nanobodies for structural biology. Nat Protoc 9:674-693 (2014)). After vector digestion, DNA amplification was performed as described above and a total of 1600 ng of DNA was used to electroporate TG1 Escherichia coli, yielding a library with an estimated size of 3.2 x 10 8 . [Figure 2] Graphical representation of VHH and various VHH-containing fusion proteins. Figure 2 illustrates a schematic of a VHH monomer and various VHH-containing fusion proteins. Free VHH binds as a monomer to one binding site on TfR1. Bivalent VHH fusion proteins were formed between two VHH and a human fragment crystallizable (Fc) region. Functional monovalent fusion proteins were produced between one VHH and a single-chain variable fragment (scFv) or between one VHH and a non-antibody-derived molecule (X). Figure 2 also shows a bivalent VHH fusion protein with two non-antibody-derived molecules (X). [Figure 3]Affinity measurement of monomeric VHH binding to hTfR1 using SPR. The figure shows representative sensorgrams of KB_B01 (A) at increasing concentrations ranging from 6.25 to 100 nM for binding to human transferrin (hTf)-loaded hTfR1 immobilized on a dextran-coated gold (CM5) chip (Cytiva) by amine coupling. As a control, the full-length monoclonal antibody BA1 (B) was run at the same concentration. [Figure 4A] Affinity measurement of dimeric VHH-Fc proteins binding to hTfR1, cTfR1, and mTfR1 using SPR. The figure shows representative sensorgrams of hTfR1 at increasing concentrations ranging from 0.16 to 100 nM binding to the Fc fusion KB_B01 loaded onto a protein A-coated chip (Cytiva). [Figure 4B] Affinity measurement of dimeric VHH-Fc proteins binding to hTfR1, cTfR1, and mTfR1 using SPR. The figure shows representative sensorgrams of cynomolgus monkey TfR1 (cTfR1) at increasing concentrations ranging from 0.16 to 100 nM binding to the Fc fusion KB_B01 loaded onto a protein A-coated chip (Cytiva). [Figure 4C] Affinity measurement of dimeric VHH-Fc proteins binding to hTfR1, cTfR1, and mTfR1 using SPR. The figure shows representative sensorgrams of mTfR1 at increasing concentrations ranging from 0.16 to 100 nM binding to the Fc fusion KB_B01 loaded onto a protein A-coated chip (Cytiva). [Figure 4D] Affinity measurement of dimeric VHH-Fc proteins binding to hTfR1, cTfR1, and mTfR1 using SPR. The figure shows representative sensorgrams of hTfR1 at increasing concentrations ranging from 0.16 to 100 nM binding to BV loaded onto a protein A-coated chip (Cytiva). [Figure 4E]Affinity measurement of dimeric VHH-Fc proteins binding to hTfR1, cTfR1, and mTfR1 using SPR. The figure shows representative sensorgrams of cynomolgus monkey TfR1 (cTfR1) at increasing concentrations ranging from 0.16 to 100 nM binding to BV loaded onto a protein A-coated chip (Cytiva). [Figure 4F] Affinity measurement of dimeric VHH-Fc proteins binding to hTfR1, cTfR1, and mTfR1 using SPR. The figure shows representative sensorgrams of mTfR1 at increasing concentrations ranging from 0.16 to 100 nM binding to BV loaded onto a protein A-coated chip (Cytiva). [Figure 4G] Affinity measurement of dimeric VHH-Fc proteins binding to hTfR1, cTfR1, and mTfR1 using SPR. The figure shows representative sensorgrams of hTfR1 at increasing concentrations ranging from 0.16 to 100 nM binding to the reference full-length monoclonal antibody BA2 loaded onto a protein A-coated chip (Cytiva). [Figure 4H] Affinity measurement of dimeric VHH-Fc proteins binding to hTfR1, cTfR1, and mTfR1 using SPR. The figure shows representative sensorgrams of cynomolgus monkey TfR1 (cTfR1) at increasing concentrations ranging from 0.16 to 100 nM binding to the reference full-length monoclonal antibody BA2 loaded onto a protein A-coated chip (Cytiva). [Figure 4I] Affinity measurement of dimeric VHH-Fc proteins binding to hTfR1, cTfR1, and mTfR1 using SPR. The figure shows representative sensorgrams of mTfR1 at increasing concentrations ranging from 0.16 to 100 nM binding to the reference full-length monoclonal antibody BA2 loaded onto a protein A-coated chip (Cytiva). [Figure 5A] Affinity measurements of dimeric KB_B01-Fc for hTfR1 and cTfR1 in the absence and presence of hTf. The figure shows a representative sensorgram of KB_B01-Fc binding to hTfR1 in the absence of an excess concentration of 250 nM hTf. [Figure 5B] Affinity measurements of dimeric KB_B01-Fc for hTfR1 and cTfR1 in the absence and presence of hTf. The figure shows a representative sensorgram of KB_B01-Fc binding to hTfR1 in the presence of an excess concentration of 250 nM hTf. [Figure 5C] Affinity measurements of dimeric KB_B01-Fc for hTfR1 and cTfR1 in the absence and presence of hTf. The figure shows a representative sensorgram of reference BV-Fc binding to hTfR1 in the absence of an excess concentration of 250 nM hTf. [Figure 5D] Affinity measurements of dimeric KB_B01-Fc for hTfR1 and cTfR1 in the absence and presence of hTf. The figure shows a representative sensorgram of reference BV-Fc binding to hTfR1 in the presence of an excess concentration of 250 nM hTf. [Figure 5E] Affinity measurements of dimeric KB_B01-Fc for hTfR1 and cTfR1 in the absence and presence of hTf. The figure shows a representative sensorgram of BA2 binding to hTfR1 in the absence of an excess concentration of 250 nM hTf. [Figure 5F] Affinity measurements of dimeric KB_B01-Fc for hTfR1 and cTfR1 in the absence and presence of hTf. The figure shows a representative sensorgram of BA2 binding to hTfR1 in the presence of an excess concentration of 250 nM hTf. [Figure 6] Sequence alignment of sequences closely related to KB_B01. The figure shows the amino acid sequences of KB_B01 (SEQ ID NO: 30) and closely related clones KB_B02 to KB_B10, as well as the reference VHH BV, in single-letter code. Dots indicate amino acids identical to the reference sequence (KB_B01), and boxes indicate the three complementarity-determining regions (CDRs). [Figure 7]Affinity measurement of VHH and scFv fusion proteins binding to hTfR1 using SPR. The figure shows representative sensorgrams of hTfR1 exposed to KB_B01 amine-coupled to the surface of a CM5 chip and genetically fused to scFv at the C-terminus of the VL (scFv-VHH, A) or the N-terminus of the VH (VHH-scFv, B). Increasing concentrations were exposed in 1:4 step increments ranging from 0.25 to 64 nM. A reference VHH BV was also fused to either end of the same scFv (scFv-VHH, C) and (VHH-scFv, D) and tested in 1:5 step increments over a concentration range of 0.08 to 50 nM. [Figure 8] hTfR1-mediated cellular uptake in HEK293T cells. HEK293T cells were cultured in 96-well plates until confluence was reached (4-5 days). KB_B01-Fc, BV-Fc, or BA1, and a negative control (hTfR1-inactive VHH-Fc), were added at 20 nM or 5 nM (diluted in DMEM), respectively, and incubated for 40 and 120 min. After incubation, the cells were washed with PBS and fixed with 4% PFA. For longer incubation times, the medium was replaced at t = 30 min by incubation with fresh DMEM. After fixation, cells were immunostained and analyzed by confocal microscopy. Representative images are shown here of cellular uptake of KB_B01-Fc, BV-Fc, and BA1, as well as the negative control, at 20 nM after 40 min, or of KB_B01-Fc, BV-Fc, and BA1 at 5 nM after 120 min of incubation. The figure shows the absence of signal using a non-hTfR1 binding VHH-Fc negative control. Images are shown as 8-bit grayscale. Scale bar 50 μM. [Figure 9]Brain and blood distribution of VHH-Fc fusions after systemic administration. Radiolabeled [I]VHH-Fc fusion proteins were injected as described in Example VIII. Brain (A) and blood (B) concentrations are shown. Blood samples were taken at t = 30 min, t = 1 h, and at termination (2.5 h). 2.5 h after injection, animals were euthanized, transcardially perfused with NaCl, and brains were excised and analyzed for radioactivity. All concentrations are expressed as % of injected dose (radioactivity) and relative to brain normalized to body weight (standardized uptake value, SUV). [Figure 10] Brain and Biodistribution after Systemic Administration of VHH-scFv Radiolabeled [I]VHH-scFv fusion proteins were injected as described in Example VIII. Brain exposure was analyzed in both wild-type mice and APPNL-GF, a mouse model of Alzheimer's disease (AD) designated "nlgf" in the figure. Brain concentrations as SUVs 2 hours after injection of KB_B01-scFv and BV-scFv fused to the N-terminus of 3D6 scFv, as described in Example IV (A). Dynamics of brain retention in APPNL-GF mice versus WT mice at 2, 6, and 24 hours after injection of KB_B01-scFv (B). Brain (C) and blood (C) concentrations 24 hours after injection. One group of APPNL-GF animals was injected with KB_B01-scFv and BV-scFv as well as a negative control VHH-scFv fusion terminated at 24 hours. Retention of AD mice versus WT is read for each construct. [Figure 11]Vascular vs. Parenchymal Distribution of scFv-VHH Fusions. As described in Example IX, one hemisphere of the brain from a mouse injected with VHH-scFv (as described in Example VIII) was cryosectioned. Representative sections were first immunostained for the vascular marker CD31, then immersed in Nuclear Track Emulsion (NTE), and subsequently developed for extended periods in a cold room. Images of both CD31 and NTE were then acquired using a wide-field Zeiss Observer microscope. Overlay images were generated as binary 8-bit images (A). From these images, white dots in the NTE indicate the localization of 125I-protein and its relationship to blood vessels. For clarity, the lower panel shows the vascular immunostaining, while the upper panel shows the overlay, with the vascular profile shown as a thin outline (white line). Scale bar: 20 μm. Quantification was performed from 20 frames of the same brain, as shown in the micrographs of the vascular profile or relative distribution within the parenchyma (B). Values ​​are presented as the mean ± standard deviation (SD). [Figure 12] Brain and Blood Distribution of VHH-Fc Fusion KB_B012 after Systemic Administration in TfR1 Extracellular Domain-Humanized Mice. Radiolabeled [I]VHH-Fc fusion protein KB_B03 (KB_B12-Fc) was injected into hTfR1 homozygous (HOM), heterozygous (HET), and wild-type (WT) genotypes as described in Example XI. (A) Blood concentration over time. Blood samples were collected at t = 5 min, t = 30 min, t = 1 h, and at termination (2.0 h). (B) Distribution in the blood compartment among leukocytes, plasma, and pellet. (C) Brain concentration at 2.0 h. Two hours after injection, animals were euthanized and transcardially perfused with NaCl, and brains were excised and analyzed for radioactivity. Blood concentrations are expressed as (% of injected dose, radioactivity) and brain uptake is expressed as standardized uptake value, SUV (% of injected dose corrected for animal weight). DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention relates to heavy chain-only variable domain (VHH) antibodies, also called single domain antibodies, in particular to such VHH antibodies capable of binding to transferrin (Tf) receptor 1 (TfR1), and to the use of such VHH antibodies for transporting molecules across the blood-brain barrier (BBB).

[0024] Brain exposure of drugs targeting central nervous system (CNS) diseases is inherently challenging because the BBB protects the brain from unwanted substances present in the peripheral circulation, including antibodies, proteins, and other molecules that may have therapeutic effects in the brain. Receptor-mediated transcytosis, a natural mechanism that uses endogenous receptors expressed on the luminal surface of the BBB, has been suggested to increase BBB exposure of drugs. One such endogenous receptor that can be used to achieve receptor-mediated transcytosis is the transferrin receptor.

[0025] The transferrin receptor (TR) is a glycoprotein that binds iron (Fe) and mediates its transport through plasma. It internalizes the transferrin-iron complex via receptor-mediated endocytosis. Humans and other mammals have two transport receptors: transferrin receptor 1 (TfR1) and transferrin receptor 2 (TfR2). While TfR1 is a high-affinity, ubiquitously expressed receptor, TfR2 expression is restricted to specific cell types and is unaffected by intracellular iron concentrations. TfR2 binds transferrin with 25-30 times lower affinity than TfR1. As used herein, the term "transferrin receptor" refers to the TfR1 homolog, also known as cluster of differentiation 71 (CD71), encoded by the TFRC gene in humans.

[0026] The extracellular domains of human TfR1 (hTfR1) and mouse TfR1 (mTfR1) were used to immunize llamas (Llama glama) to generate a library of VHH antibody-expressing clones. These VHH antibodies were screened for binding to hTfR1, and a lead VHH antibody, KB_B01, with desirable binding properties was selected. Further, VHH antibodies were identified that exhibit sequence similarity to KB_B01. The VHH antibodies of the present invention specifically bind to hTfR1 and also to mTfR1 with binding affinity to hTfR1 and binding properties favorable for endocytosis, including binding, without interfering with transferrin (Tf) binding to hTfR1. As shown in the experimental section, these VHH antibodies retain TfR1-binding properties when present as VHH-containing fusion proteins, including bivalent VHH fusion proteins formed between two VHH antibodies and a human constant antibody fragment crystallizable region (Fc), and functional fusion proteins fused to drug molecules, such as single-chain variable fragments (scFv). Experimental data further demonstrate that VHH antibodies, including VHH-containing fusion proteins, were internalized by human cells expressing hTfR1 and transcytosed through human brain-like endothelial cell monolayers, used as an in vitro model of the BBB. The brain distribution of the VHH antibodies was validated in vivo and demonstrated superior brain distribution to reference antibodies, including reference VHH antibodies.

[0027] The VHH antibodies of the present invention further have advantages over the VHH antibodies disclosed in WO2020 / 144233. First, fusion proteins of VHH antibodies and scFv molecules were distributed to the brain parenchyma without being trapped in blood vessels, which was superior to fusion proteins of the VHH antibodies of WO2020 / 144233 with the same scFv molecules. More specifically, fusion proteins of the VHH antibodies of the present invention showed preferential distribution to the brain parenchyma of nearly 80%, whereas the corresponding figure for the VHH antibodies of WO2020 / 144233 was only slightly above 50%. Another advantage of the VHH antibodies of the present invention over the VHH antibodies of WO2020 / 144233 is that the VHH antibodies of the present invention have a higher brain-to-blood ratio compared to the negative control. This means that the VHH antibodies of the present invention have less nonspecific binding in other compartments in the body and are suitable for specifically targeting disease-related proteins in the brain.

[0028] An embodiment of the present invention relates to a heavy chain-only variable domain (VHH) antibody that specifically binds to transferrin receptor 1 (TfR1). The VHH antibody comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence GX1X2FX3X4X5Y, where X1 is T or N, X2 is P, D, or R, X3 is S, T, or G, X4 is N, L, M, or I, and X5 is N or E. The VHH antibody also comprises a CDR2 consisting of the amino acid sequence FTX6X7GX8T as defined in SEQ ID NO: 1, where X6 is S, T, or A, X7 is G, A, H, S, or T, and X8 is S, N, or D. The VHH antibody further comprises a CDR2 consisting of the amino acid sequence X9X 10 LX 11 X 12 wherein X is Y or H, and X 10 is F or Y and X 11 is D or G, and X 12 is V, N or D.

[0029] In one embodiment, CDR1 consists of the amino acid sequence GTX2FX3X4NY as defined in SEQ ID NO: 2, where X2 is P or D, X3 is S or T, and X4 is N, L, or M. In this embodiment, CDR3 consists of the amino acid sequence X9FLX 11 X 12 wherein X9 is Y or H; 11 is D or G, and X 12 is V, N or D.

[0030] In one embodiment, CDR1 consists of the amino acid sequence GTDFSX4NY as defined in SEQ ID NO: 3, where X4 is L or M. In this embodiment, CDR2 consists of the amino acid sequence FTX6X7GST as defined in SEQ ID NO: 4, where X6 is S or A and X7 is S or T. In this embodiment, CDR3 consists of the amino acid sequence HFLGD as defined in SEQ ID NO: 5.

[0031] In one embodiment, CDR1 consists of the amino acid sequence GTDFSLNY as defined in SEQ ID NO: 6. In this embodiment, CDR2 consists of the amino acid sequence FTAX7GST as defined in SEQ ID NO: 7, where X7 is S or T. In this embodiment, CDR3 consists of the amino acid sequence HFLGD as defined in SEQ ID NO: 5.

[0032] In a particular embodiment, CDR1 consists of the amino acid sequence GTDFSLNY as defined in SEQ ID NO: 6, CDR2 consists of the amino acid sequence FTATGST as defined in SEQ ID NO: 8, and CDR3 consists of the amino acid sequence HFLGD as defined in SEQ ID NO: 5. A VHH antibody having CDR regions according to this particular embodiment is designated herein as KB_B06.

[0033] In another particular embodiment, CDR1 consists of the amino acid sequence GTDFSLNY as defined in SEQ ID NO: 6, CDR2 consists of the amino acid sequence FTASGST as defined in SEQ ID NO: 9, and CDR3 consists of the amino acid sequence HFLGD as defined in SEQ ID NO: 5. A VHH antibody having CDR regions according to this particular embodiment is designated herein as KB_B07.

[0034] In a further particular embodiment, CDR1 consists of the amino acid sequence GTDFSMNY as defined in SEQ ID NO: 10, CDR2 consists of the amino acid sequence FTSTGST as defined in SEQ ID NO: 11, and CDR3 consists of the amino acid sequence HFLGD as defined in SEQ ID NO: 5. A VHH antibody having CDR regions according to this particular embodiment is designated herein as KB_B05.

[0035] In one embodiment, CDR1 consists of the amino acid sequence GTX2FX3X4NY as defined in SEQ ID NO: 2, where X2 is P or D, X3 is S or T, and X4 is N, L, or M. In this embodiment, CDR2 consists of the amino acid sequence FTX6X7GX8T as defined in SEQ ID NO: 1, where X6 is S or T, X7 is G, A, H, or S, and X8 is S, N, or D. In this embodiment, CDR3 consists of the amino acid sequence YFLDX as defined in SEQ ID NO: 12. 12 where X 12 is V or N.

[0036] In one embodiment, CDR1 consists of the amino acid sequence GTPFX3NNY as defined in SEQ ID NO: 13, wherein X3 is S or T. In this embodiment, CDR2 consists of the amino acid sequence FTSX7GX8T as defined in SEQ ID NO: 14, wherein X7 is G or A and X8 is S or N. In this embodiment, CDR3 consists of the amino acid sequence YFLDV as defined in SEQ ID NO: 15.

[0037] In a particular embodiment, CDR1 consists of the amino acid sequence GTPFSNNY as defined in SEQ ID NO: 16, CDR2 consists of the amino acid sequence FTSGGST as defined in SEQ ID NO: 17, and CDR3 consists of the amino acid sequence YFLDV as defined in SEQ ID NO: 15. A VHH antibody having CDR regions according to this particular embodiment is designated herein as KB_B01.

[0038] In another particular embodiment, CDR1 consists of the amino acid sequence GTPFTNNY as defined in SEQ ID NO: 18, CDR2 consists of the amino acid sequence FTSAGNT as defined in SEQ ID NO: 19, and CDR3 consists of the amino acid sequence YFLDV as defined in SEQ ID NO: 15. A VHH antibody having CDR regions according to this particular embodiment is designated herein as KB_B02.

[0039] In one embodiment, CDR1 consists of the amino acid sequence GTDFSX4NY as defined in SEQ ID NO: 3, where X4 is L or M. In this embodiment, CDR2 consists of the amino acid sequence FTX6X7GX8T as defined in SEQ ID NO: 1, where X6 is S or T, X7 is H or S, and X8 is N or D. In this embodiment, CDR3 consists of the amino acid sequence YFLDN as defined in SEQ ID NO: 20.

[0040] In a particular embodiment, CDR1 consists of the amino acid sequence GTDFSLNY as defined in SEQ ID NO: 6, CDR2 consists of the amino acid sequence FTTHGDT as defined in SEQ ID NO: 21, and CDR3 consists of the amino acid sequence YFLDN as defined in SEQ ID NO: 20. A VHH antibody having CDR regions according to this particular embodiment is designated herein as KB_B03.

[0041] In another particular embodiment, CDR1 consists of the amino acid sequence GTDFSMNY as defined in SEQ ID NO: 10, CDR2 consists of the amino acid sequence FTSSGNT as defined in SEQ ID NO: 22, and CDR3 consists of the amino acid sequence YFLDN as defined in SEQ ID NO: 20. A VHH antibody having CDR regions according to this particular embodiment is designated herein as KB_B04.

[0042] In a further particular embodiment, CDR1 consists of the amino acid sequence GNRFGIEY as defined in SEQ ID NO: 23, CDR2 consists of the amino acid sequence FTSAGST as defined in SEQ ID NO: 24, and CDR3 consists of the amino acid sequence HYLGD as defined in SEQ ID NO: 25. A VHH antibody having CDR regions according to this particular embodiment is designated herein as KB_B08.

[0043] In one embodiment, the VHH antibody is of the formula: framework region 1 (FR1)-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0044] In one embodiment, FR1 has the amino acid sequence QVQLQESGX as defined in SEQ ID NO:26. 14 GX 15 VQX 16 GGSLRLSCX 17 X 18 S, where X 14 is R or G, and X 15 is L or V, and X 16 is A or T, and X 17 is A, T or V, and X 18 is A, L or V.

[0045] In certain embodiments, FR1 has the amino acid sequence QVQLQESGGGLVQX as defined in SEQ ID NO:77. 16 GGSLRLSCX 17 X 18 S, where X 16 is A or T, and X 17 is A, T or V, and X 18 is A, L or V.

[0046] In one embodiment, FR2 comprises the amino acid sequence X defined in SEQ ID NO:27. 19 RWYRQAPGX 20 QREX 21 VAG, where X 19 is M or L, and X 20 is K or N, and X 21 is W or F.

[0047] In certain embodiments, FR2 comprises the amino acid sequence X as defined in SEQ ID NO:78. 19 RWYRQAPGX 20 QREWVAG, where X 19 is M or L, and X 20 is K or N.

[0048] In one embodiment, FR3 comprises the amino acid sequence NYX as defined in SEQ ID NO:28. 20 DSX 21 KGRFTIX 22 RDNAX 23 X 24 TVYLQMDX 25 LX 26 PEDTAVYX 27 C, where X 20 is A, P or G, and X 21 is V or M, and X 22 is S or G, and X 23 is E or K, and X 24 is G or N, and X 25 is N or S, and X 26 is K, I or T, and X 27 is Y or F.

[0049] In certain embodiments, FR3 comprises the amino acid sequence NYX as defined in SEQ ID NO:28. 20 DSX 21 KGRFTIX 22 RDNAX 23 X 24 TVYLQMDX 25 LX 26 PEDTAVYX 27C, where X 20 is A or P, and X 21 is V or M, and X 22 is S or G, and X 23 is E or K, and X 24 is G or N, and X 25 is N or S, and X 26 is K or T, and X 27 is Y or F.

[0050] In one embodiment, FR4 has the amino acid sequence WGQGTQVTVSS as defined in SEQ ID NO:29.

[0051] In certain embodiments, the VHH antibody has the amino acid sequences of FR1, FR2, FR3 and FR4 defined in SEQ ID NOs: 26 or 77, 27 or 78, 28 and 29.

[0052] The present invention also encompasses VHH antibodies having an FR1 amino acid sequence comprising or consisting of SEQ ID NO: 26 or 77, or a variant thereof having at least 88% sequence identity to SEQ ID NO: 26 or 77, preferably at least 92% sequence identity, more preferably at least 96% sequence identity.

[0053] The present invention also encompasses VHH antibodies having an FR2 amino acid sequence comprising or consisting of SEQ ID NO: 27 or 78, or a variant thereof having at least 82% sequence identity to SEQ ID NO: 27 or 78, preferably at least 88% sequence identity, more preferably at least 94% sequence identity.

[0054] The present invention also encompasses VHH antibodies having an FR3 amino acid sequence comprising or consisting of SEQ ID NO: 28, or a variant thereof having at least 92% sequence identity to SEQ ID NO: 28, preferably at least 94% sequence identity, more preferably at least 97% sequence identity.

[0055] The present invention also encompasses VHH antibodies having an FR4 amino acid sequence comprising or consisting of SEQ ID NO: 29, or a variant thereof having at least 72% sequence identity to SEQ ID NO: 29, preferably at least 81% sequence identity, more preferably at least 90% sequence identity.

[0056] As used herein, the term "% sequence identity" can be determined using methods well known in the art. For example, the % sequence identity is calculated as follows: The query sequence is aligned to the target sequence using the CLUSTAL W algorithm. Comparison is performed over a window corresponding to the shortest of the aligned sequences. The shortest of the aligned sequences may in some cases be the target sequence. In other examples, the query sequence may constitute the shortest of the aligned sequences. The amino acid residues at each position are compared, and the percentage of positions in the query sequence that have the same correspondence in the target sequence is reported as the % sequence identity.

[0057] An amino acid sequence having a specified % sequence identity with a reference amino acid sequence is preferably obtained by amino acid substitutions, such as conservative amino acid substitutions, also called conservative amino acid substitutions or mutations, which are amino acid substitutions in an amino acid sequence that change a given amino acid into a different amino acid with similar biochemical, structural and / or chemical properties.

[0058] For example, amino acids can be divided into six major classes based on their structure and the general chemical properties of their side chains (R groups): Aliphatic: Isoleucine (I), Leucine (L), Glycine (G), Alanine (A), Valine (V) Hydroxyl or sulfur / selenium-containing: serine (S), cysteine ​​(C), threonine (T), methionine (M) Cyclic: Proline (P) Aromatic: phenylalanine (F), tyrosine (Y), tryptophan (W) Basic: histidine (H), lysine (K), arginine (R); and · Acids and their amides: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q).

[0059] This means that an amino acid sequence having a specified % sequence identity with a reference amino acid sequence is obtained by one or more conservative amino acid substitutions of one or more amino acid residues in the reference amino acid sequence, preferably with a respective amino acid from the same R group as the given amino acid residue, as represented above.

[0060] In one embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO: 30. Such a VHH antibody is designated herein as KB_B01.

[0061] In another embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO: 31. Such a VHH antibody is designated herein as KB_B02.

[0062] In a further embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO: 32. Such a VHH antibody is designated herein as KB_B03.

[0063] In yet another embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO: 33. Such a VHH antibody is designated herein as KB_B04.

[0064] In another embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO: 34. Such a VHH antibody is designated herein as KB_B05.

[0065] In a further embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO: 35. Such a VHH antibody is designated herein as KB_B06.

[0066] In yet another embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO: 36. Such a VHH antibody is designated herein as KB_B07.

[0067] In another embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO: 37. Such a VHH antibody is designated herein as KB_B08.

[0068] In one embodiment, the VHH antibody has an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-37, preferably selected from the group consisting of SEQ ID NOs: 30-36.

[0069] Another aspect of the present invention relates to a VHH antibody that specifically binds to TfR1. The VHH antibody has the amino acid sequence DSAFX as defined in SEQ ID NO: 38. 28 MNT, wherein X 28 is S or N. The VHH antibody also comprises a CDR2 consisting of the amino acid sequence IVSDDNT as defined in SEQ ID NO: 39. The VHH antibody further comprises a CDR3 consisting of the amino acid sequence KGDVV as defined in SEQ ID NO: 40.

[0070] In a particular embodiment, CDR1 consists of the amino acid sequence DSAFSMNT as defined in SEQ ID NO: 41, CDR2 consists of the amino acid sequence IVSDDNT as defined in SEQ ID NO: 39, and CDR3 consists of the amino acid sequence KGDVV as defined in SEQ ID NO: 40. A VHH antibody having CDR regions according to this particular embodiment is designated herein as KB_B09.

[0071] In another specific embodiment, CDR1 consists of the amino acid sequence DSAFNMNT as defined in SEQ ID NO: 42, CDR2 consists of the amino acid sequence IVSDDNT as defined in SEQ ID NO: 39, and CDR3 consists of the amino acid sequence KGDVV as defined in SEQ ID NO: 40. A VHH antibody having CDR regions according to this specific embodiment is designated herein as KB_B10.

[0072] In one embodiment, the VHH antibody is of the formula: framework region 1 (FR1)-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0073] In one embodiment, FR1 has the amino acid sequence QVQLQESGGGLVQVGGSLRLSCAAS as defined in SEQ ID NO:43.

[0074] In one embodiment, FR2 comprises the amino acid sequence MYWYRQAPGKX as defined in SEQ ID NO:44. 29 REFVAX 30 where X 29 is Q or S, and X 30 is Y or W.

[0075] In one embodiment, FR3 comprises the amino acid sequence X defined in SEQ ID NO:45. 31 YADSVKGRFTISRDNAKNTVYLQMNX 32 LKPEDTAX 33 YYC, where X 31 is R or Q, and X 32 is S or N, and X 33 is V or G.

[0076] In one embodiment, FR4 has the amino acid sequence WGQGTQVTVSS as defined in SEQ ID NO:29.

[0077] In certain embodiments, the VHH antibody has the amino acid sequences of FR1, FR2, FR3 and FR4 defined in SEQ ID NOs: 43, 44, 45 and 29.

[0078] The present invention also encompasses VHH antibodies having an FR1 amino acid sequence comprising or consisting of SEQ ID NO: 43, or a variant thereof having at least 88% sequence identity to SEQ ID NO: 43, preferably at least 92% sequence identity, more preferably at least 96% sequence identity.

[0079] The present invention also encompasses VHH antibodies having an FR2 amino acid sequence comprising or consisting of SEQ ID NO: 44, or a variant thereof having at least 82% sequence identity to SEQ ID NO: 44, preferably at least 88% sequence identity, more preferably at least 94% sequence identity.

[0080] The present invention also encompasses VHH antibodies having an FR3 amino acid sequence comprising or consisting of SEQ ID NO: 45, or a variant thereof having at least 92% sequence identity to SEQ ID NO: 45, preferably at least 94% sequence identity, more preferably at least 97% sequence identity.

[0081] The present invention also encompasses VHH antibodies having an FR4 amino acid sequence comprising or consisting of SEQ ID NO: 29, or a variant thereof having at least 72% sequence identity to SEQ ID NO: 29, preferably at least 81% sequence identity, more preferably at least 90% sequence identity.

[0082] In one embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO: 46. Such a VHH antibody is designated herein as KB_B09.

[0083] In another embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO: 47. Such a VHH antibody is designated herein as KB_B10.

[0084] In one embodiment, the VHH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 46-47.

[0085] In another embodiment, the VHH antibody has an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-37, 46-47, preferably selected from the group consisting of SEQ ID NOs: 30-36, 46-47.

[0086] Figure 6 shows a sequence alignment of closely related VHH antibodies KB_B01 to KB_B10.

[0087] The VHH antibodies of the present invention specifically bind to hTfR1.

[0088] VHH antibodies specifically bind to hTfR1 both as monomeric VHH antibodies (see Table 2) and in fusion molecules conjugated to another molecule (see Tables 3, 5, and 7). In the latter case, binding to hTfR1 by VHH antibodies in fusion molecules is not affected regardless of whether the other molecule is conjugated to the N-terminus or C-terminus of the VHH antibody (see Table 7). Furthermore, as shown in Table 4, binding of VHH antibodies of the present invention to hTfR1 is not significantly affected by the presence or absence of transferrin. This is in stark contrast to the reference VHH antibody BV, whose affinity for hTfR1 changed dramatically in the presence and absence of 250 nM transferrin, as shown in Table 4.

[0089] "Specifically binds to" and similar expressions mean that a molecule of interest, such as a VHH antibody, specifically binds to a target antigen without significantly binding to other molecules. The specificity of an antibody can be determined based on affinity and / or avidity. The equilibrium dissociation constant (K D Affinity, expressed as K, is a measure of the binding strength between an antigenic determinant, i.e., an epitope, and the antigen-binding site of the antibody. D The smaller the value of K, the stronger the binding strength between the antigenic determinant and the antibody. Alternatively, affinity can be expressed as 1 / K D The equilibrium binding constant (K A As will be apparent to those skilled in the art, affinity can be determined in a manner known per se depending on the particular antigen of interest.

[0090] Typically, antibodies have an equilibrium dissociation constant (K D )10 -5 ~10 -12 moles / liter (M) or less, and preferably 10 -7 ~10 -12 M or less, and more preferably 10 -8 ~10 -12M, i.e., the affinity constant (K A )10 5 ~10 12 M -1 or more, and preferably 10 7 ~10 12 M -1 More preferably, 10 8 ~10 12 M -1 Generally, 10 -4 Any K greater than M D value (or 10 4 M -1 Any K less than A values) are considered to represent nonspecific binding.

[0091] Specific binding of an antibody to an antigen or antigenic determinant can be determined by any suitable method known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, surface plasmon resonance (SPR), biolayer interferometry (BLI), and various variants thereof known per se in the art.

[0092] The affinity of VHH antibodies for TfR1 needs to be adjusted to be within a certain range to achieve efficient BBB penetration and high relative uptake in the brain. Generally, if the affinity of the antibody for TfR1 is too low, e.g., a K above about 1 μM, D In the case of , the affinity of the antibody is too low to efficiently bind to TfR1 and be taken up by the endothelial cells of the BBB, meaning that most of the antibody remains in the peripheral blood system. Correspondingly, if the affinity of the antibody is too high, e.g., K D If the affinity (K) is much lower than 1 nM (<<1 nM), the antibody will be endocytosed but will instead undergo lysosomal degradation in the endothelial cells of the BBB. This suggests that VHH antibodies should have an affinity range (K) of low nM to nM for optimal receptor-mediated endocytosis, i.e., receptor-mediated cellular uptake, and transcytosis, i.e., transport across the interior of the cell. D) (Bien-Ly N, et al., Transferrin receptor (TfR) trafficking determines brain uptake of TfR antibody affinity variants. J Exp Med. 211(2):233-244(2014), WO2016 / 081643).

[0093] As shown in Table 2, the VHH antibody KB_B01 of the invention has an affinity (K D ) Furthermore, K D VHH-containing fusion proteins (see Figure 2), such as dimer-VHH Fc fusion proteins (see Table 3) with a K D When included in a fusion with an scFv (see Table 7) with a K of 0.85-1.84 nM, the affinity still remains in the desired low-nM to nM range. Additionally, other VHH antibodies of the present invention, when tested as dimeric Fc fusions, have affinities for hTfR1 in the sub-nM range, making them suitable for crossing the BBB by receptor-mediated endocytosis and transcytosis, with K values ​​in the range of less than 0.0031 to 0.451 nM as dimeric-VHH Fc fusion proteins when bound to hTfR1. D and a 5-6000 higher K when binding to mTfR. D See Table 5 for more information.

[0094] In one embodiment, the VHH antibodies of the invention specifically bind to human TfR1 (hTfR1).

[0095] In one embodiment, the VHH antibodies of the present invention specifically bind to mTfR1. In a specific embodiment, the VHH antibodies of the present invention specifically bind to not only hTfR1 but also mTfR1. See Table 5.

[0096] In one embodiment, the VHH antibodies of the invention specifically bind to cTfR1. In a particular embodiment, the VHH antibodies of the invention specifically bind to cTfR1 in addition to hTfR1 and mTfR. See Table 3.

[0097] Thus, in a preferred embodiment, the VHH antibodies exhibit broad species cross-reactivity, i.e., they bind not only to hTfR1 but also to mTfR1 and cTfR1 with the desired affinity. This property of VHH antibodies is highly desirable from a translational perspective in predicting both pharmacokinetics and pharmacodynamics from mouse or monkey to human.

[0098] In one embodiment, the VHH antibody of the present invention has an affinity (K D In a specific embodiment, the VHH antibodies of the invention in monovalent form specifically bind to mTfR1 at a K selected within the range of 1 to 150 nM, preferably within the range of 1 to 100 nM, more preferably within the range of 1 to 50 nM. D It specifically binds to mTfR1.

[0099] In one embodiment, the VHH antibodies of the invention in monovalent or monomeric form have an affinity (K D ) and specifically binds to hTfR1.

[0100] Structurally, TfR1 is a dimeric transmembrane glycoprotein with a large ectodomain (residues 90-760), an intramembrane region (residues 62-89), and the remaining 61 residues in the cytoplasm. The ectodomain contains three domains: a helical domain (residues 606-760), a protease-like domain (residues 121-183, 384-605), and an apical domain (residues 184-383). The helical domain is involved in receptor dimerization. Transferrin binds to the helical and protease-like domains.

[0101] The VHH antibodies of the present invention can inhibit the binding of other VHH antibodies of the present invention to hTfR1, thereby binding to the same epitope on hTfR1, as shown in Table 6. As shown in Table 6, the VHH antibodies do not prevent human or mouse transferrin from binding to hTfR1. Thus, the VHH antibodies of the present invention do not interfere with transferrin binding for iron uptake by cells that use TfR1.

[0102] In one embodiment, the VHH antibody of the invention is a Camelid VHH antibody.

[0103] In another embodiment, the VHH antibody of the present invention is a humanized VHH antibody. For example, the CDR regions of a VHH antibody can be grafted onto a human skeleton. Soler et al., "Effect of Humanizing Mutations on the Stability of the Llama Single-Domain Variable Region," Biomolecules 11(2):163 (2021), identified several amino acid positions and an N-terminal Gln as hot spots for converting camelid VHHs to human consensus germline sequences.

[0104] Hereinafter, a sequence alignment between the framework regions of KB_B01, the reference human VH (sVH), the universal VHH (uVHH), and the humanized VHH sequence (hVHH) as disclosed in the above Biomolecules article is presented.

[0105] QVQLQESGGGLVQAGGSLRLSCAAS KB_B01 FR1 ----VQ-------P------------ sVH FR1 ----V---------P---------T-- uVHH FR1 ----V---------P------------ hVHH FR1 MRWYRQAPGKQREWVAG KB_B01 FR2 -SV------GL---SP sVH FR2 L---------E--A--A uVHH FR2 L--------GL-A--A hVHH FR2 NYADSVKGRFTISRDNAEGTVYLQMDNLKPEDTAVYYC KB_B01 FR3 Y--------------SKN-L----NT-RA------- sVH FR3 Y---KN--T---N--------I--- uVHH FR3 Y---------------SKN-L----NS-RA------- hVHH FR2 WGQGTQVTVSS KB_B01 FR4 -----M----- sVH FR4 ----------- UVHH FR4 -----L----- sVH FR4

[0106] The VHH antibodies of the present invention can be humanized by modification, for example, amino acid substitution, in FR1, FR2, FR3, and / or FR4. The amino acid positions in FR1 to FR4 referred to herein are relative to the amino acid positions in KB_B01 (SEQ ID NO: 30) shown in Figure 6.

[0107] As an example, the humanized positions in FR1 can be selected from E1 or Q1; V5; Q6 or E6, S11 or L11, P14; and / or R19. In one embodiment, any one of these humanized positions in FR1 is used for humanized FR1, or any combination of two to all of these positions is humanized. As an example, humanized FR1 can be based on SEQ ID NO: 26 or 77, but Q1 is replaced with E, Q5 is replaced with V, or two or all of these amino acids are replaced.

[0108] The humanized positions in FR2 may be selected from L34 or M34 (the first amino acid position of FR2), S35 or G35 (the second amino acid position of FR2), V37 or F37 (the fourth amino acid position of FR2), Q43 or K43 (the tenth amino acid position of FR2), G44 (the eleventh amino acid position of FR2), L45 (the twelfth amino acid position of FR2), E46 (the thirteenth amino acid position of FR2), W47 or A47 (the fourteenth amino acid position of FR2), S49 (the sixteenth amino acid position of FR2), and / or P50, A50, V50, or G50 (the seventeenth amino acid position of FR2). In one embodiment, any one of these humanized positions in FR2 is used for the humanized FR2, or any combination of two up to all of these positions are humanized. As an illustrative example, the humanized positions in FR2 can be V37, G44, L45 and W47, or F37, G44, L45 and A47.

[0109] The humanized positions in FR3 can be selected from Y58 (the 1st amino acid position of FR3), S74 (the 17th amino acid position of FR3), K75 (the 18th amino acid position of FR3), N76 (the 19th amino acid position of FR3), L78 or I78 (the 21st amino acid position of FR3), N84, S84 or T84 (the 27th amino acid position of FR3), R86 (the 29th amino acid position of FR3), A87 (the 30th amino acid position of FR3), A96 (the 39th amino acid position of FR3), and / or R97 or A97 (the 40th amino acid position of FR3). In one embodiment, any one of these humanized positions in FR3 is used for the humanized FR3, or any combination of two up to all of these positions are humanized.

[0110] The humanized position in FR4 can be L106 or M106 (the sixth amino acid position in FR4).

[0111] I78 also has a stabilizing effect on VHH. Thus, Ile at position 78 stabilizes VHH.

[0112] In one embodiment, a VHH antibody of the invention binds to Protein A. In certain embodiments, a VHH antibody of the invention binds to a Protein A-based resin, such as a Protein A chromatography resin, such as a MabSelect PrismA™ resin.

[0113] Protein A is a 42 kDa surface protein originally found in the cell wall of the bacterium Staphylococcus aureus. It is encoded by the spa gene. It has found use in biochemical research due to its ability to bind immunoglobulins. It is composed of five homologous Ig-binding domains, each folded into a three-helical bundle. Each domain can bind proteins from many mammalian species, most notably IgG. It binds to the heavy chains within the Fc region of most immunoglobulins and, in the case of the human VH3 family, also within the Fab region. Protein A generally does not bind, or only weakly binds, camelid VHH antibodies.

[0114] Henry KA, et al., A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies, PLoS One 11(9):e0163113 (2016) discloses methods for generating non-Protein A binding VHH antibodies that can bind to Protein A. See Table 5.

[0115] More specifically, amino acid residue 15 should be G or D, amino acid residue 17 should be S or A, and amino acid residue 19 should be R. The FR1 sequence of KB_B01 is the protein A binding FR1 region, as shown below.

[0116] QVQLQESGGGLVQA G G S L RLSCAAS KB_B01 FR1 ----------S-------------- Protein A binding FR1

[0117] Furthermore, amino acid residue 59 should be Y, amino acid residue 64 should be K or E, amino acid residue 65 should be G, amino acid residue 66 should be R, amino acid residue 68 should be T or A, amino acid residue 70 should be S, amino acid residue 75 should be A, E, K, Q, or R, amino acid residue 81 should be Q, amino acid residue 83 should be N, and amino acid residue 84 should be S, N, or G. The FR3 region of KB_B01 is protein A-binding by substituting amino acid residue 83 from D to N. However, in optional embodiments, the N at amino acid residue 84 may be substituted with S.

[0118] N Y ADSV KGR F T I S RDNA E GTVYL Q M DN LKPEDTAVYYC KB_B01 FR3 E----------------KN------NS----------- Protein A binding FR3

[0119] In one embodiment, the FR1 region has an R at amino acid residue 19, and the FR3 region has an N at amino acid position 83 and an S at amino acid position 84.

[0120] In one embodiment, the VHH antibody of the invention is an isolated VHH antibody.

[0121] The term "isolated," when used in connection with a VHH antibody, such as the expression "isolated VHH antibody" and similar expressions, means that the VHH antibody has been purified and removed from its original environment. As used herein, an isolated VHH antibody is intended to refer to a VHH antibody that is substantially free of other antibodies with different antigen specificities; for example, an isolated VHH antibody that specifically binds to TfR1, particularly human TfR1 (hTfR1), is substantially free of antibodies that specifically bind to antigens other than TfR1. However, an isolated VHH antibody that specifically binds to hTfR1 may have cross-reactivity to other antigens, such as TfR1 molecules from other species, such as cTfR1 and mTfR1. Furthermore, an isolated VHH antibody may be substantially free of other cellular material and / or chemicals. For example, isolated VHH antibodies may be purified to greater than 95% or 99% purity, as determined, for example, by electrophoretic methods (e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF), capillary electrophoresis) or chromatographic methods (e.g., ion exchange or reverse-phase high performance liquid chromatography (HPLC)). One of skill in the art will understand that isolated VHH antibodies are referred to herein, even though the word "isolated" is not explicitly mentioned every time the term "VHH antibody" or the like is used.

[0122] Further aspects of the embodiments include nucleic acid molecules encoding VHH antibodies according to the embodiments or fusion molecules according to the embodiments, see further below. As used herein, a nucleic acid molecule includes polynucleotides, oligonucleotides, and nucleic acid sequences, and generally refers to a polymer of DNA or RNA, which may be single-stranded or double-stranded, and which may contain natural, non-natural, or modified nucleotides, and which may contain natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages in place of the phosphodiesters found between nucleotides in unmodified oligonucleotides. The term "nucleic acid molecule" also includes complementary DNA (cDNA) and messenger RNA (mRNA).

[0123] In one embodiment, the nucleic acid molecule is an isolated nucleic acid molecule encoding a VHH antibody according to the embodiments, or a fusion molecule according to the embodiments.

[0124] The nucleic acid molecule may encode a single VHH antibody according to the embodiments, multiple copies of a single VVH antibody according to the embodiments, or one or more copies of different VHH antibodies according to the embodiments.

[0125] The nucleic acid molecule may also encode other molecules than the VHH antibodies of the embodiments, for example VHH antibodies genetically fused to another molecule, i.e. in the form of a VHH-containing fusion protein as further described herein.

[0126] Another aspect of the embodiments relates to vectors comprising nucleic acid molecules according to the embodiments.

[0127] The vector is preferably an expression vector, i.e., a vector comprising at least one nucleic acid molecule comprising a coding sequence capable of being expressed, e.g., transcribed and translated, in a host cell comprising the expression vector. Thus, an expression vector comprises a nucleic acid molecule according to an embodiment operably linked to a promoter.

[0128] As used herein, operably linked means that the nucleic acid molecule is in the correct functional location and / or orientation with respect to the promoter to allow expression of the nucleic acid molecule in a host cell, i.e., the promoter controls transcription of the nucleic acid molecule (constitutively or inducibly).

[0129] In one embodiment, the expression vector is selected from a DNA molecule, an RNA molecule, a plasmid, an episomal plasmid, and a viral vector. In one such embodiment, the expression vector contains, in addition to a nucleic acid sequence encoding a VHH antibody, regulatory sequences necessary for producing the VHH antibody in a host cell. For example, the nucleic acid sequence encoding the VHH antibody is under the transcriptional control of a promoter sequence included in the expression vector. A promoter is a DNA sequence to which a protein binds and initiates transcription of an RNA molecule from the DNA (gene) downstream. The promoter is preferably selected based on the specific host cell in which the VHH antibody will be expressed, such as T5, T7, lac, or BL21 promoters for expression of VHH antibodies in bacterial cells, GAL1, MET25, CUP1, LAC4, ADH2, SUC2, or GAPDH promoters for expression of VHH antibodies in yeast, and EF1α, CMV, or CAG promoters for expression in mammalian cells such as human cells. The promoter can be a constitutive promoter or an inducible promoter. A constitutive promoter, also known as a constitutively active promoter, is active under all conditions within the host cell. Inducible promoters, also known as inducibly active promoters, are regulated and activated in host cells only in response to specific stimuli, such as chemically inducible promoters, temperature-inducible promoters, or light-inducible promoters. Expression vectors may optionally contain other regulatory sequences, such as enhancers. Enhancers are short regions of DNA that can be bound by activators to increase the likelihood of transcription of a particular gene. An optional signal peptide may be provided at the N- or C-terminus of the polypeptide encoded by the expression vector.

[0130] A further aspect of the embodiment relates to a host cell comprising a nucleic acid molecule or an expression vector according to the embodiment.

[0131] The nucleic acid molecule or expression vector can then be transcribed in a host cell to produce the VHH antibody intracellularly.

[0132] In one embodiment, the cell is selected from the group consisting of a bacterial cell, a yeast cell, and a mammalian cell.

[0133] Yet another aspect of this embodiment relates to a VHH antibody according to the present invention linked to at least one molecule. Accordingly, this aspect of the present invention relates to a fusion molecule or protein between a VHH antibody and at least one other molecule. The fusion molecule or protein is preferably in the form of a genetic fusion between the VHH antibody and at least one other molecule. In such cases, the nucleic acid molecule encodes not only the VHH antibody but also at least one other molecule. For example, the nucleic acid molecule may comprise, from its 5'-end to its 3'-end, a nucleic acid sequence encoding a VHH molecule and a nucleic acid sequence encoding at least one other molecule, or a nucleic acid sequence encoding at least one other molecule and a nucleic acid sequence encoding a VHH molecule. Thus, a VHH molecule may be covalently linked to at least one other molecule at its N-terminus or C-terminus. See Figure 2.

[0134] Furthermore, it is possible to generate fusion molecules that comprise multiple molecules in addition to a VHH antibody, for example, multiple different molecules or multiple copies of a single molecule may be attached or linked to the N-terminus of a VHH antibody, the C-terminus of a VHH antibody, or at least one molecule may be attached or linked to the N-terminus of a VHH antibody and at least one molecule is attached or linked to the C-terminus of a VHH molecule.

[0135] In one embodiment, the fusion molecule comprises at least one other molecule attached or bound to the C-terminus of the VHH antibody.

[0136] As mentioned above, the VHH antibody is preferably linked to at least one other molecule by being genetically fused to the other molecule. In such embodiments, the nucleic acid molecule encodes the VHH antibody and at least one other molecule as a VHH-containing fusion protein. This means that the nucleic acid molecule comprises a nucleic acid sequence encoding the VHH antibody connected or linked to a nucleic acid sequence encoding at least one other molecule, and these nucleic acid sequences are under the transcriptional control of the same promoter.

[0137] However, a VHH antibody may be linked, connected, bonded, or conjugated to at least one molecule by means other than genetic fusion, for example, by chemically connecting the VHH antibody to at least one molecule. For example, a VHH antibody may be covalently attached to at least one molecule by reaction between a maleimide and an amine or thiol, often referred to as thiol-maleimide or amine-maleimide click chemistry. In such embodiments, additional amino acid residues, such as cysteine, arginine, or lysine, may be added to the N- and / or C-terminus of the VHH antibody to enable such reaction with a maleimide.

[0138] The VHH antibody and at least one molecule can be enzymatically linked, for example, by the enzyme transglutaminase (TGase), which catalyzes the formation of an isopeptide bond between the γ-carboxamide group (-(C=O)NH2) of the side chain of a glutamine residue and the ε-amino group (-NH2) of the side chain of a lysine residue, followed by the release of ammonia (NH3). In one such embodiment, additional amino acid residues, e.g., glutamine or lysine, may be added to the N-terminus and / or C-terminus of the VHH antibody to enable such enzymatic reaction.

[0139] In one embodiment, the VHH antibody according to the invention is covalently bound to at least one molecule.

[0140] The VHH antibody can be directly linked, e.g., fused, to at least one molecule. Alternatively, the VHH antibody is covalently linked to at least one molecule via a linker. Various such linkers, particularly peptide linkers, can be used according to embodiments, including, but not limited to, G n linker (where n is an integer of 1 or more, typically an integer of 10 or less), S m Liker (where m is an integer of 1 or more, and is usually 10 or less), A q Linkers, (where q is an integer equal to or greater than 1, and usually equal to or less than 10), include various GS-linkers or GA-linkers, i.e., combinations of one or more G with one or more S or one or more A, such as (G n S m ) p or (S m G n ) p or (G n A q ) p or (A q G n ) p (where p is an integer equal to or greater than 1 and typically equal to or less than 10). For example, linkers such as G4A, G4S, G3S, and combinations thereof, such as G4A-G4A-G4S or G4S-G3S, can be used. Other commonly used peptide linkers are disclosed in Table 1 of Vishnu Priyanka Reddy Chichil, et al., Linkers in the structural biology of protein-protein interactions, Protein Science 22("):153-167 (2013), the linkers listed in Table 1 on pages 156-157 of which are incorporated herein by reference as illustrative but non-limiting examples of peptide linkers that can be used to covalently link a VHH antibody to at least one molecule.

[0141] The fusion molecules or VHH antibodies of the present invention may comprise one or more tags, including but not limited to affinity purification tags such as His-tag, C-tag, Q-tag, and / or myc-tag. The tags may then be present at the N-terminus and / or C-terminus of the fusion molecule or VHH antibody. The addition of tags to enable purification may also be accompanied by a protease site, such as but not limited to a TEV site, for efficient subsequent removal of the purification tag from the VHH antibody or fusion molecule.

[0142] The fusion molecule may also comprise multiple VHH antibodies in addition to at least one molecule shown in FIG.

[0143] In one embodiment, the fusion molecule is monovalent with respect to the VHH antibody, i.e., preferably comprises only a single VHH antibody in addition to the last molecule. There is in vitro and in vivo evidence that monovalent binding modes facilitate transcytosis, whereas bivalent binding modes result in lysosomal sorting (Niewoehner et al., Increased brain penetration and potency of a therapeutic antibody using a monovalent molecular shuttle, Neuron. 81(1):49-60(2014)).

[0144] The at least one molecule can be any molecule, such as a drug or pharmaceutical agent, a diagnostic agent, an imaging agent, a tracer, a half-life extender, etc. Examples of such molecules include, but are not limited to, antibiotics, antivirals, immunomodulators, antitumor agents, anti-inflammatory agents, adjuvants, peptides, polypeptides and proteins, such as enzymes, hormones, neurotrophic factors, neuropeptides, cytokines, apolipoproteins, growth factors, antigens, antibodies or antibody portions, adjuvants, etc., nucleic acids, such as RNA or DNA, including encoding genes, ribozymes, antisense, interfering nucleic acids, complete genomes or portions thereof, inhibitory nucleic acids, such as plasmids.

[0145] In one embodiment, at least one molecule of the fusion molecule is selected from the group consisting of a therapeutic agent and an imaging agent.

[0146] As used herein, an imaging agent refers to an agent or molecule that is administered to a patient and used to visualize the imaging agent during the imaging process, such as when taken up by cells. Examples of such imaging agents include radioactive atoms or isotopes, e.g., radioactive tracers such as position emission tomography (PET) tracers, e.g., 18 F-containing PET tracer, 11 C-containing PET tracer, 64 Radioactive atoms or isotopes such as Cu-containing PET tracers, or e.g. 99m Tc or 111 Included are agents or molecules containing single-photon emission computed tomography (SPECT) tracers, such as In-containing SPECT tracers. Other examples of imaging agents include fluorescent probes, luminescent probes, metal complex-containing probes, near-infrared (NIR) fluorescent probes, etc.

[0147] In general, there is an urgent need for diagnostic tools that allow early detection of diseases or medical conditions, particularly in brain compartments, which would facilitate efficient disease-modifying therapies, particularly for evaluating therapeutic efficacy in preclinical and clinical trials of new drug candidates.

[0148] Specific targeting of disease-causing proteins, including but not limited to amyloid beta, tau, and alpha-synuclein, or false markers of disease, such as inflammation, is difficult with classical PET ligands or tracers based on small molecules that can cross the BBB. Generally, it is preferable to use PET ligands or tracers based on specific binding molecules, such as antibodies. However, monoclonal antibody (mAb)-based PET tracers generally have a long circulatory half-life, making them unsuitable for any use as PET ligands or tracers. This means that radioactivity generated from radiolabeled mAbs in the cerebral blood circulation interferes with specific signals at target sites in the brain. Therefore, to be useful in PET applications, antibody-based PET ligands or tracers must have a high blood-to-brain ratio and rapid blood clearance. Smaller antibody domains or fragments, such as VHH antibodies (average molecular weight ≤60 kDa), are excreted through the kidneys and are therefore excreted more rapidly than mAbs. Therefore, due to their smaller size compared to mAbs, VHH antibodies are suitable as PET ligands or tracers (Syvaenen, et al., A bispecific tribody PET radioligand for visualization of amyloid-beta protofibrils—a new concept for neuroimaging. Neuroimage 148:55-63. (2017); Vandesquille, et al., Chemically-defined camelid antibody bioconjugate for the magnetic resonance imaging of Alzheimer's disease. mAbs 9:1016-1027 (2017)).

[0149] In these embodiments, the fusion molecules can be used as diagnostic agents for the diagnosis of various diseases or disorders.

[0150] For example, a VHH antibody of the embodiments may be linked to another antibody, antigen-binding fragment or domain thereof, including another VHH antibody that specifically binds to a disease-specific marker. In such cases, the other antibody is preferably labeled to detect its binding to the disease-specific marker upon delivery, such as to a brain compartment, by receptor-mediated transcytosis using the VHH antibody as a BBB transporter.

[0151] As used herein, an antigen-binding fragment or domain of an antibody may be selected from the group consisting of a single chain antibody, an Fv fragment, an scFv fragment, an Fab fragment, an F(ab')2 fragment, an Fab' fragment, an Fd fragment, a single domain antibody (sdAb), an scFv-Fc fragment, a di-scFv fragment, and two or more CDR regions.

[0152] For therapeutic purposes, the fusion molecule may contain, in addition to the therapeutic agent, a half-life extender or group, also known as a stabilizer or stabilizing group. Such a half-life extender is then included to increase the half-life of the fusion molecule when administered to a patient. Any such half-life extender that can be linked to a VHH antibody without adverse biological effects can be used, including, but not limited to, serum proteins such as the Fc fragment of IgG, human serum albumin (HSA), albumin-binding protein scaffolds, IgG, various polyethylene glycol (PEG) molecules, or unstructured polypeptides such as XTEN, or PAS as an alternative to PEGylation.

[0153] The present invention also relates to a pharmaceutical composition comprising the fusion molecules defined above, wherein at least one molecule is a therapeutic agent. The pharmaceutical composition also further comprises a pharmaceutically acceptable vehicle or excipient.

[0154] The pharmaceutically acceptable vehicle can be any pharmaceutically acceptable vehicle or carrier that is compatible with the other component(s) of the pharmaceutical composition.The pharmaceutically acceptable vehicle can be selected from conventionally used vehicles according to each mode of administration.The pharmaceutical composition can be a solid pharmaceutical composition such as a tablet, pill, powder, or granule, a semi-solid pharmaceutical composition such as a suppository, or a liquid pharmaceutical composition such as a soft capsule or an injection solution.

[0155] Pharmaceutically acceptable vehicles or excipients include, but are not limited to, diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine; lubricants such as silica, talc, stearic acid (including its salts), and polyethylene glycol; binders such as magnesium and aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone; disintegrating agents such as starch, agar, alginic acid, and sodium alginate; absorbents; dyes; flavors; sweeteners; polypropylene glycol; liquid vehicles such as water, saline, aqueous dextrose, glycerol, ethanol, and oils.

[0156] Embodiments also relate to fusion molecules according to the above for use as a medicament, wherein at least one molecule is a therapeutic molecule.

[0157] In one embodiment, the therapeutic agent is capable of treating a disease or disorder of the CNS.

[0158] In certain embodiments, the fusion molecules described above are for use in treating a disease or disorder of the CNS. In such embodiments, at least one molecule is a therapeutic agent capable of treating a disease or disorder of the CNS.

[0159] A further embodiment is directed to a method for treating a CNS disease or disorder in a patient. The method comprises administering to the patient an effective amount of a fusion molecule according to the above or a pharmaceutical composition according to the above. In such embodiments, at least one molecule is a therapeutic agent capable of treating a CNS disease or disorder.

[0160] Illustrative, but non-limiting examples of diseases or disorders of the CNS include Alzheimer's disease (AD), Bell's palsy, cerebral palsy, epilepsy, motor neuron diseases (MND) such as amyotrophic lateral sclerosis (ALS), progressive bulbar palsy (PBP), pseudobulbar palsy, progressive muscular atrophy (PMA), primary lateral sclerosis (PLS), spinal muscular atrophy (SMA) and monomeric muscular atrophy (MMA), multiple sclerosis (MS), neurofibromatosis, Parkinson's disease (PD), lysosomal storage diseases, neuropathic lysosomal storage diseases, ischemic stroke, intracerebral hemorrhage, traumatic brain injury (TBI), vascular dementia, frontotemporal dementia, amyloidosis, tauopathy, Creutzfeldt-Jakob disease, neuroinflammation and neuropathic pain.

[0161] Malignant cells often overexpress TfR1, and increased expression can be associated with poor prognosis in different types of cancer (Candelaria et al., Antibodies Targeting the Transferrin Receptor 1 (TfR1) as Direct Anti-cancer Agents. Front. Immunol. 12: 607692 (2021)). TfR1 is overexpressed in many different types of cancer cells, often at levels several times higher than in normal cells. Indeed, TfR1 has been identified as a universal cancer marker. Increased expression of TfR1 correlates with advanced stage and / or poor prognosis in several cancers, including solid tumors such as esophageal squamous cell carcinoma, breast cancer, ovarian cancer, lung cancer, cervical cancer, bladder cancer, osteosarcoma, pancreatic cancer, cholangiocarcinoma, renal cell carcinoma, hepatocellular carcinoma, adrenocortical carcinoma, glioblastoma multiforme (GBM), and cancers of the nervous system, as well as hematopoietic malignancies such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin's lymphoma (NHL) (Candelaria et al., Antibodies Targeting the Transferrin Receptor 1 (TfR1) as Direct Anti-cancer Agents. Front. Immunol. 12:607692 (2021); Ramalho et al., Transferrin Receptor-Targeted Nanocarriers: Overcoming Barriers to Treat Glioblastoma. Pharmaceutics 14(2): 279(2022)).

[0162] The elevated expression levels of TfR1 on malignant cells, along with its extracellular accessibility, ability to be internalized, and central role in cancer cell pathology, make this receptor an attractive target for antibody-mediated therapy. TfR1 can be targeted by the VHH antibodies of the present invention for cancer therapy by using VHH antibodies conjugated to anticancer drugs that are internalized by receptor-mediated endocytosis.

[0163] In another embodiment, the therapeutic agent is capable of treating cancer.

[0164] In certain embodiments, the fusion molecules according to the above are for use in the treatment of cancer, in such embodiments, at least one molecule is a therapeutic, immunotherapeutic and / or adjunctive therapeutic agent capable of treating cancer.

[0165] A further embodiment is directed to a method of treating cancer in a patient. The method comprises administering to the patient an effective amount of a fusion molecule according to the above, or a pharmaceutical composition according to the above. In such embodiments, at least one molecule is a therapeutic agent capable of treating cancer.

[0166] hTfR1 is also a target for delivering therapeutic agents, including oligonucleotides, to muscle (Desjardins et al., Enhanced exon skipping and prolonged dystrophin restoration achieved by TfR1-targeted delivery of antisense oligonucleotides using FORCE TM (Sugo et al., Development of antibody-siRNA conjugate targeted to cardiac and skeletal muscles, J Control Release 237:1-13(2016)). Therefore, the fusion molecules of the present invention can be used to treat various muscle diseases, particularly muscular dystrophies.

[0167] The fusion molecules could also be used to treat muscular dystrophies such as Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Mitton muscular dystrophy (DM), limb-girdle muscular dystrophy (LGMD), facioscapulohumeral muscular dystrophy (FSHD), congenital muscular dystrophy (CMD), distal muscular dystrophy (DD), oculopharyngeal muscular dystrophy (OPMD), or Emery-Dreifuss muscular dystrophy (EDMD), particularly DMD, FSHD, or DM.

[0168] In a further embodiment, the therapeutic agent can affect muscular dystrophy.

[0169] In certain embodiments, the fusion molecules according to the above are for use in treating muscular dystrophy. In such embodiments, at least one molecule is a therapeutic agent capable of treating muscular dystrophy.

[0170] A further embodiment is directed to a method for treating muscular dystrophy in a patient. The method comprises administering to the patient an effective amount of a fusion molecule according to the above or a pharmaceutical composition according to the above. In such embodiments, at least one molecule is a therapeutic agent capable of treating muscular dystrophy.

[0171] As used herein, an effective amount refers to an amount effective at a dosage and for a period of time necessary to achieve a desired therapeutic result. An effective amount may vary depending on factors such as the state of the disease, age, sex, and weight of the patient. As used herein and as is well understood in the art, treating or treatment refers to an approach for obtaining beneficial or desired results, such as clinical results. Beneficial or desired clinical results may include, for example, alleviation or amelioration of one or more symptoms or conditions, reduction in the extent of disease, stable disease, i.e., prevention of disease worsening, prevention of disease spread, delay or slowing of disease progression, improvement or palliation of the condition, reduction in disease recurrence, and remission. Treating or treatment may also prolong survival compared to the expected survival without treatment.

[0172] As used herein, and as well understood in the art, prevention or prophylaxis refers to an approach in which the risk of developing a disease or condition is reduced or prevented, including prolonging or delaying the onset of the disease. For example, a patient who is predisposed to developing a disease, such as due to a genetic or inherited predisposition, may benefit from administration of a fusion molecule or pharmaceutical composition to prevent, reduce the risk of, delay and / or delay the onset of the disease.

[0173] The patient is preferably a human patient, however, embodiments may also be applied to veterinary applications, i.e., non-human patients, such as non-human mammals, including primates, monkeys, apes, cows, sheep, pigs, goats, horses, cats, dogs, mice, rats, and guinea pigs.

[0174] Fusion molecules or pharmaceutical compositions according to embodiments can be administered to patients by a variety of routes, including, for example, parenteral routes such as subcutaneous, intravenous, intraperitoneal, or intramuscular injection, oral, rectal, topical, intranasal, perilingual, or intraocular routes. In specific embodiments, the route of administration is subcutaneous or intravenous. In specific embodiments, the route of administration is subcutaneous or intravenous. [Example]

[0175] Example I - Protein production for llama immunization and assay The extracellular domains of hTfR1 (amino acids R121 to F760, SEQ ID NO: 48), mTfR1 (S122 to F763, SEQ ID NO: 49), and cTfR1 (R121 to F760, SEQ ID NO: 50) were cloned into the pCDNA3.4-TOPO plasmid optimized for expression in Critulus griseus using 5' cloning sites: HindIII, KpnI, and NheI, and 3' cloning sites: XhoI and PacI, stop codons: TGA, TGA, and a Kozak sequence (generated with the Geneart tool (Thermo Fischer Scientific)). An N-terminal signal peptide (MDWLRNLLFLMAAAQSINA, SEQ ID NO: 51), followed by a single amino acid linker "A," a histidine tag (6xHis, SEQ ID NO: 56), and an SG linker (hTfR1, cTfR1) or a G linker (mTfR1) were used for expression. Plasmids were ordered from Geneart (Thermo Fischer Scientific, Germany). Transient transfection of Chinese hamster ovary (CHO) cells was performed according to standard procedures. Cultures were harvested on day 6. The supernatant was filtered (0.45 μm syringe filter) and then purified by immobilized metal ion chelate (IMAC) using a HisTrapExcel 5 mL column (Cytiva). The following buffers were used: equilibration / wash (50 mM phosphate, 500 mM NaCl, pH 8), primary wash buffer (50 mM phosphate, 500 mM NaCl, 20 mM imidazole, pH 8), and elution buffer (50 mM phosphate, 500 mM, 500 mM NaCl, imidazole, pH 8). The eluted fraction was concentrated using an Amicon Ultra 10000 MWCO spin concentrator. The protein was dialyzed overnight with 10% glycerol (Medicago, tablets, glycerol diluted from an 80% stock solution) in PBS with a 10 kDa cutoff (Pierce) at 4°C (3 ml cassette slide alyzer). PBS was set to pH 8. Protein concentration was measured using absorbance at 280 nm.

[0176] Example II - Generation and selection of a VHH library that binds to human TfR1 Immunizations for antibody generation in llama (Lama glama) animals (N=2) were performed at Modiquest Research BV (The Netherlands) using the ModiPhage™ method. Based on monitoring the specific immune response to the immunogens hTfR1 and mTfR1 (produced as described in Example I), primary immunizations were performed in two llama animals using an adaptive immunization protocol. Immunizations were administered intramuscularly (i.m.) with a dose of 500 μg of protein on days 1 (hTfR1), 21 (mTfR1), and 42 (hTfR1), as shown in Figure 1A. Blood was collected on day 54, and the immune response was tested by ELISA. Serum samples were serially diluted in 3-fold steps (1:100 to 1:72900) and screened against plate-immobilized hTfR1, mTfR1, and the control protein bovine serum albumin (BSA) (all at 1 μg / ml). Specific binding of anti-llama IgG1, IgG2 / IgG3 antibodies was detected with goat anti-rabbit horseradish peroxidase (HRP) and goat anti-mouse HRP, respectively, and detected with 3,3',5,5'-tetramethylbenzidine (TMB) substrate at 480 nm absorbance in a spectrophotometric plate reader. Two additional doses with 500 μg of hTfR1 were delivered on days 86 and 107. Blood was collected on day 117, and ELISA was performed as described above. One animal was selected for the final collection and also received an additional boost dose with a 1:1 mixture of hTfR1 and mTfR1 (250 μg + 250 μg) on ​​day 120. Four days later (day 124), approximately 1.5 × 10 9 Peripheral blood was collected for PBL isolation by density gradient centrifugation using TRIzol (Thermo Fischer Scientific). RNA extraction was performed using approximately 0.7 × 10 9 Reverse transcription into cDNA from PBLs was performed by oligo(dT)-primed reverse transcription of 400 μg of freshly isolated RNA.

[0177] All subsequent steps for constructing a phage display library of VHHs were carried out as described in Pardon et al., A general protocol for the generation of nanobodies for structural biology. Nat Protoc 9:674-693 (2014). See Figure 1B. More specifically, the above cDNA was used as a template in PCR reactions to amplify the IgG2 / 3 repertoire. A first PCR reaction was performed to amplify all antibodies (IgG1 and IgG2 / 3), followed by a second nested PCR reaction using primers CALL001 (SEQ ID NO: 52) and CALL002 (SEQ ID NO: 53) to amplify and isolate the VHH repertoire, including overhanging regions for subcloning into the phagemid pADL10b. A 3 μg quantity of cDNA template was used in a ThermoFischer Scientific High Fidelity® kit: 10 μl of 5× High Fidelity buffer, 1 μl of dNTPs (dinucleotide triphosphates; each 10 mM, 0.2 mM final concentration), 25 pmol per primer (0.5 μM final primer concentration), 0.5 μl of Phusion® polymerase (1 U / reaction), 50 μl of ddHO (double-distilled water), 1-2 μl of template, and a cycling program of 98°C for 5 min, 98°C for 30 s (30×), 72°C for 30 s (30×), 72°C for 10 min, and storage at 4°C. A total of 36 wells were pooled and then purified to remove PCR dimers using Genejet Enzyme Cleanup (ThermoFischer, catalog no. K0832). The mixture was purified using four columns and eluted with 4 x 20 μl of elution buffer. The eluate was loaded onto a 1% agarose gel with SYBRSafe DNA GelStain (Sigma-Aldrich) for gel extraction, and the extracted product was used as a template in the subsequent PCR reaction. Next, a nested PCR reaction containing the pADL10 cloning site was used for amplification using the following oligonucleotides: VHH-R-SfiI (SEQ ID NO: 54) and VHH-F-SfiI (SEQ ID NO: 55).Two PCR mixtures were prepared with 0.5 μM each primer (VHH-F-SfiI and VHH-R-SfiI) and 0.1 μM each primer, and thermocycling was performed with 1 μl of template as follows: 98°C for 5 min, 98°C for 30 s (20x), 72°C for 1 min (20x), 72°C for 10 min, and storage at 4°C. Approximately 50 ng of amplified and gel-extracted heavy chain antibody was used as template in each well. After amplification, all wells were pooled and purified using Genejet PCR cleanup according to the manufacturer's protocol, followed by elution with 10 mM Tris-HCl pH 8.5. The PCR product concentration was measured using a Nanodrop (result: 160 ng / μl). The product was digested with SfiI restriction enzyme (Thermo Fischer Scientific). Using the Genejet gel extract, a 400 bp DNA fragment was extracted on a 2% agarose gel.

[0178] The pADL10b vector was digested with SfiI restriction enzyme (Thermo Fischer Scientific) overnight at 37°C according to the manufacturer's instructions. The digested vector was purified on a 1% agarose gel and visualized using Sybr Safe (Thermo Fischer Scientific). This procedure was repeated to a final concentration of 154.6 ng / μl.

[0179] After PCR amplification, the library was digested (using Sfil) at 50°C for 2 hours. The enzyme was removed using a Genejet PCR cleanup column as described above. The resulting material was eluted with 20 μl of elution buffer (10 mM Tris-HCl pH 8.5). The DNA concentration was measured to be 73 ng / μl. The mixture was then religated. First, the material was heated at 50°C, slowly cooled to room temperature (RT, 20-25°C), and then incubated overnight at 16°C with shaking. Cleanup was performed as described above and eluted with 10 mM Tris-HCl pH 8.5. A total of 1600 ng of DNA was used. 1 or 2 μl of the ligation mixture was used to electroporate 10 μl of TG1 Escherichia coli in a total reaction volume of 25 μl (5.48 ms / 1.92 kV and 5.48 ms / 1.92 kV, respectively). Cells were removed from the -70°C freezer and thawed on ice immediately before use. Transformants were distributed onto large agar plates (20 cm) and grown under standard conditions until colonies were present and ready to be picked.

[0180] The cells were harvested by adding Luria Broth (LB) medium. Colonies were counted, and the library size was 3.2 × 10 8 After overnight growth on agar plates, cultures were prepared from the final library pool. The cultures were shaken at 200 revolutions per minute (rpm) for 2 hours at 37°C. Hyperphage was added at a multiplicity of infection (MOI) of 2 for 30 minutes at 37°C without shaking. The medium was removed by centrifugation at 4500 g for 5 minutes in a Falcon tube. The cells were then resuspended in an equal volume of Terrific Broth (TB) medium supplemented with 100 μg / ml ampicillin, 50 μg / ml kanamycin, and 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). The cultures were shaken at 200 rpm overnight at 30°C.

[0181] A 5 mL culture was prepared in LB from an overnight culture of the selected clone in a 15 mL Falcon tube. 100 μg / mL ampicillin was added and glucose was omitted. The overnight culture was diluted 1:100 into 5 mL of LB and grown for 2 hours at 150 rpm until the culture became turbid. 1 μL of Hyperphage helper phage (Hyperphage, titer: 1.4 × 10) was added. 12 cfu / ml (Progen) was added to each culture. Helper phage was allowed to infect for 30 minutes at 37°C. The phage-containing LB was discarded by centrifugation at 4500g. The bacterial pellet was resuspended in 5 ml of TB medium supplemented with 100 μg / ml ampicillin, 50 μg / ml kanamycin, and 1 mM IPTG. The culture was shaken at 200 rpm at 30°C overnight. The culture was centrifuged at 4500g for 4 minutes to remove bacteria, and 0.25×V volume of 20% PEG8000 / 2.5M NaCl was added and the centrifuge tube was incubated on ice for 2 hours. The phage / bacteria were pelleted at 15000g for 10 minutes and resuspended in 0.5 ml of PBS. The phage-containing supernatant was transferred to a separate microcentrifuge tube preblocked with PBS / casein.

[0182] Panning with Dynabeads® using biotinylated TfR1 Biotinylation of hTfR1, cTfR1, and mTfR1 was performed according to standard procedures using the Biotinylation Kit EZ-link Sulfo NHS PEG4 (Thermo Fisher Scientific) with a mixture of 1 mg of target protein and 2 mM biotin at a 1:1 molar ratio at pH 8. Free biotin was removed by dialysis against 1 L of PBS buffer, pH 8, for 3 x 1 h at 4 °C in a Pierce 3 mL 10 kDa MWCO (molecular weight cut-off) dialysis unit. Protein was concentrated to 850 μl using an Amicon spin concentrate with a 10 kDa MWCO. Phage supernatants were preblocked with PBS / casein and mixed with 100 nM biotinylated hTfR1, cTfR1, or mTfR1 and incubated for 1 h. Bead blocking was performed with 50 μl of Streptavidin Dynabeads® (M280, Invitrogen, Thermo Fisher Scientific) using hTfR1 (1.1 mg / mL) and mTfR1 (1 mg / mL) and washed with PBS containing 0.15% Tween® 20 (PBST). The beads were resuspended in 1 ml of ELISA blocker (PBS / casein ELISA blocker (Thermo Scientific)) and incubated at room temperature for 30 minutes on a rotating mixer. The blocking was replaced with the phage + target mixture and incubated at room temperature for 30 minutes with gentle rotation to capture biotinylated TfR1 on the beads. The beads were washed five times with PBS. Both mTfR1 and hTfR1 captures were eluted with 400 μl of PBS using 0.25 mg / mL trypsin.

[0183] Phages were trypsinized for 30 minutes at room temperature before titration and rescue. TG1 Eschericia coli cultures were initiated by inoculating a 10 ml culture in 10 ml LB medium with a single colony of TG1 grown on an M9 minimal agar plate. The culture was incubated at OD 600 until the OD reaches the log phase (approximately 4 hours). 600The phage input was trypsinized by mixing 75 μl of phage stock with 25 μl of TG1 (1 mg / ml trypsin) and incubated at room temperature for 30 minutes before dilution and titration. The remaining phage output was mixed with 2 ml of TG1 E. coli and incubated at room temperature for 5 minutes without shaking. The mixtures were plated onto two large LB-Lennox agar plates (Merck-Sigma-Aldrich) and allowed to dry completely before being incubated at 37°C.

[0184] The procedure for selection (and subsequent rescue) with capture Dynabeads was repeated in three subsequent rounds using either hTfR1, cTfR1, or mTfR1 at 100 nM, 25 nM, 10 nM, and 5 nM, respectively, according to Table 1 below (the last two rounds in the presence of 1 μM human transferrin (recombinant human transferrin produced in rice, Sigma-Aldrich)). After the fourth selection round, 25 colonies representing each track and elution method (trypsin or citrate / phosphate buffer, pH 5.5, containing 0.15% Tween® 20) were randomly picked from the agar plates and sent for sequencing with primer pADL 10fwd at the Karolinska Institutet (Sweden) Genetic Analysis Core Facility.

[0185] [Table 1]

[0186] Example III - Binding with ELISA to select VHHs that bind to hTfR1 Prior to ELISA, selected phage samples were diluted 1:10 in blocking buffer (casein ELISA blocker) containing 20% ​​v / v PEG8000 in 2.5 M NaCl. Targets (hTfR1, cTfR1, mTfR1) were coated onto 96-well untreated Greiner plates (100 μl) at 1 μg / ml in PBS, pH 7.4, overnight at 4°C. The coating buffer was discarded and replaced with 100 μl of blocking solution. The plates were covered and incubated at room temperature for 2 hours at 900 rpm. Anti-human TfR1 antibody BA3 (MEM189, Sigma, 1 mg / ml) and anti-mTfR1 (8D3, ​​Novus biologicals, 1 mg / ml) were diluted in blocking buffer to a starting concentration of 100 nM and serially diluted 1:2 by transferring 100 μl to 100 μl of blocking buffer. Plates were incubated at 900 rpm at room temperature for 1 hour, and wells were washed 3 times with 300 μl of PBST (PBS containing 0.1% Tween® 20) using a 50TS microplate washer (Biotek). 100 μl of anti-M13 antibody (anti-PVIII GE Healthcare) diluted 1:4000 in blocking buffer was added to the target-coated wells, followed by incubation at 900 rpm at room temperature for 1 hour. Washing with PBST followed. Secondary anti-mouse antibody (Sigma) 1:4000 in blocking buffer was incubated at 900 rpm at room temperature for 1 hour. After washing in PBST, color development was performed with TMB substrate. The reaction was stopped by adding 2 M H2SO4 to each well, and absorbance readings were taken at 450 nm using a SpectraMax 3000 (Molecular Devices). One clone (KB_B01) used in the following examples was selected based on cross-reactive binding in ELISA to both cTfR1 and hTfR1. The selection criteria were >10-fold reactivity to both human and cynomolgus receptors over the blank (negative control). Selection criteria in this workflow were also based on non-competition with Tf for the Tf binding site (see Example II), which is not the preferred binding site for transport of therapeutic agents to the brain.

[0187] Example IV - Production of free VHH or fusion variants The selected VHH clone (KB_B01) was produced as a single VHH unit (12-14 kDa) (see Figure 2). A C-terminal 6xHis tag (SEQ ID NO: 56) followed by a "C-tag" (C-terminal amino acids EPEA, SEQ ID NO: 57) was ordered and codon-optimized from Genscript Inc. using the expression vector pEt-22b(+). The protein was obtained from the periplasmic space of Escherichia coli using standard methods and purified using a one-step purification on a Ni-column.

[0188] The human Fc region was genetically fused to the VHH using a GG spacer to create a bivalent TfR1-binding functional entity (see Figure 2).

[0189] More specifically, a VHH-Fc fusion protein (SEQ ID NO: 59) between human IgG1 Fc (SEQ ID NO: 58) and KB_B01 (SEQ ID NO: 30), containing a signal peptide from the mouse IgG kappa light chain (SEQ ID NO: 81), was cloned into the vector pcDNA3.4 backbone containing codon-optimized sequences (performed by GeneArt, Thermo Fischer Scientific) and transiently expressed in 200 mL cultures of Expi293™ cells (Thermo Fischer Scientific), grown under standard conditions, and harvested 6 days post-transfection. Purification of the culture medium was performed using MabSelect Sure™ and polished on HiLoad Superdex 200, yielding a fraction of dimeric VHH-Fc fusion with a purity of >99% as verified by analytical size-exclusion chromatography (SEC). The same process was performed to generate fusion proteins for a reference VHH (SEQ ID NO: 80) and human IgG1 Fc as disclosed in WO2020 / 144233. A VHH-containing fusion protein according to this standard is referred to herein as a BV.

[0190] Furthermore, KB_B02 (SEQ ID NO: 31), KB_B03 (SEQ ID NO: 32), KB_B04 (SEQ ID NO: 33), KB_B05 (SEQ ID NO: 34), KB_B06 (SEQ ID NO: 35), KB_B07 (SEQ ID NO: 36), KB_B08 (SEQ ID NO: 37), KB_B09 (SEQ ID NO: 46), and KB_B10 (SEQ ID NO: 47) were sequence-optimized and cloned into a proprietary plasmid in a volume of 4 mL at GenScript Biotech Corporation based on the same human IgG1 Fc and GG linker sequences and signal peptide (SEQ ID NO: 79) as above, and then transformed into Turbo-CHO™ High The following Fc fusions were produced using the Performance platform system: KB_B02-Fc (SEQ ID NO: 60), KB_B03-Fc (SEQ ID NO: 61), KB_B04-Fc (SEQ ID NO: 62), KB_B05-Fc (SEQ ID NO: 63), KB_B06-Fc (SEQ ID NO: 64), KB_B07-Fc (SEQ ID NO: 65), KB_B08-Fc (SEQ ID NO: 66), KB_B09-Fc (SEQ ID NO: 67), and KB_B10-Fc (SEQ ID NO: 82). Final samples were collected 6 days post-transfection and purified using a Genscript protein A-based system, yielding 95-99% purity as verified by either SDS-PAGE or analytical size-exclusion chromatography (SEC).

[0191] Functional fusion proteins were produced in the form of fusions with VHH and single-chain variable domains (scFv). Such fusion proteins have monomeric TfR1 binding but have the potential for bispecific binding due to the presence of scFv domains that can bind to targets different from TfR1 or different epitopes on the same target. The amyloid beta-binding murine antibody 3D6 was selected as a functional representative scFv unit. The scFv of the 3D6 antibody was composed of a VH (SEQ ID NO: 68) and a VL (SEQ ID NO: 69) and was fused to either the N- or C-terminus of KB_B01 and BV, linked by a 3×(G4S) linker (SEQ ID NO: 70), resulting in KB_B01-scFv (SEQ ID NO: 71), scFv-BV (SEQ ID NO: 72), BV-scFv (SEQ ID NO: 73), or scFv-KB_B01 (SEQ ID NO: 74). The gene construct was sequence-optimized and synthesized by GenScript Biotech Corporation, cloned into a custom plasmid, and expressed in transiently transfected TurboCHO™ High Performance platform cells. Purification was performed using HiTrap™ FF Crude (Cytiva) followed by HiLoad™ 26 / 600 Superdex (Cytiva) according to standard procedures.

[0192] Reference antibody 128.1 (BA1) was expressed as full-length human IgG1 (SEQ ID NO: 75) in transiently transfected FreeStyle™-293 cells using the vector pcDNA3.4, recovered from the culture medium, and purified by one-step purification on Protein A resin. Reference antibody JCR-IgG (BA2) (SEQ ID NO: 76) was designed based on information from US 9,994,641 as an IgG1 antibody that reacts with hTfR1 through its Fab'2 (SEQ ID NO: 51). The heavy chain (HC) and light chain (LC) sequences were optimized, cloned into separate plasmids, transfected into transiently transfected ExpiCHO™ cells using standard reagents, expressed, and harvested on day 8. Proteins were purified using MabSelect Sure™ (Cytiva) followed by Superdex 16 / 60 (Cytiva) and analyzed according to standard procedures known to those skilled in the art.

[0193] Binding characteristics of Example V-KB_B01 Binding of llama VHH KB_B01 to hTfR1 and cTfR1 was identified during phage display panning toward hTfR1 and cTfR1 by ELISA. Binding kinetics were further analyzed by surface plasmon resonance (SPR) and biolayer interferometry (BLI). VHH KB_B01 was tested for binding affinity to the target human protein and for the lack of interference with affinity by transferrin (Tf). SPR analysis was performed on a Biacore T200 or Biacore 8K instrument (Cytiva, Sweden). SPR Series S Chip SA and Series S CM5 chips, or Series S Protein A chips (Cytiva, Uppsala, Sweden) were used. Biotinylated transferrin was coupled to the SA chip.

[0194] Human TfR1, mTfR1, and cTfR1 were all biotinylated using a biotinylation kit (EZ-Link NHS-PEG4-Biotin, No-Weigh Format (Thermo Fisher Scientific)) according to the manufacturer's instructions. A vial of biotin stock solution was dissolved in DMSO at a concentration of 20 nM and stored at -70°C. The tube was thawed, freshly diluted to 2 mM with ddH2O, and mixed with TfR1 protein in a 2-fold molar excess of biotin in PBS in a total volume of 200 μL. The mixture was incubated with shaking at 25°C for 45 minutes, followed by overnight incubation at 4°C. Buffer exchange was performed using a Nap5 column (Cytiva) according to the manufacturer's instructions. The final elution volume was 750 μL. PBS buffer, pH 8, was used for buffer exchange. Protein concentration was determined using absorbance at 280, and the yield was >75%.

[0195] The binding characteristics of monomeric VHH KB_B01 and reference full-length antibody BA1 were evaluated by SPR on a Biacore T200 instrument using single-cycle kinetics. Human Tf was amine-coupled (to approximately 3000 RU) on a CM5 chip, and then hTfR1 was captured on immobilized human transferrin (hTf). Human TfR1 was injected at 100 nM, followed by injections of increasing concentrations of analyte (VHH KB_B01 or BA1) ranging from 6.25 to 100 nM in two-fold increments. The contact time was 150 seconds for each concentration at 30 μL / s, with a final dissociation step of 500 seconds. At the end of each single-cycle run, the hTf surface was fully regenerated by injecting 10 mM glycine pH 2 for 30 seconds. The association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (K) were measured. D ) were evaluated using the Biacore T200 software evaluation tool, applying the Langmuir 1:1 model.

[0196] Monomeric VHH KB_B01 and reference antibody BA1 bound to hTfR1 (Table 2 and Figures 3A and 3B), confirming human Tf-free binding. Because hTfR1 was immobilized by loading onto hTf, analyte binding meant that neither KB_B01 nor BA1 competed with hTf for binding to hTfR1. Furthermore, a comparison of KB_B01 and BA1 in terms of affinity and binding kinetics revealed that monomeric KB_B01 bound to bivalent BA1 (K D = 0.0199 nM), a 166-fold lower affinity (K ) of 3.31 nM compared to D ) bound to the precomplex hTfR1-Tf at a significantly higher affinity than BA1, which was due to a dramatic difference in dissociation rate (kd). It has been shown that too high an affinity can result in retention of TfR1-binding antibodies and any proteins fused to them within the vasculature and / or lysosomal degradation of the TfR1-anti-TfR1 complex (Bien-Ly N, et al., Transferrin receptor (TfR) trafficking determines brain uptake of TfR antibody affinity variants. J Exp Med. 211(2):233-244(2014)). Therefore, VHH KB_B01 has lower affinity for hTfR1 compared to BA1, and therefore has more favorable binding properties as a brain drug transporter.

[0197] [Table 2]

[0198] Example VI - Characterization of Fc-fused VHH KB_B01 Affinity measurements were performed by SPR on a Biacore T200 (Cytiva) by loading KB_B01-Fc, BV-Fc, and BA2 (Example IV) onto a Protein A chip (Cytiva) at high temperature (100 nM for 60 seconds). These were then exposed to increasing concentrations of hTfR1 ranging from 0.16 to 100 nM in 5-fold increments. The data are summarized in Table 3, and sensorgrams are presented in Figures 4A-4I. Dimeric KB_B01-Fc bound to immobilized hTfR1 (K D = 3.31 nM, see Table 2) compared to the free monomer KB_B01. D BA2 bound to hTfR1 with an affinity (K) of 0.176 nM (Table 3, Figure 4A), i.e., 19-fold higher. As controls, the reference BV-Fc and BA2 were run under the same conditions and bound to hTfR1 with an affinity (K) of 0.00228 nM (Figure 4D) and 0.0249 nM (Figure 4G), respectively. D Cynomolgus monkey and mouse TfR1 were also tested under the same conditions, and KB_B01-Fc bound to mTfR1 with an affinity of 2.82 nM (Figure 4C). The reference BV-Fc bound to mTfR1 with a high affinity (K D ) bound to mTfR1. This meant that when both were in the form of Fc fusions, KB-B01 bound to mTfR1 with 6.4-fold lower affinity than the reference antibody BA2. In contrast, the reference antibody BA2 did not bind to mTfR1 with a detectable signal (Figure 4I). This experiment demonstrated that KB_B01 possesses the desired attributes of a low nM affinity range, as well as a K for receptor release (off). D It shows that it has an interesting profile.

[0199] Furthermore, KB_B01 exhibited broad species cross-reactivity, a highly desirable property from a translational perspective in predicting both pharmacokinetics and pharmacodynamics from mice to humans.

[0200] [Table 3]

[0201] To further investigate the effect of soluble human transferrin on binding to human TfR1, the above-described SPR was performed in the presence and absence of human Tf. The KB_B01-Fc fusion protein, reference BV-Fc, BA1, and BA2 were immobilized on a Protein A sensor (Cytiva) and then exposed to increasing concentrations of receptor ranging from 0.156 to 40 nM (4-fold increments) in the absence or presence of a saturating concentration of 250 nM hTf. The high affinity for hTfR1 was maintained even in the presence of 250 nM hTf, suggesting that KB_B01-Fc and the two reference antibodies BA1 and BA2 do not interfere with Tf binding to human TfR1 (Table 4, Figures 5A–5F). This experiment demonstrates that KB_B01 possesses desirable and unique attributes for using the TfR1 molecule in humans to increase the delivery of therapeutic or diagnostic proteins to the CNS.

[0202] An interesting difference between KB_B01 and the reference VHH BV was that the binding of BV-Fc to hTfR1 was more affected by the presence of hTf than that of KB_B01-Fc: the affinity to hTfR1 was reduced nearly 390-fold for BV-Fc but only 10-fold for KB_B01-Fc in the presence of 250 nM hTf versus its absence.

[0203] [Table 4]

[0204] Example VIII - Bioinformatics and analysis of additional clones KB_B01 was aligned to all sequenced clones from the initial screening (panning) of clones using the Jalview Multiple Sequence Alignment Editor and Analysis Workbench version 2 (Burton Group, University of Dundee) software. Based on sequence identity to KB_B01 and positive binding confirmed by ELISA, nine additional clones, KB_B02 to KB_B10 (see Figure 6 for alignment), were selected and produced as fusions to human IgG1 Fc (Example IV). One clone known for its high interference with Tf binding was selected for use as the reference, KB_BC.

[0205] Based on the above, KB_B02-KB_B10 were generated as fusions to human IgG1 Fc (see Example IV) and tested for affinity to hTfR1 and mTfR1 using SPR. The dimeric Fc-fused VHHs were immobilized on a Protein A chip as described above. Briefly, using a Biacore 8K and single-cycle kinetics, the VHH-Fc constructs were diluted to 5 nM and captured on a Protein A chip (Cytiva) in eight increments at 5 μL / s for 120 s, followed by injections of receptor concentrations ranging from 0.8 to 500 nM in 5-fold increments (contact time 120 s, dissociation time 600 s). After each complete single-cycle kinetics cycle, the surface was regenerated with 10 mM glycine, pH 1.5, at 30 μL / s for 30 s. The kinetic data for KB_B02-KB_B10 as Fc fusions are presented in Table 5. KB_B02~KB_B10 are all K D It bound to hTfR1 with high affinity, defined as values ​​ranging from <0.00309 nM to 0.451 nM (Table 5). The affinity for mTfR1 ranged from <0.0171 nM to 89.7 nM.

[0206] [Table 5]

[0207] Epitope binning was then performed by combinatorially testing hTfR1 binders pairwise using SPR on a Biacore 8K. KB_B01, KB_B04, KB_B05, KB_B08, KB_B09, and KB_B10 were immobilized along with reference antibodies BA1 and BA2 on a CM5 chip series S (Cytiva) by amine coupling. An additional VHH-Fc known to bind to a distinct epitope, KB_BC, was also immobilized. The immobilized protein was diluted to 25 μg / mL in sodium acetate at pH 5.5, resulting in an SPR signal of approximately 6000 resonance units (RU). 50 nM of hTfR1 ectodomain was used in the first injection (contact time 120 s, 10 μL / s), and 100 nM of each analyte was used in the second injection: KB_B01-KB_B10, BA1, BA2, KB_BC, mouse transferrin (mTf), and human transferrin (hTf) with a contact time of 150 s, 10 μL / s. After each cycle, the surface was regenerated with 10 mM glycine, pH 2.1, at 30 μL / s for 30 s. Data were analyzed using the Biacore Insight evaluation package version 4.0.8.20368. The results show that KB_B01-KB_B10 all compete with each other for binding to hTfR1, thus forming distinct epitopes (Table 6). Antibody BA1, known to bind to the apical domain of hTfR1 (Helguera G. et al., An antibody recognizing the apical domain of human transferrin receptor 1 efficiently inhibits the entry of all new world hemorrhagic fever arenaviruses. J Virol. 86(7):4024-4028(2012)), competed with BA2 but not with KB_B01 to KB_B10. The control VHH-Fc molecule KB_BC did not block any of the other anti-TfR1 molecules, nor BA1 or BA2; instead, it completely prevented mTf and hTf from binding to hTfR1.The KB_BC control shows that all others do not compete with Tf, and therefore KB_B01 to KB_B10 have the desired properties for transport to the brain.

[0208] [Table 6]

[0209] To demonstrate functionality when fused to non-Fc proteins, KB_B01 and reference BV were generated as fusions with single-chain variable domains (VH-VL type scFvs with the heavy chain located at the amino terminus and the light chain located at the carboxy terminus) at either the amino terminus (scFv-KB_B01) or the carboxy terminus (KB_B01-scFv). For details, see Example IV and Figure 2. To verify that these fusion proteins still bind to hTfR1, the binding kinetics were examined by SPR using an 8K Biacore. Briefly, the receptor hTfR1 was immobilized on a CM5 chip (Cytiva) by amine coupling and exposed to increasing concentrations of either scFv-KB_B01 or KB_B01-scFv in four-fold increments ranging from 0.25 to 64 nM using single-cycle kinetics. Similarly, the reference VHH BVs were produced as similar fusion proteins, scFc-BV and BV-scFv, respectively, and tested in the same experiments, but at a slightly wider concentration range, from 0.08 nM to 50 nM in five-fold increments. The kinetic binding data and sensorgrams are presented in Table 7 and Figures 7A-7D. Thus, KB_B01 exhibited a significant increase in affinity for hTfR1, with the amino-terminus (scFv-KB_B01, K D 1.80 nM) or the carboxy terminus (KB_B01-scFv, K D It can be concluded that the KB_B1 complex can bind to another protein with either a K of 0.845 nM or 0.845 nM. In fact, the affinity is slightly higher (K ​​of 0.845 nM) compared to the monomeric KB_B1 complexed with hTf. D 3.31 nM, Table 2), suggesting that KB_B01 can be used as a BBB transport vehicle fused with therapeutic molecules without loss of function.

[0210] [Table 7]

[0211] Example VII - TfR1-mediated uptake in human cells Next, we tested whether the KB_B01-Fc fusion protein could be internalized into human TfR1-expressing cells. Adherent HEK293T cells (ATCC), known to express TfR1, were grown in collagen-coated 96-well plates (ThermoFischer Scientific) and seeded at a density of 30,000 cells / well. Cells were grown in DMEM (Dulbecco's Modified Eagle's Medium) for 3-5 days until approximately 80-90% confluence. KB_B01, as well as a reference BV-Fc fusion and the known reference binder BA1, and a negative control (a similar VHH-Fc that lacks hTfR1 binding) were used in this assay.

[0212] The cell assay was established based on titration of test items and negative controls ranging from 0.85 nM to 82.5 nM for 5, 15, 30, and 45 minutes at 37°C in 5% CO2. To compare the cell fate of added proteins after a prolonged exposure (set at 120 minutes), after a 30-minute incubation, the medium was replaced with fresh DMEM for 90 minutes (incubated at 37°C in 5% CO2), followed by two medium changes. After 120 minutes, the cells were transferred to the bench, immediately washed with PBS (1x), and fixed with 4% paraformaldehyde for 10–15 minutes. After fixation, the cells were washed with PBS, permeabilized with 0.1% Triton-X100™ in PBS, and then blocked with 1% bovine serum albumin (BSA) in PBS. Following this, Alexa Fluor-488-anti-human IgG (Fc fragment-specific, stock 0.75 mg / ml) (Jackson Immunoresearch) was added. 300 U of Texas Red-phalloidin (1:200 dilution of a 10 μg / mL stock) was added to the wells during the final 20 minutes of secondary antibody incubation. After this, cells were washed at least three times with PBS and stored at 4°C until imaging. Cells were imaged on an inverted Zeiss 710 laser scanning confocal microscope using lasers 488, 555, and 647 and a 20x air objective.

[0213] Results from these experiments demonstrated that KB_B01, as well as BV and BA1, were readily internalized into HEK293T cells at the various concentrations tested (0.7–85 nM) and incubation times from 5 to 240 minutes. Representative of this, Figure 8 shows that KB_B01-Fc was internalized as efficiently as BV-Fc at 20 nM for 40 minutes. At longer time points, i.e., 120 minutes of incubation, comparable amounts of KB_B01-Fc, BV-Fc, and BA1 were detected after 30 minutes of rinsing to remove excess test protein in the medium. A negative control (VHH-Fc, not binding to hTfR1) did not accumulate intracellularly (Figure 8) or show any presence at any time point or concentration tested, indicating that the assay and internalization were TfR1-dependent.

[0214] Example VIII - In vivo and ex vivo evaluation of VHH-Fc and scFv fusions To evaluate the properties of KB_B01 for in vivo brain targeting, KB_B01-Fc was compared to one equivalent VHH-Fc (KB-negative control) lacking mTfR1 binding in wild-type (WT) mice. Next, scFv-VHH fusions (described in Examples IV, VI, and Table 7) were compared to the negative control based on the N-terminal versus C-terminal orientation of the fusion partner. All fusion proteins were prepared according to the method described in Greenwood et al., The preparation of 131 Iodine-125 (I-labeled human growth hormone of high specific radioactivity) was measured using the chloramine T method as described in Biochem J 89:114-123 (1963). 125 Protein amounts of 15–80 μg were radiolabeled with 260 ± 26 MBq / μg or 61 ± 2.4 kBq / μg stocks. 125 Radiolabeled samples were immediately purified on a 7 kDa cutoff Zeba-column (ThermoFischer).

[0215] The retained binding to mTfR1 and hTfR1 was verified using ELISA. Briefly, high-binding half-area 96-well plates (Costar 3690, Merck) were coated with 1-4 μg / mL (in PBS pH 7.4) of hTfR1 and mTfR1 ectodomains and left overnight at 4°C. The plates were blocked for 1 hour using 1% BSA 0.05% Tween® 20 (Merck) in PBS. After washing (PBS wash buffer), the radiolabeled samples (or original samples) were incubated for 2 hours or overnight (4°C) at a serial dilution of 1:5 in dilution buffer (0.1% BSA in PBS-Tween® 20 0.05%), followed by four repeated washes and incubation with a secondary antibody detecting VHH (anti-VHH secondary antibody (A01861, Genscript Biotech Corporation) at a dilution of 1:6000). In all reported experiments, binding to the target (mTfR1, hTfR1) was confirmed.

[0216] In vivo experiments were performed in 12-week-old C57BL / 6 mice. Additionally, to examine the retention of amyloid beta by bispecific VHH-scFv fusions with the amyloid beta-binding 3D6 scFv, 13- to 15-month-old mice were used to treat AD APP. NL-G-F The model (Nilsson P et al., ACS Chem. Neurosci. 2014, 5, 7, 499-502) was used. Immediately after radiolabeling, mice were intravenously administered either VHH-Fc (5 nmol / kg, 3.4 MBq / nmol) or VHH-scFv (5 nmol / kg, 6.8 ± 0.5 MBq / nmol) fusion protein via the tail vein. Blood samples (8 μL) were obtained from the tail vein at 5 min, 0.5, 1, 2, 4, and 9 h post-injection. Mice were euthanized by cardiac withdrawal prior to transcardial perfusion with 40 mL of NaCl for 2.5 min. Post-injection euthanasia time points were 2.5 h post-injection for VHH-Fc constructs in WT mice, and 9 h post-injection for WT and APP mice. NL-G-FIn mice, brain retention was examined after 2, 6, or 24 hours for the VHH-scFv fusions. Only KB_B01-scFv was used at the 6-hour time point. Brains and major organs were dissected. Blood was separated into plasma and a blood cell pellet by centrifugation at 10,000 × g for 5 minutes. The brain was separated into left and right hemispheres. The cerebellum was removed from the left hemisphere. The remaining tissues of the left hemisphere are hereafter referred to as the "brain." Radioactivity was then measured in the brain, blood fractions, and major organs using a γ counter (2480 Wizard™, Wallac Oy PerkinElmer, Turku, Finland). As shown in Figure 9A, at 2.5 hours postinjection, the distribution in the brain of WT mice showed a clearly improved SUV for the dimeric VHH-Fc fusion KB_B01 compared with the negative control VHH-Fc fusion, suggesting a functional brain targeting mechanism in vivo. The blood levels of the non-mTfR1-binding negative control were higher than those of KB_B01 as a VHH-Fc fusion (Figure 9B). These experiments demonstrate that KB_B01 as an Fc fusion targets brain compartments depending on its mTfR1-binding properties.

[0217] Next, the fate of scFv-VHH fusions was investigated at 2 and 24 hours post-injection using both N- and C-terminal constructs. Relative to KB_B01, the N-terminal configuration of VHH (KB_B01-scFv) showed favorable brain concentrations at 2 hours (Table 8). The reference BV showed a similar relationship between the direction of brain concentration at 2 hours and total brain uptake (Table 8).

[0218] [Table 8]

[0219] Next, WT and APP mice were analyzed at 2 and 24 hours after injection. NL-G-FBrain concentrations were assessed in mice. Protein retention through binding to amyloid beta is important for both the diagnostic and therapeutic efficacy of the active moiety / conjugate. As shown in Figure 10A, at 2 hours post-injection, brain distribution was higher for BV-scFv than for KB_B01-scFv. At 6 hours, only KB-B01 was examined, and KB-B01 significantly increased the brain distribution of APP. NL-G-F Both KB_B01 and BV showed clear brain parenchymal retention in WT mice (Figure 10B). At 24 hours, both KB_B01 and BV showed lower brain concentrations than at the 2-hour time point (Figure 10C). Here, nonspecific retention in the brain (not binding to amyloid beta) is advantageous, as reflected in the low levels in WT brain, particularly for KB_B01-scFv. As can be seen in Table 9, KB_B01-scFv showed the lowest detectable levels of injected radiolabeled fusion protein in the brains of WT mice at 24 hours. Also, at 24 hours, APP NL-G-F The brain concentration ratio between KB_B01-scFv and WT mice was higher for KB_B01-scFv compared to the reference BV-scFv (17.2 vs. 13.4, WT vs. APP NL-G-F (See Table 9 for fold differences between mice.) Furthermore, looking more closely at peripheral pharmacokinetics, specific distribution to red blood cells (erythrocytes, i.e., pellets) was higher for BV-scFv than for KB_B01-scFv 24 hours after injection (Figure 10D).

[0220] [Table 9]

[0221] Example IX - Ex vivo characterization of VHH-scFv fusions To examine VHH distribution between the vasculature and brain parenchyma, 20 μm sagittal cryosections were fixed in 4% paraformaldehyde (PFA) for 10 minutes, washed in PBS, and blocked with 5% normal goat serum. The sections were incubated in PBS with 0.1% Tween® 20 for 15 minutes, followed by overnight incubation at 4°C with rat anti-mouse CD31 antibody (BD, no. 553370) and rabbit anti-Aβ42 antibody (Agrisera). The next day, the sections were washed in PBS and incubated with goat anti-rat antibody (Alexa 647, Molecular Probes, Thermo Fischer Scientific) and goat anti-rabbit antibody (Alexa 488, Molecular Probes, Thermo Fischer Scientific). The sections were stored in PBS until the nuclear track emulsion (NTE) procedure (described below) was performed the same day.

[0222] NTE was performed according to a previously published procedure (Gustavsson et al., SPECT imaging of distribution and retention of a brain-penetrating bispecific amyloid-β antibody in a mouse model of Alzheimer's disease, Transl Neurodegener 9: 37 (2020)). According to the manufacturer's instructions, Ilford K5 emulsion (Ilford Photo, Harman Technologies) was melted in a 40°C water bath and prepared as a 50% solution with MQ-H2O under darkened conditions with safelight. Brain sections were immersed in the emulsion for 10 seconds and then air-dried at room temperature for 2 hours. Sections were stored at 4°C in a light-tight box for 4 weeks. Development was performed according to the manufacturer's instructions. After NTE, sections with immunostained and developed emulsions were visualized using either wide-field illumination (brightfield for NTE) or fluorescence (CD31) with a filter block for emission wavelength 647 using a Zeiss Observer Z.1 microscope (Carl Zeiss Microimaging GmbH, Jena, Germany) with ZEN software, and systematic images were acquired. Images were then overlaid (merged), and NTE puncta within vasculature or parenchymal space were quantified based on macro-based image analysis. Relative parenchymal-to-capillary signal (% parenchymal) in NTE images was analyzed with n = 20 images per animal from one animal per group with the median (i.e., most representative) concentration of all brain puncta. Quantification was performed using a standardized macro in Fiji (ImageJ) as previously described (Faresjoe et al., Brain pharmacokinetics of two BBB penetrating bispecific antibodies of different sizes, Fluids and Barriers of the CNS 18: 26 (2021)).

[0223] As shown in Figure 11A, KB_B01-scFv showed superior distribution to the brain parenchyma without vascular trapping at 2 h postinjection compared to the reference BV-scFv. Figure 11A shows representative vascular structures outlined by thin white lines to indicate vascular versus parenchymal localization of NTE points (white dots). Quantification revealed preferential distribution to the brain parenchyma: 78 ± 6% for KB_B01-scFv versus 52 ± 10% for BV-scFv (Figure 11B). This means that each dose of KB_B01-scFv more efficiently reached its target site (epitope) in the brain, making it the most suitable construct tested for use in PET imaging diagnostics for specific imaging of disease biomarkers in situ. For contrast agents used in PET, nonspecific retention within blood vessels is undesirable, and better parenchymal targeting is also a priority for therapeutic agents to reach their target.

[0224] Example X - Generation of Protein A Binding Antibodies Although Llama VHHs do not normally bind to the Protein A-based resin PrismA™, amino acids within the framework sequences can be mutated to generate VHHs that will bind PrismA™ and thereby bind Protein A. To investigate the potential of KB_B01 and KB_B03, and fusion proteins comprising KB_B01 and KB_B03, to enable Protein A binding, these two VHHs were mutated according to the consensus Protein A binding sequence by Henry et al., A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies, PLoS One 11(9):e0163113 (2016). As free VHHs without a His6 tag, KB_B01 was mutated from glutamic acid to lysine at position 75, aspartic acid to asparagine at position 83, and asparagine to serine at position 84 (KB_B01: E75K, D83N, N84S, designated as KB_B11 SEQ ID NO: 83), and KB_B03 was mutated from aspartic acid to asparagine at position 83 (KB_B03: D83N, designated as KB_B12 SEQ ID NO: 84). KB_B12 was also produced as a fusion protein with an scFv (SEQ ID NO: 85) fused to a His6 tag via a short Gly2 linker (GGHHHHHH, SEQ ID NO: 86), allowing Ni-NTA-based purification in addition to PrismA™-based purification. A positive control known to bind Protein A (SEQ ID NO: 87) was also included.

[0225] Proteins were produced in CHO cells by ProteoGenix SAS, France. cDNAs encoding VHHs (and fusions with scFvs) were chemically synthesized and optimized for expression in CHO cells and subcloned into ProteoGenix's proprietary mammalian cell expression vector. The vectors were transfected into XtenCHO™ cells using the XtenCHO transfection protocol. A total of 3.5 mL of medium was collected 8 days after transfection and purified using Mabselect PrismA resin (Cytiva). The medium was clarified by filtration (0.22 μm) and loaded onto pre-equilibrated (PBS pH 7.5) PrismA™ resin. Bound VHHs were eluted by pH shift using citric acid according to the resin manufacturer's instructions, and the eluate was immediately neutralized by the addition of 1 M Tris-HCl pH 9.0. Protein concentration was determined by A280 spectrophotometry. The flow-through fraction for the scFv fusion construct was then purified by Ni-NTA-based purification using IMAC high-binding-capacity nickel resin. The equilibration and binding buffer was PBS pH 7.5, and washing and elution were performed by imidazole shift. Protein concentration was determined by A280 nm spectrophotometry and qualified and quantified by SDS-PAGE.

[0226] Protein concentrations after PrismA™ elution were generally low at approximately 5, 8, and 7 μg / mL culture for KB_B11, KB_B12, and the scFv fusion KB_B12-scFv, respectively. Subsequent purification of the scFv fusion proteins by Ni-NTS IMAC revealed the absence of material in the flow-through fractions, demonstrating the functionality of the PrismA™ resin-based purification for this protein construct.

[0227] PrismA™ resin was used to purify KB_B011, KB_B12, and scFv fusions with KB_B12. Binding to PrismA™ resin for scFv fusions with KB_B011, KB_B12, and KB_B12, as well as a ProA positive control (SEQ ID NO: 90), was verified by SPR using a precoated chip (PrismA chip, Cytiva). A negative control VHH with a sequence not optimized for ProA was also included. The positive and negative controls were produced in E. coli and contained a C-terminal His tag (SEQ ID NO: 86). KB_B11, KB_B12, and the positive control bound with signals reaching 1232, 651, and 1193 RU, respectively, at the highest concentration (500 nM), and KB_B12-scFv bound with a signal reaching 545 RU at the highest concentration tested (125 nM), confirming the functionality of VHH-mediated binding to PrismA™ resin (Table 10). No signal above baseline was recorded with the negative control.

[0228] [Table 10]

[0229] Example XI - In vivo and ex vivo evaluation of VHH-Fc of KB_B03 in TfR1 extracellular domain humanized mice (hECD-TfR1-mice) To evaluate the properties of KB_B03 for in vivo brain targeting, the distribution characteristics of KB_B03 were examined in homozygous, heterozygous, and wild-type hECD-TfR1 mice. Homozygous mice express only partially humanized TfR1, i.e., human-mouse chimeric TfR1. Humanized mice expressing a chimeric receptor containing the extracellular domain of hTfR1 and the intramembrane and transmembrane domains of mTfR1 were generated at Taconic Biosciences GmbH, Leverkusen, Germany.

[0230] A genetically engineered mouse model was generated by Taconic Biosciences GmbH, Leverkusen, Germany, in which the endogenous mouse Tfrc gene was partially humanized, resulting in the engineered mice producing a chimeric TFRC protein containing the human TFRC extracellular domain. Targeting vectors were constructed using methods known to those skilled in the art using BAC-derived mouse and human genomic DNA fragments and including standard selection cassettes.

[0231] Embryonic stem cells (ES) derived from C57BL / 6NTac mice were grown on a mitotically inactivated feeder cell layer composed of mouse embryonic fibroblasts in ES cell medium containing leukemia inhibitory factor and fetal bovine serum. Cells were electroporated with a linearized DNA targeting vector according to methods known to those skilled in the art. Homologous recombinant clones were isolated using methods known to those skilled in the art. ES cell colonies (ES clones) with distinct morphologies were isolated and analyzed by Southern blotting and / or PCR. Homologous recombination at the 3' and 5' sites and single integration were confirmed by PCR and Southern blot using conventional methods known to those skilled in the art. Homologous recombinant ES cell clones were expanded and frozen in liquid nitrogen.

[0232] Generation of chimeras and heterozygotes: After hormone administration, superovulated BALB / c females were mated with BALB / c males. Blastocysts were removed from the uterus at 3.5 days post-transplant (dpc) and placed in a drop of DMEM containing 15% fetal calf serum (FCS) under mineral oil. Using a microinjection pipette, 10–15 targeted ES cells were injected into each blastocyst. After retrieval, approximately eight injected blastocysts were transferred into each uterine horn of pseudopregnant NMRI females. The degree of chimera formation was assessed in chimeras (F0) by observing the contribution of ES cell coat color to the BALB / c host (black / white). In vitro fertilization was performed using oocytes from superovulated C57BL / 6NTac females and thawed semen from previously cryopreserved sperm from male chimeras. Fertilized embryos were then transferred into pseudopregnant Swiss-Webster recipient females. Germline transmission was initially identified by the presence of black-coated pups (C57BL / 6NTac strain) and confirmed by PCR-mediated genotyping of the black pups. F1 mice heterozygous for the modified Tfrc allele were subsequently bred to generate homozygous (HOM), heterozygous (HET), and wild-type (WT) genotypes, which were used in the following studies.

[0233] KB_B03-Fc was purified by iodine-125 ( 125The mice were radiolabeled with KB_B03-Fc (5 nmol / kg, 3.4 MBq / nmol). In vivo experiments were performed in 6- to 10-week-old C57BL / 6 mice. Immediately after radiolabeling, the mice were intravenously administered KB_B03-Fc (5 nmol / kg, 3.4 MBq / nmol) via the tail vein. Blood samples (8 μL) were obtained from the tail vein at 5 min, 0.5 h, 1 h, and finally 2 h after injection. Mice were euthanized 2 h after injection by cardiac blood collection followed by transcardial perfusion with 40 mL of NaCl for 2.5 min. The brain and major organs were dissected. Blood was separated into plasma and a blood cell pellet by centrifugation at 10,000 × g for 5 min. The brain was separated into left and right hemispheres. The cerebellum was removed from the left hemisphere. The remaining tissue of the left hemisphere will hereafter be referred to as the "brain." Radioactivity was then measured in the brain, blood compartments, and major organs using a g-counter (2480 Wizard™, Wallac Oy PerkinElmer, Turku, Finland). As shown in Figures 12A and 12B, the blood distribution profiles of hECD-TfR1 mice indicated that blood levels declined more rapidly from 5 to 30 minutes in HOM mice compared to HET and WT mice, whereas blood levels were similar from 30 to 120 minutes. Blood compartment analysis indicated slightly higher binding to the pellet in HOM mice due to a slightly higher affinity of KB_B_03 for hTfR1 than mTfR1 (Example VIII, Table 5). Normalized brain uptake (Figure 12C) demonstrated high uptake in all genotypes, as expected due to the cross-specific properties of KB_B03. The results demonstrate the brain-targeting ability of KB_B03 in functional hECD-TfR1 in vivo.

[0234] [Table 11] JPEG2025539339000013.jpg199159JPEG2025539339000014.jpg198156JPEG2025539339000015.jpg197159 JPEG2025539339000016.jpg179159JPEG2025539339000017.jpg200159JPEG2025539339 000018.jpg198159JPEG2025539339000019.jpg212159JPEG2025539339000020.jpg20315 9JPEG2025539339000021.jpg207159JPEG2025539339000022.jpg202159JPEG2025539339 000023.jpg205159JPEG2025539339000024.jpg199159JPEG2025539339000025.jpg60159

[0235] The above-described embodiments should be understood as a few illustrative examples of the present invention. Those skilled in the art will understand that various modifications, combinations, and changes can be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in different embodiments can be combined in other configurations, if technically possible. However, the scope of the present invention is defined by the appended claims.

Claims

1. A heavy chain-only variable domain (VHH) antibody that specifically binds to transferrin receptor 1 (TfR1), Amino acid sequence GX １ X ２ FX ３ X ４ X ５ Y, wherein X １ is T or N, and X ２ is P, D or R, and X ３ is S, T or G, and X ４ is N, L, M or I, and X ５ is N or E; and The amino acid sequence FTX defined in SEQ ID NO: 1 ６ X ７ GX ８ T, wherein X ６ is S, T or A, and X ７ is G, A, H, S or T, and X ８ is S, N or D; and Amino acid sequence X ９ X １０ LX １１ X １２ wherein X ９ is Y or H, and X １０ is F or Y, and X １１ is D or G, and X １２ is V, N or D; and The VHH antibody comprising:

2. The CDR1 has the amino acid sequence GTX defined in SEQ ID NO:2 ２ FX ３ X ４ NY, where X ２ is P or D, and X ３ is S or T, and X ４ is N, L or M, The CDR3 has the amino acid sequence X ９ FLX １１ X １２ where X ９ is Y or H, and X １１ is D or G, and X １２ The VHH antibody of claim 1, wherein is V, N or D.

3. The CDR1 has the amino acid sequence GTDFSX defined in SEQ ID NO: 3 ４ NY, where X ４ is L or M, The CDR2 has the amino acid sequence FTX defined in SEQ ID NO:4 ６ X ７ GST, where X ６ is S or A, and X ７ is S or T, The VHH antibody according to claim 2, wherein the CDR3 consists of the amino acid sequence HFLGD defined in SEQ ID NO:

5.

4. The CDR1 consists of the amino acid sequence GTDFSLNY defined in SEQ ID NO: 6; The CDR2 has the amino acid sequence FTAX defined in SEQ ID NO:7 ７ GST, where X ７ is S or T, The VHH antibody according to claim 3 , wherein the CDR3 consists of the amino acid sequence HFLGD defined in SEQ ID NO:

5.

5. The CDR1 consists of the amino acid sequence GTDFSLNY defined in SEQ ID NO: 6; the CDR2 consists of the amino acid sequence FTATGST defined in SEQ ID NO: 8; The VHH antibody according to claim 4, wherein the CDR3 consists of the amino acid sequence HFLGD defined in SEQ ID NO:

5.

6. The CDR1 consists of the amino acid sequence GTDFSLNY defined in SEQ ID NO: 6; the CDR2 consists of the amino acid sequence FTASGST defined in SEQ ID NO: 9; The VHH antibody according to claim 4, wherein the CDR3 consists of the amino acid sequence HFLGD defined in SEQ ID NO:

5.

7. The CDR1 consists of the amino acid sequence GTDFSMNY defined in SEQ ID NO: 10; the CDR2 consists of the amino acid sequence FTSTGST defined in SEQ ID NO: 11; The VHH antibody according to claim 3 , wherein the CDR3 consists of the amino acid sequence HFLGD defined in SEQ ID NO:

5.

8. The CDR1 has the amino acid sequence GTX defined in SEQ ID NO:2 ２ FX ３ X ４ NY, where X ２ is P or D, and X ３ is S or T, and X ４ is N, L or M, The CDR2 has the amino acid sequence FTX defined in SEQ ID NO: 1 ６ X ７ GX ８ T, where X ６ is S or T, and X ７ is G, A, H or S, and X ８ is S, N or D, The CDR3 has the amino acid sequence YFLDX defined in SEQ ID NO: 12 １２ where X １２ The VHH antibody of claim 2, wherein is V or N.

9. The CDR1 has the amino acid sequence GTPFX defined in SEQ ID NO: 13 ３ NNY, where X ３ is S or T, The CDR2 has the amino acid sequence FTSX defined in SEQ ID NO: 14 ７ GX ８ T, where X ７ is G or A, and X ８ is S or N, The VHH antibody of claim 8, wherein the CDR3 consists of the amino acid sequence YFLDV defined in SEQ ID NO:

15.

10. The CDR1 consists of the amino acid sequence GTPFSNNY defined in SEQ ID NO: 16; the CDR2 consists of the amino acid sequence FTSGGST defined in SEQ ID NO: 17; The VHH antibody of claim 9 , wherein the CDR3 consists of the amino acid sequence YFLDV defined in SEQ ID NO:

15.

11. The CDR1 consists of the amino acid sequence GTPFTNNY defined in SEQ ID NO: 18; the CDR2 consists of the amino acid sequence FTSAGNT defined in SEQ ID NO: 19; The VHH antibody of claim 9 , wherein the CDR3 consists of the amino acid sequence YFLDV defined in SEQ ID NO:

15.

12. The CDR1 has the amino acid sequence GTDFSX defined in SEQ ID NO: 3 ４ NY, where X ４ is L or M, The CDR2 has the amino acid sequence FTX defined in SEQ ID NO: 1 ６ X ７ GX ８ T, where X ６ is S or T, and X ７ is H or S, and X ８ is N or D, The VHH antibody of claim 8, wherein the CDR3 consists of the amino acid sequence YFLDN defined in SEQ ID NO:

20.

13. The CDR1 consists of the amino acid sequence GTDFSLNY defined in SEQ ID NO: 6; the CDR2 consists of the amino acid sequence FTTHGDT as defined in SEQ ID NO: 21; The VHH antibody of claim 12, wherein the CDR3 consists of the amino acid sequence YFLDN defined in SEQ ID NO:

20.

14. The CDR1 consists of the amino acid sequence GTDFSMNY defined in SEQ ID NO: 10; the CDR2 consists of the amino acid sequence FTSSGNT defined in SEQ ID NO: 22; The VHH antibody of claim 12, wherein the CDR3 consists of the amino acid sequence YFLDN defined in SEQ ID NO:

20.

15. the CDR1 consists of the amino acid sequence GNRFGIEY as defined in SEQ ID NO: 23; the CDR2 consists of the amino acid sequence FTSAGST defined in SEQ ID NO: 24; The VHH antibody of claim 1, wherein the CDR3 consists of the amino acid sequence HYLGD defined in SEQ ID NO:

25.

16. said VHH antibody is of the formula: framework region 1 (FR1)-CDR1-FR2-CDR2-FR3-CDR3-FR4, FR1 has the amino acid sequence QVQLQESGX defined in SEQ ID NO:26 １４ GX １５ VQX １６ GGSLRLSCX １７ X １８ S, where X １４ is R or G, and X １５ is L or V, and X １６ is A or T, and X １７ is A, T or V, and X １８ is A, L or V, FR2 is the amino acid sequence X defined in SEQ ID NO:27 １９ RWYRQAPGX ２０ QREX ２１ VAG, where X １９ is M or L, and X ２０ is K or N, and X ２１ is W or F, FR3 is the amino acid sequence NYX defined in SEQ ID NO:28 ２０ DSX ２１ KGRFTIX ２２ RDNAX ２３ X ２４ TVYLQMDX ２５ LX ２６ PEDTAVYX ２７ C, where X ２０ is A, P or G, and X ２１ is V or M, and X ２２ is S or G, and X ２３ is E or K, and X ２４ is G or N, and X ２５ is N or S, and X ２６ is K, I or T, and X ２７ is Y or F, A VHH antibody according to any one of claims 1 to 15, wherein FR4 has the amino acid sequence WGQGTQVTVSS defined in SEQ ID NO:

29.

17. The VHH antibody of claim 1, wherein the VHH antibody has an amino acid sequence selected from the group consisting of SEQ ID NOs: 30 to 37.

18. A heavy chain-only variable domain (VHH) antibody that specifically binds to transferrin receptor 1 (TfR1), The amino acid sequence DSAFX defined in SEQ ID NO: 38 ２８ MNT, wherein X ２８ is S or N; and CDR2 consisting of the amino acid sequence IVSDDNT defined in SEQ ID NO: 39; CDR3 consisting of the amino acid sequence KGDVV defined in SEQ ID NO: 40; The VHH antibody comprising:

19. The CDR1 consists of the amino acid sequence DSAFSMNT defined in SEQ ID NO: 41; The CDR2 consists of the amino acid sequence IVSDDNT defined in SEQ ID NO: 39; The VHH antibody of claim 18, wherein the CDR3 consists of the amino acid sequence KGDVV defined in SEQ ID NO:

40.

20. the CDR1 consists of the amino acid sequence DSAFNMNT defined in SEQ ID NO: 42; The CDR2 consists of the amino acid sequence IVSDDNT defined in SEQ ID NO: 39; The VHH antibody of claim 18, wherein the CDR3 consists of the amino acid sequence KGDVV defined in SEQ ID NO:

40.

21. said VHH antibody is of the formula: framework region 1 (FR1)-CDR1-FR2-CDR2-FR3-CDR3-FR4, FR1 has the amino acid sequence QVQLQESGGGLVQVGGSLRLSCAAS as defined in SEQ ID NO: 43; FR2 is the amino acid sequence MYWYRQAPGKX defined in SEQ ID NO:44 ２９ REFVAX ３０ where X ２９ is Q or S, and X ３０ is Y or W, FR3 is the amino acid sequence X defined in SEQ ID NO:45 ３１ YADSVKGRFTISRDNAKNTVYLQMNX ３２ LKPEDTAX ３３ YYC, where X ３１ is R or Q, and X ３２ is S or N, and X ３３ is V or G, The VHH antibody according to any one of claims 18 to 20, wherein FR4 has the amino acid sequence WGQGTQVTVSS defined in SEQ ID NO:

29.

22. The VHH antibody of claim 18, wherein the VHH antibody has an amino acid sequence selected from the group consisting of SEQ ID NOs: 46 to 47.

23. The VHH antibody according to any one of claims 1 to 22, which specifically binds to human TfR1.

24. The VHH antibody according to any one of claims 1 to 23, which specifically binds to mouse TfR1.

25. The VHH antibody according to any one of claims 1 to 24, which specifically binds to cynomolgus monkey TfR1.

26. The VHH antibody according to any one of claims 1 to 25, wherein the VHH antibody is a Camelidae VHH antibody.

27. The VHH antibody according to any one of claims 1 to 25, wherein the VHH antibody is a humanized VHH antibody.

28. The VHH antibody according to any one of claims 1 to 27, wherein the VHH antibody binds to Protein A.

29. A fusion molecule comprising a VHH antibody according to any one of claims 1 to 28 linked to at least one molecule.

30. The fusion molecule of claim 29, wherein the fusion protein comprises a VHH antibody of any one of claims 1 to 28 covalently linked to the at least one molecule via a linker.

31. 31. The fusion molecule of claim 29 or 30, wherein the at least one molecule is selected from the group consisting of a therapeutic agent and an imaging agent.

32. 32. The fusion molecule of claim 31, wherein the therapeutic agent is capable of treating a disease or disorder of the central nervous system.

33. 32. The fusion molecule of claim 31, wherein the imaging agent is selected from the group consisting of a position emission tomography tracer, a single photon emission computed tomography tracer, a fluorescent probe, a luminescent probe, a metal complex-containing probe, and a near-infrared fluorescent probe.

34. The fusion molecule of any one of claims 29 to 32, wherein the at least one molecule is a therapeutic agent; a pharmaceutically acceptable vehicle; and A pharmaceutical composition comprising:

35. A nucleic acid molecule encoding a VHH antibody according to any one of claims 1 to 28 or a fusion molecule according to any one of claims 29 to 32.

36. 36. An expression vector comprising the nucleic acid molecule of claim 35 operably linked to a promoter.

37. 37. A host cell comprising the nucleic acid molecule of claim 35 or the expression vector of claim 36.

38. The fusion molecule of any one of claims 29 to 32 for use as a medicament, wherein said at least one molecule is a therapeutic agent.

39. 33. The fusion molecule of any one of claims 29 to 32 for use in treating a disease or disorder of the central nervous system (CNS), wherein said at least one molecule is a therapeutic agent capable of treating said disease or disorder of the CNS.

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