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 into cells and across the blood brain barrier.

Description

Technical Field

[0001] The present invention particularly relates to heavy chain only variable domain (VHH) antibodies that can bind 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 Art

[0002] Drugs targeting central nervous system (CNS) diseases are inherently difficult to expose in the brain 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. There are also some small molecules that are excluded from the cerebral ventricles due to their properties (such as lack of lipophilicity or substrates of efflux pumps). Generally, compounds with a molecular weight less than 600 daltons (Da) can pass through the BBB without the exclusion of other forces. Without specific active transport assistance, larger molecules (such as proteins) cannot easily enter the brain in large quantities. For therapeutic antibodies and protein-based drugs, it is estimated that less than 0.1% of the systemically injected therapeutic antibodies reach the cerebral ventricles. Several strategies to overcome this strict barrier have been tested and evaluated.

[0003] The BBB is composed of brain endothelial cells (BECs) and is the first barrier to enter the brain. Other cells of the so-called neurovascular unit (NVU) are also important for the transport and interaction of target cells in the brain parenchyma. In the human brain, the total span of cerebral blood vessels is 20m 2 , representing a large surface area, which can provide opportunities for cerebral exposure of circulating therapeutic agents.

[0004] Therapeutic antibodies or other protein-based drugs have great potential in the treatment of CNS diseases. However, the low availability of such therapeutic molecules in the cerebral ventricles is a major problem. Recently, clinical effects have been reported for therapeutic monoclonal antibodies with brain targets such as amyloid-β fibrils. Nevertheless, the exposure of these therapeutic molecules in the human cerebral ventricles after each administration is not considered to be in an optimal state.

[0005] Therefore, it is necessary to increase the exposure of therapeutic molecules to the brain to improve the safety, dosage, and total cost of CNS therapeutic agents.

[0006] Receptor-mediated transcytosis is a natural mechanism that uses endogenous receptors expressed on the luminal surface of the BBB. It has been reported that it successfully increases the cerebral exposure of therapeutic molecules and has clinical effectiveness and safety.

[0007] WO 2020 / 144233 discloses camelid heavy chain variable domain (VHH) molecules that bind to the transferrin receptor (TfR), and their use in transporting molecules with drug or diagnostic significance to cells and organs under pathological conditions including cancer.

[0008] WO 2016 / 077840, WO 2019 / 089395, WO 2020 / 056327, and WO 2022 / 103769 disclose TfR-specific binding moieties that can be used to carry biomolecules across membranes, including the BBB and the gastrointestinal tract. These TfR-specific binding moieties include single-domain nurse shark new antigen receptor variable domain (VNAR) antibodies that bind to TfR.

[0009] WO 2016 / 081643 relates to anti-transferrin receptor antibodies and methods of using the same.

[0010] There is still a need for effective therapeutic molecule transporters into the brain. These transporters should be able to conjugate or fuse with therapeutic or diagnostic molecules in a manner that does not affect the target engagement of the transporter or the therapeutic efficacy of the therapeutic molecule. SUMMARY OF THE INVENTION

[0011] The general objective is to provide VHH molecules that are specific for transferrin receptor 1 and can effectively transport cargo to the desired compartment via receptor-mediated transcytosis.

[0012] Embodiments of the present invention meet these and other objectives.

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

[0014] One aspect of the present invention relates to a variable heavy chain only (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 further 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 CDR3 consisting of the amino acid sequence X9X 10 LX 11 X 12 where X9 is Y or H, X 10 is F or Y, 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 comprises the amino acid sequence DSAFX as defined in SEQ ID NO:38 28CDR1 consisting of MNT, where X 28 is S or N. The VHH antibody also comprises a CDR2 consisting of the amino acid sequence IVSDDNT defined in SEQ ID NO:39. The VHH also comprises a CDR3 consisting of the amino acid sequence KGDVV defined in SEQ ID NO:40.

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

[0017] Related aspects of the present invention define an above-mentioned fusion molecule for use as a medicament, wherein at least one molecule is a therapeutic agent, or for the treatment of a central nervous system (CNS) disease or disorder, wherein at least one molecule is a therapeutic agent capable of treating a CNS disease or disorder.

[0018] Another aspect of the present invention relates to a pharmaceutical composition comprising the above fusion molecule and a pharmaceutically acceptable carrier. In such a pharmaceutical composition, at least one molecule is a therapeutic agent.

[0019] Other aspects of the present invention relate to a nucleic acid molecule encoding the above VHH antibody or fusion molecule, an expression vector comprising the above nucleic acid molecule operably linked to a promoter, and a host cell comprising the above nucleic acid molecule or the above expression vector.

[0020] The VHH antibody of the present invention specifically binds to TfR1 without interfering with the binding of transferrin to TfR1. The binding characteristics of the VHH antibody are tailored to be most suitable for transcytosis across endothelial cells at the BBB. Thus, the VHH antibody can serve as a transporter for various molecules, including therapeutic agents or diagnostic imaging agents, to enter the ventricles upon systemic administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The embodiments, as well as additional objects and advantages thereof, may best be understood by reference to the following description taken in conjunction with the drawings, in which:

[0022] Figure 1. Strategy for establishing TfR1-binding VHH

[0023] Graphical illustration of immunization of llamas with an immunogen (1A). According to ModiPhage of Modiquest Research BV (the Netherlands) TMThe method immunized llamas (Llama glama) (N = 2). On day 1, a primary immunization was performed using the extracellular domain of human transferrin receptor 1 (hTfR1) (500 μg protein + complete Freund's adjuvant (CFA), intramuscularly (i.m.)). On day 21, a booster dose of mouse TfR1 (mTfR1) was given (500 μg protein + incomplete Freund's adjuvant (IFA), i.m.), and on day 42, a booster dose of hTfR1 was given. On day 54, peripheral blood was drawn, and the immune response was studied by enzyme-linked immunosorbent assay (ELISA) to test for the presence of immunoglobulin G1 (IgG1) and IgG2 / 3 antibodies and their reactivity to mTfR1 and hTfR1. Immunizations were repeated on day 86 (hTfR1 + IFA) and day 107 (hTfR1 + IFA). On day 117, a new peripheral blood sample was analyzed. On day 120, an additional pre-harvest booster dose was given, a 1:1 mixture of hTfR1 and mTfR1 (250 μg + 250 μg + IFA), and then peripheral blood lymphocytes (PBL) were harvested on day 124.

[0024] Graphical overview of phage library construction (1B). PBL were separated by density gradient centrifugation using Ficoll-Paque TM PLUS ~1.5×10 9 . RNA was extracted and then reverse transcribed into complementary deoxyribonucleic acid (cDNA). Subsequently, the cDNA was used as a template for polymerase chain reaction (PCR) to amplify the IgG library (IgG2 / 3, from the variable heavy chain (VH) domain to the constant heavy chain 2 (CH2), i.e., 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 library (as described by 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 DNA was electroporated into TG1 Escherichia coli to obtain a library with an estimated size of 3.2×10 8 .

[0025] Figure 2 . Graphical representation of VHH and various VHH-containing fusion proteins

[0026] Figure 2Schematically illustrates VHH monomers and various VHH-containing fusion proteins. The free VHH binds as a monomer to one binding site on TfR1. A bivalent VHH fusion protein is formed between two VHHs and one human fragment crystallizable (Fc) region. A functional monovalent fusion protein is generated between one VHH and one single-chain variable fragment (scFv) or between one VHH and a non-antibody-derived molecule (X). Figure 2 Also shown is a bivalent VHH fusion protein with two non-antibody-derived molecules (X).

[0027] Figure 3. Measurement of the binding affinity of monomeric VHH to hTfR1 using SPR

[0028] This figure shows a representative sensorgram of the binding of increasing concentrations of KB_B01(3A) in the range from 6.25 to 100 nM to hTfR1 loaded on human transferrin (hTf), which is immobilized on a dextran-coated gold (CM5) chip (Cytiva) by amine coupling. As a control benchmark, the full-length monoclonal antibody BA1(3B) was run at the same concentrations.

[0029] Figure 4. Measurement of the binding affinity of dimeric VHH-Fc proteins to hTfR1, cTfR1, and mTfR1 using SPR

[0030] This figure shows representative sensorgrams of the binding of increasing concentrations of hTfR1 (A, D, and G), cynomolgus monkey TfR1 (cTfR1) (B, E, and H), or mTfR1 (C, F, and I) in the range from 0.16 to 100 nM to Fc-fused KB_B01(4A - 4C), BV(4D - 4F), and the benchmark full-length monoclonal antibody BA2(4G - 4I) loaded on a protein A-coated chip (Cytiva).

[0031] Figure 5. Measurement of the binding affinity of dimeric KB_B01-Fc to hTfR1 and cTfR1 in the absence and presence of hTf

[0032] This figure shows representative sensorgrams of the binding of KB_B01-Fc(5A - 5B), the benchmark BV-Fc(5C - D), and BA2(5E - 5F) to hTfR1 in the absence (5A, 5C, and 5E) and presence (5B, 5D, and 5F) of an excess concentration of 250 nM hTf.

[0033] Figure 6 . Sequence alignment of sequences closely related to KB_B01

[0034] This 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 represent amino acids identical to the reference sequence (KB_B01), and boxes represent the three complementarity-determining regions (CDRs).

[0035] Figure 7. Affinity of VHH and scFv fusion proteins for hTfR1 binding measured using SPR

[0036] This figure shows representative sensorgrams of hTfR1 amine of KB_B01 fused to the scFv gene conjugated to the CM5 chip surface and exposed to the C-terminus of VL (scFv-VHH, 7A) or the N-terminus of VH (VHH-scFv, 7B). The exposure was increased in 1:4 step increments in the concentration range of 0.25 to 64 nM. The reference VHH BV was also fused to both ends of the same scFv (scFv-VHH, 7C) and (VHH-scFv, 7D) and tested in 1:5 increments in the concentration range of 0.08 to 50 nM.

[0037] Figure 8 . Cellular uptake via hTfR1 in HEK293T cells

[0038] HEK293T cells were cultured in 96-well plates until confluence (4 - 5 days). 20 nM or 5 nM (diluted in DMEM) of KB_B01-Fc, BV-Fc, or BA1 and the negative control (hTfR1-inert VHH-Fc) were added and incubated for 40 and 120 minutes respectively, then rinsed with PBS and fixed in 4% PFA. For longer incubation times, the medium was replaced by incubation with fresh DMEM at t = 30 minutes. After fixation, the cells were immunostained and analyzed using a confocal microscope. Shown here are representative images of cellular uptake of KB_B01-Fc, BV-Fc, and BA1 and the negative control after incubation at 20 nM for 40 minutes, or representative images of cellular uptake of KB_B01-Fc, BV-Fc, and BA1 after incubation at 5 nM for 120 minutes. The figure also shows no signal when using the non-hTfR1-binding VHH-Fc negative control. The images are shown in 8-bit grayscale. Scale bar 50 μM.

[0039] Figure 9. Brain and blood distribution after systemic administration of VHH-Fc fusion proteins.

[0040] Injected radiolabeled as described in Example VIII 125I] VHH-Fc fusion protein. Brain concentration (9A) and blood concentration (9B) are shown. Blood samples were collected at t = 30 minutes, t = 1 hour, and at the end (2.5 hours). At 2.5 hours after injection, the animals were euthanized, perfused transcardially with NaCl, the brains were excised and subjected to radioanalysis. All concentrations are expressed as % of the injected dose (radioactivity), and for the brain, are normalized to body weight (standardized uptake value SUV).

[0041] Figure 10. Brain distribution and biodistribution after systemic administration of VHH-scFvs

[0042] Inject the radiolabeled 125 I] VHH-scFv fusion protein as described in Example VIII. The brain exposure of wild-type mice and Alzheimer's disease (AD) mouse model APP NL-GF (named "nlgf" in the figure) was analyzed. Brain concentration in SUV form 2 hours after injection of KB_B01-scFv and BV-scFv fused to the N-terminus of 3D6 scFv, as described in Example IV (10A). Brain retention kinetics of APP NL-G-F mice and WT mice 2, 6, and 24 hours after injection of KB_B01-scFv (10B). Brain (10C) and blood (10C) concentrations 24 hours after injection. A group of APP NL-G-F animals were also injected with a negative control VHH-scFv fusion terminated at 24 hours, as well as KB_B01-scFv and BV-scFv. The retention rates of AD mice versus WT were read from each construct.

[0043] Figure 11. Vascular and parenchymal distribution of scFv-VHH fusions

[0044] As described in Example IX, cryosections of one brain hemisphere of mice injected with VHH-scFv (as described in Example VIII) were prepared. Representative sections were first immunostained with the vascular marker CD31 and then immersed in nuclear track emulsion (NTE), followed by long-term development in a cold room. Thereafter, images of CD31 and NTE were acquired using a wide-field Zeiss Observer Microscope. Overlay images were made into binary 8-bit images (11A). From these images, NTE white dots show the 125 localization of I protein and its relationship to blood vessels. For clarity, the bottom panel shows the vascular immunostaining, and the top panel shows the overlay, where the vascular profile is shown as a thin contour (white line). Scale bar 20 μm. 20 frames of images from the same brain were quantified, as shown in micrographs of the relative distribution in the vascular profile or parenchyma (11B). Values are shown as mean ± standard deviation (SD).

[0045] Figure 12. Brain and blood distribution after systemic administration of the VHH-Fc fusion KB_B012 to humanized mice with the extracellular domain of TfR1

[0046] As described in Example XI, the radiolabeled 125 I] VHH-Fc fusion protein KB_B03 (KB_B12-Fc) was injected into homozygous (HOM), heterozygous (HET), and wild-type (WT) genotypes of hTfR1. (12A) shows the blood concentration over time. Blood samples were collected at t = 5 minutes, t = 30 minutes, t = 1 hour, and final (2.0 hours). (12B) Distribution of leukocytes, plasma, and pellet in the blood compartment. (12C) Brain concentration at 2.0 hours. Two hours after injection, the animals were euthanized, perfused transcardially with NaCl, the brains were excised and analyzed for radioactivity. Blood concentration is expressed as (% of injected dose, radioactivity), while brain uptake is expressed as standardized uptake value SUV (% of injected dose corrected for animal body weight). DETAILED DESCRIPTION

[0047] The present invention relates to single heavy chain variable domain (VHH) antibodies, also known as single domain antibodies, in particular VHH antibodies capable of binding transferrin (Tf) receptor 1 (TfR1), and the use of such VHH antibodies to transport molecules across the blood-brain barrier (BBB).

[0048] Drug exposure in the brain for drugs targeting central nervous system (CNS) diseases is inherently difficult 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 is a natural mechanism that utilizes endogenous receptors expressed on the luminal surface of the BBB and is thought to increase drug exposure across the BBB. One endogenous receptor that can be used to achieve receptor-mediated transcytosis is the transferrin receptor.

[0049] Transferrin receptor is the carrier protein for transferrin, a glycoprotein that binds to plasma and mediates the transport of iron (Fe) in plasma. Transferrin receptor internalizes the transferrin-iron complex by receptor-mediated endocytosis, thereby importing iron. In humans and other mammals, there are two transfer receptors: transferrin receptor 1 (TfR1) and transferrin receptor 2 (TfR2). TfR1 is a high-affinity, ubiquitously expressed receptor, while the expression of TfR2 is restricted to certain cell types and is not affected by intracellular iron concentration. The binding affinity of TfR2 for transferrin is 25 to 30 times lower than that of TfR1. As used herein, transferrin receptor refers to the TfR1 homolog, also known as cluster of differentiation 71 (CD71), which is encoded by the human TFRC gene.

[0050] The extracellular domains of human TfR1 (hTfR1) and mouse TfR1 (mTfR1) were used to immunize llama animals and create a clone library expressing VHH antibodies. These VHH antibodies were screened for binding to hTfR1, and the key VHH antibody KB_B01 with the desired binding characteristics was selected. VHH antibodies showing sequence similarity to KB_B01 were further identified. The VHH antibodies of the present invention specifically bind to hTfR1 as well as mTfR1 and have binding characteristics suitable for endocytosis, including binding affinity and binding to hTfR1 without interfering with the binding of transferrin (Tf) to hTfR1. As shown in the experimental section, these VHH antibodies retained their TfR1-binding characteristics when presented in the form of VHH-containing fusion proteins, including bivalent VHH fusion proteins formed between two VHH antibodies and the crystallizable fragment (Fc) of the human constant antibody fragment, and functional fusion proteins fused to drug molecules, such as single-chain variable fragments (scFv). The experimental data further showed that the VHH antibodies (including VHH-containing fusion proteins) were taken up by human cells expressing hTfR1 and transcytosed through a monolayer of human brain-like endothelial cells used as an in vitro model of the BBB. The distribution of the VHH antibodies in the brain was verified in vivo and showed a broader brain distribution than the reference antibodies (including reference VHH antibodies).

[0051] Compared with the VHH antibodies disclosed in WO 2020 / 144233, the VHH antibodies of the present invention also have advantages. First, compared with the fusion protein between the VHH antibody of WO 2020 / 144233 and the same scFv molecule, the fusion protein between the VHH antibody and the scFv molecule has a better distribution in the brain parenchyma and does not accumulate in blood vessels. More specifically, the fusion protein with the VHH antibody of the present invention preferentially distributes in the brain parenchyma, approaching 80%, while the corresponding figure for the VHH antibody of WO 2020 / 144233 is only slightly higher than 50%. Another advantage of the VHH antibodies of the present invention over the VHH antibodies of WO 2020 / 144233 is that the VHH antibodies of the present invention have a higher brain-blood ratio compared to the negative control. This means that the VHH antibodies of the present invention are suitable for specifically targeting disease-related proteins in the brain and have less non-specific binding in other compartments of the body.

[0052] One aspect 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 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 further comprises 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 CDR3 consisting of the amino acid sequence X9X 10 LX 11 X 12 where X9 is Y or H, X 10 is F or Y, X 11 is D or G, and X 12 is V, N or D.

[0053] 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 where X9 is Y or H, X 11 is D or G, and X 12 is V, N or D.

[0054] 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.

[0055] 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.

[0056] In a specific embodiment, CDR1 consists of the amino acid sequence GTDFSLNY defined in SEQ ID NO:6, CDR2 consists of the amino acid sequence FTATGST defined in SEQ ID NO:8, and CDR3 consists of the amino acid sequence HFLGD defined in SEQ ID NO:5. The VHH antibody having the CDR regions according to this specific embodiment is designated as KB_B06 herein.

[0057] In another specific embodiment, CDR1 consists of the amino acid sequence GTDFSLNY defined in SEQ ID NO:6, CDR2 consists of the amino acid sequence FTASGST defined in SEQ ID NO:9, and CDR3 consists of the amino acid sequence HFLGD defined in SEQ ID NO:5. The VHH antibody having the CDR regions according to this specific embodiment is designated as KB_B07 herein.

[0058] In another specific embodiment, CDR1 consists of the amino acid sequence GTDFSMNY defined in SEQ ID NO:10, CDR2 consists of the amino acid sequence FTSTGST defined in SEQ ID NO:11, and CDR3 consists of the amino acid sequence HFLGD defined in SEQ ID NO:5. The VHH antibody having the CDR regions according to this specific embodiment is designated as KB_B05 herein.

[0059] In one embodiment, CDR1 consists of the amino acid sequence GTX2FX3X4NY 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 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 12 consisting of, where X 12 is V or N.

[0060] In one embodiment, CDR1 consists of the amino acid sequence GTPFX3NNY defined in SEQ ID NO:13, where X3 is S or T. In this embodiment, CDR 2 consists of the amino acid sequence FTSX7GX8T defined in SEQ ID NO:14, where X7 is G or A, and X8 is S or N. In this embodiment, CDR3 consists of the amino acid sequence YFLDV defined in SEQ ID NO:15.

[0061] In a specific embodiment, CDR1 consists of the amino acid sequence GTPFSNNY defined in SEQ ID NO:16, CDR2 consists of the amino acid sequence FTSGGST defined in SEQ ID NO:17, and CDR3 consists of the amino acid sequence YFLDV defined in SEQ ID NO:15. The VHH antibody having the CDR regions according to this specific embodiment is designated herein as KB_B01.

[0062] In another specific embodiment, CDR1 consists of the amino acid sequence GTPFTNNY defined in SEQ ID NO:18, CDR2 consists of the amino acid sequence FTSAGNT defined in SEQ ID NO:19, and CDR3 consists of the amino acid sequence YFLDV defined in SEQ ID NO:15. The VHH antibody having the CDR regions according to this specific embodiment is designated herein as KB_B02.

[0063] In one embodiment, CDR1 consists of the amino acid sequence GTDFSX4NY defined in SEQ ID NO:3, where X4 is L or M. In this embodiment, CDR2 consists of the amino acid sequence FTX6X7GX8T 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 defined in SEQ ID NO:20.

[0064] In a specific embodiment, CDR1 consists of the amino acid sequence GTDFSLNY defined in SEQ ID NO:6, CDR2 consists of the amino acid sequence FTTHGDT defined in SEQ ID NO:21, and CDR3 consists of the amino acid sequence YFLDN defined in SEQ ID NO:20. The VHH antibody having the CDR regions according to this specific embodiment is designated herein as KB_B03.

[0065] In another specific embodiment, CDR1 consists of the amino acid sequence GTDFSMNY defined in SEQ ID NO:10, CDR2 consists of the amino acid sequence FTSSGNT defined in SEQ ID NO:22, and CDR3 consists of the amino acid sequence YFLDN defined in SEQ ID NO:20. The VHH antibody having the CDR regions according to this specific embodiment is designated herein as KB_B04.

[0066] In another specific embodiment, CDR1 consists of the amino acid sequence GNRFGIEY defined in SEQ ID NO:23, CDR2 consists of the amino acid sequence FTSAGST defined in SEQ ID NO:24, and CDR3 consists of the amino acid sequence HYLGD defined in SEQ ID NO:25. The VHH antibody having CDR regions according to this specific embodiment is designated as KB_B08 herein.

[0067] In one embodiment, the VHH antibody has the following formula: framework region 1 (FR1)-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0068] In one embodiment, 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, X 15 is L or V, X 16 is A or T, X 17 is A, T or V, and X 18 is A, L or V.

[0069] In a specific embodiment, FR1 has the amino acid sequence QVQLQESGGGLVQX defined in SEQ ID NO:77 16 GGSLRLSCX 17 X 18 S, where X 16 is A or T, X 17 is A, T or V, and X 18 is A, L or V.

[0070] In one embodiment, FR2 has the amino acid sequence X defined in SEQ ID NO:27 19 RWYRQAPGX 20 QREX 21 VAG, where X 19 is M or L, X 20 is K or N, and X 21 is W or F.

[0071] In a specific embodiment, FR2 has the amino acid sequence X defined in SEQ ID NO:78 19 RWYRQAPGX 20 QREWVAG, where X 19 is M or L, and X 20is K or N.

[0072] In one embodiment, FR3 has the amino acid sequence NYX defined in SEQ ID NO:28 20 DSX 21 KGRFTIX 22 RDNAX 23 X 24 TVYLQMDX 25 LX 26 PEDTA VYX 27 C, where X 20 is A, P or G, X 21 is V or M, X 22 is S or G, X 23 is E or K, X 24 is G or N, X 25 is N or S, X 26 is K, I or T, and X 27 is Y or F.

[0073] In a specific embodiment, FR3 has the amino acid sequence NYX defined in SEQ ID NO:28 20 DSX 21 KGRFTIX 22 RDNAX 23 X 24 TVYLQMDX 25 LX 26 PE DTAVYX 27 C, where X 20 is A or P, X 21 is V or M, X 22 is S or G, X 23 is E or K, X 24 is G or N, X 25 is N or S, X 26 is K or T, and X 27 is Y or F.

[0074] In one embodiment, FR4 has the amino acid sequence WGQGTQVTVSS defined in SEQ ID NO:29.

[0075] In a specific embodiment, the VHH antibody has the amino acid sequences of FR1, FR2, FR3 and FR4 defined in SEQ ID NO:26 or 77, 27 or 78, 28 and 29.

[0076] The present invention also encompasses VHH antibodies having an FR1 amino acid sequence comprising or consisting of SEQ ID NO: 26 or 77, or variants thereof having at least 88% sequence identity, preferably at least 92% sequence identity and more preferably at least 96% sequence identity with SEQ ID NO: 26 or 77.

[0077] The present invention also encompasses VHH antibodies having an FR2 amino acid sequence comprising or consisting of SEQ ID NO: 27 or 78, or variants thereof having at least 82% sequence identity, preferably at least 88% sequence identity and more preferably at least 94% sequence identity with SEQ ID NO: 27 or 78.

[0078] The present invention also encompasses VHH antibodies having an FR3 amino acid sequence comprising or consisting of SEQ ID NO: 28, or variants thereof having at least 92% sequence identity, preferably at least 94% sequence identity and more preferably at least 97% sequence identity with SEQ ID NO: 28.

[0079] The present invention also encompasses VHH antibodies having an FR4 amino acid sequence comprising or consisting of SEQ ID NO: 29, or variants thereof having at least 72% sequence identity, preferably at least 81% sequence identity and more preferably at least 90% sequence identity with SEQ ID NO: 29.

[0080] As used herein, the term "sequence identity %" can be determined using methods well known in the art. For example, sequence identity % can be calculated as follows. Align the query sequence with the target sequence using the CLUSTAL W algorithm. Compare the window corresponding to the shortest of the aligned sequences. In some cases, the shortest of the aligned sequences may be the target sequence. In other cases, the query sequence may constitute the shortest of the aligned sequences. Compare the amino acid residues at each position and report the percentage of positions in the query sequence that have the same correspondence to the target sequence as sequence identity %.

[0081] Amino acid sequences having a determined sequence identity % to a reference amino acid sequence are preferably obtained by amino acid substitutions, for example by conservative amino acid replacements. Conservative amino acid replacements, also known as conservative amino acid substitutions or mutations, are amino acid substitutions in an amino acid sequence that change a given amino acid to a different amino acid having similar biochemical, structural, and / or chemical properties.

[0082] For example, based on the general chemical characteristics of the structure and side chain (R group) of amino acids, amino acids can be divided into six major groups:

[0083] · Aliphatic: isoleucine (I), leucine (L), glycine (G), alanine (A), valine (V)

[0084] · Containing hydroxyl or sulfur / selenium: serine (S), cysteine (C), threonine (T), methionine (M)

[0085] · Cyclic: proline (P)

[0086] · Aromatic: phenylalanine (F), tyrosine (Y), tryptophan (W)

[0087] · Basic: histidine (H), lysine (K), arginine (R); and

[0088] · Acidic and its amides: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q).

[0089] This means that an amino acid sequence having a defined % sequence identity to a reference amino acid sequence is preferably obtained by making one or more conservative amino acid substitutions of one or more amino acid residues in the reference amino acid sequence with the corresponding amino acids from the above-listed R groups that are the same as the given amino acid residue.

[0090] In one embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO:30. Such a VHH antibody is referred to herein as KB_B01.

[0091] In another embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO:31. Such a VHH antibody is referred to herein as KB_B02.

[0092] In another embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO:32. Such a VHH antibody is referred to herein as KB_B03.

[0093] In another embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO:33. Such a VHH antibody is referred to herein as KB_B04.

[0094] In another embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO:34. Such a VHH antibody is referred to herein as KB_B05.

[0095] In another embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO:35. Such a VHH antibody is referred to herein as KB_B06.

[0096] In another embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO:36. Such a VHH antibody is referred to herein as KB_B07.

[0097] In another embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO: 37. Such a VHH antibody is referred to herein as KB_B08.

[0098] In one embodiment, the VHH antibody has an amino acid sequence selected from the group consisting of SEQ ID NO: 30 to 37, preferably an amino acid sequence selected from the group consisting of SEQ ID NO: 30 to 36.

[0099] Another aspect of the invention relates to a VHH antibody that specifically binds to TfR1. The VHH antibody comprises a CDR1 consisting of the amino acid sequence DSAFX 28 MNT defined in SEQ ID NO: 38, where X 28 is S or N. The VHH antibody further comprises a CDR2 consisting of the amino acid sequence IVSDDNT defined in SEQ ID NO: 39. The VHH antibody further comprises a CDR3 consisting of the amino acid sequence KGDVV defined in SEQ ID NO: 40.

[0100] In a specific embodiment, CDR1 consists of the amino acid sequence DSAFSMNT defined in SEQ ID NO: 41, CDR2 consists of the amino acid sequence IVSDDNT defined in SEQ ID NO: 39, and CDR3 consists of the amino acid sequence KGDVV defined in SEQ ID NO: 40. The VHH antibody having the CDR regions according to this specific embodiment is denoted herein as KB_B09.

[0101] In a specific embodiment, CDR1 consists of the amino acid sequence DSAFNMNT defined in SEQ ID NO: 42, CDR2 consists of the amino acid sequence IVSDDNT defined in SEQ ID NO: 39, and CDR3 consists of the amino acid sequence KGDVV defined in SEQ ID NO: 40. The VHH antibody having the CDR regions according to this specific embodiment is denoted herein as KB_B10.

[0102] In one embodiment, the VHH antibody has the following formula: framework region 1 (FR1)-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0103] In one embodiment, FR1 has the amino acid sequence QVQLQESGGGLVQVGGSLRLSCAAS defined in SEQ ID NO: 43.

[0104] In one embodiment, FR2 has the amino acid sequence MYWYRQAPGKX 29REFVAX 30 , wherein X 29 is Q or S, and X 30 is Y or W.

[0105] In one embodiment, FR3 has the amino acid sequence defined in SEQ ID NO:45 X 31 YADSVKGRFTISRDNAKNTVYLQMNX 32 LKPEDTAX 33 YYC, wherein X 31 is R or Q, X 32 is S or N, and X 33 is V or G.

[0106] In one embodiment, FR4 has the amino acid sequence WGQGTQVTVSS defined in SEQ ID NO:29.

[0107] In a specific embodiment, the VHH antibody has the amino acid sequences of FR1, FR2, FR3 and FR4 defined in SEQ ID NO:43, 44, 45 and 29.

[0108] 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, preferably at least 92% sequence identity and more preferably at least 96% sequence identity with SEQ ID NO:43.

[0109] 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, preferably at least 88% sequence identity and more preferably at least 94% sequence identity with SEQ ID NO:44.

[0110] 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, preferably at least 94% sequence identity and more preferably at least 97% sequence identity with SEQ ID NO:45.

[0111] 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, preferably at least 81% sequence identity and more preferably at least 90% sequence identity with SEQ ID NO:29.

[0112] In one embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO: 46. Such a VHH antibody is referred to herein as KB_B09.

[0113] In another embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO: 47. Such a VHH antibody is referred to herein as KB_B10.

[0114] In one embodiment, the VHH antibody has an amino acid sequence selected from the group consisting of SEQ ID NOs: 46 to 47.

[0115] 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 30 - 36, 46 - 47.

[0116] Figure 6 A sequence alignment of closely related VHH antibodies KB_B01 to KB_B10 is shown.

[0117] The VHH antibodies of the present invention specifically bind to hTfR1.

[0118] The VHH antibody can specifically bind to hTfR1 either in the form of a monomeric VHH antibody, see Table 2, or in the form of a fusion molecule linked to another molecule, see Tables 3, 5, and 7. In the latter case, the binding of the VHH antibody to hTfR1 in the fusion molecule is not affected regardless of whether the other molecule is linked to the N - terminus or the C - terminus of the VHH antibody, see Table 7. In addition, as shown in Table 4, the binding of the VHH antibody of the present invention to hTfR1 is not greatly affected by the presence or absence of transferrin. This is in sharp contrast to the reference VHH antibody BV, as shown in Table 4, where the affinity of the reference VHH antibody for hTfR1 changes significantly in the presence of 250 nM transferrin compared to the absence of transferrin.

[0119] "Specifically bind" and similar expressions mean that the molecule in question (e.g., the VHH antibody) specifically binds to the target antigen without any significant binding to other molecules. Antibody specificity can be determined based on affinity and / or avidity. The affinity, represented by the equilibrium dissociation constant (K D ) of the antigen and the antibody, is a measure of the binding strength between the antigenic determinant (i.e., the epitope) and the antigen - binding site on the antibody. The lower the K D value, the stronger the binding strength between the antigenic determinant and the antibody. Alternatively, affinity can also be expressed as the equilibrium association constant (K A ), which is 1 / K DAs will be clear to the skilled person, affinity can be determined in a manner known per se depending on the specific antigen of interest.

[0120] Typically, antibodies will be present at 10 -5 Up to 10 -12 mol / L (M) or less, and preferably 10 -7 Up to 10 -12 M or less and more preferably 10 -8 Up to 10 -12 The equilibrium dissociation constant (K D ), that is, 10 5 Up to 10 12 M -1 or greater, and preferably 10 7 Up to 10 12 M -1 or greater and more preferably 10 8 Up to 10 12 M -1 The affinity constant (K A ) binds to its antigen. Generally speaking, greater than 10 -4 Any K of M D Value (or less than 10 4 M -1 Any K A Values ) were considered to indicate nonspecific binding.

[0121] Specific binding of an antibody to an antigen or antigenic determinant can be determined in any suitable manner known per se including, for example, Scatchard analysis and / or competition binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competition assays, surface plasmon resonance (SPR), biolayer interferometry (BLI) and different variations thereof known per se in the art.

[0122] The affinity of VHH antibodies for TfR1 should be adjusted to a specific range to achieve efficient BBB crossing and high relative uptake in the brain. In general, if the affinity of the antibody for TfR1 is too low, e.g., K D If the affinity of the antibody is greater than about 1 μM, the affinity of the antibody is too low to effectively bind to TfR1 and be taken up by BBB endothelial cells. This means that most of the antibody will remain in the peripheral blood system. Correspondingly, if the affinity of the antibody is too high, such as K D At concentrations well below 1 nM (<<1 nM), antibodies are endocytosed but instead undergo lysosomal degradation in BBB endothelial cells. This means that for optimal receptor-mediated endocytosis (i.e., receptor-mediated cellular uptake) and transcytosis (i.e., transport across the cell interior), VHH antibodies should have affinities (K) in the low nM to nM range. 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), WO 2016 / 081643).

[0123] As shown in Table 2, the affinity (K D ) of the VHH antibody KB_B01 of the present invention in monomeric form is 3.3 nM, i.e., within the nM range. In addition, when incorporated into a VHH-containing fusion protein, the affinity remains within the desired low nM to nM range (see Figure 2 ), for example, the dimeric-VHH Fc fusion protein with K D of 0.18 nM (see Table 3), or the fusion with scFv with K D of 0.85 - 1.84 nM (see Table 7). In addition, other VHH antibodies of the present invention have sub-nM range affinity for TfR1 when tested as dimeric Fc fusions, and thus are suitable for BBB crossing by receptor-mediated endocytosis and transcytosis (see Table 5), where when binding to hTfR1, the K D range of the dimeric VHH Fc fusion protein is from 0.0031 to 0.451 nM, and when binding to mTfR, K D is 5 to 6000 times higher.

[0124] In one embodiment, the VHH antibody of the present invention specifically binds to human TfR1 (hTfR1).

[0125] In one embodiment, the VHH antibody of the present invention specifically binds to mTfR1. In a specific embodiment, the VHH antibody of the present invention not only specifically binds to hTfR1, but also binds to mTfR1, see Table 5.

[0126] In one embodiment, the VHH antibody of the present invention specifically binds to cTfR1. In a specific embodiment, the VHH antibody of the present invention specifically binds to cTfR1 in addition to binding to hTfR1 and mTfR, see Table 3.

[0127] Therefore, in a preferred embodiment, the VHH antibody exhibits broad species cross-reactivity, i.e., it binds not only to hTfR1 but also to mTfR1 and cTfR1 with the desired affinity. From a translational perspective, this feature of the VHH antibody is highly desirable in predicting pharmacokinetics and pharmacodynamics from mouse or monkey to human.

[0128] In one embodiment, the VHH antibody of the present invention binds specifically to mTfR1 with an affinity (K D ) selected in the range of 0.1 to 150 nM, preferably in the range of 0.1 to 100 nM. In a specific embodiment, the VHH antibody in the monomeric form of the present invention binds specifically to mTfR1, where K D is selected from the range of 1 to 150 nM, preferably 1 to 100 nM, and more preferably 1 to 50 nM.

[0129] In one embodiment, the VHH antibody in the monomeric form of the present invention binds specifically to hTfR1 with an affinity (K D ) selected in the range of 0.01 to 150 nM, preferably in the range of 0.01 to 100 nM.

[0130] Structurally, TfR1 is a dimeric transmembrane glycoprotein with a large extracellular domain (residues 90 - 760), a transmembrane region (residues 62 - 89), and the remaining 61 residues in the cytoplasm. The extracellular domain has three domains, namely the helical domain (residues 606 - 760), the protease - like domain (residues

[0131] 121 - 183, 384 - 605), and the apical domain (residues 184 - 383). The helical domain is responsible for receptor dimerization. Transferrin binds to the helical and protease - like domains.

[0132] As shown in Table 6, the VHH antibodies of the present invention bind to similar epitopes on hTfR1 by being able to block the binding of other VHH antibodies of the present invention to hTfR1. 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 the cell's use of TfR1 for transferrin binding to uptake iron.

[0133] In one embodiment, the VHH antibody of the present invention is a camelid VHH antibody.

[0134] In another embodiment, the VHH antibody of the present invention is a humanized VHH antibody. For example, the CDR regions of the VHH antibody can be transplanted onto a human framework. 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 the N - terminal Gln as popular choices for converting camelid VHHs into human consensus germline sequences.

[0135] The following shows the sequence alignment between the KB_B01 framework region, the reference human VH (sVH), the general VHH (uVHH), and the humanized VHH sequence (hVHH), as disclosed in the above-mentioned Biomolecules article.

[0136] QVQLQESGGGLVQAGGSLRLSCAAS KB_B01 FR1

[0137] ----VQ-------P-----------sVH FR1

[0138] ----V--------P--------T--uVHH FR1

[0139] ----V--------P-----------hVHH FR1

[0140] MRWYRQAPGKQREWVAG KB_B01 FR2

[0141] -S-V------GL---SP sVH FR2

[0142] L---------E--A--A uVHH FR2

[0143] L---------GL-A--A hVHH FR2

[0144] NYADSVKGRFTISRDNAEGTVYLQMDNLKPEDTAVYYC KB_B01 FR3

[0145] Y---------------SKN-L----NT-RA--------sVH FR3

[0146] Y----------------KN--T---N--------I---uVHH FR3

[0147] Y---------------SKN-L----NS-RA--------hVHH FR2

[0148] WGQGTQVTVSS KB_B01 FR4

[0149] -----M-----sVH FR4

[0150] -----------UVHH FR4

[0151] -----L-----sVH FR4

[0152] The VHH antibody of the present invention can be humanized by modifications in FR1, FR2, FR3, and / or FR4, such as amino acid substitutions. The amino acid positions in FR1-FR4 referred to herein are the amino acid positions in KB_B01 (SEQ ID NO: 30) shown in Figure 6 .

[0153] For 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, or a combination of any two positions, is used to humanize FR1 until all of these positions are humanized. For example, humanized FR1 can be based on SEQ ID NO: 26 or 77, but Q1 is replaced by E, and Q5 is replaced by V, or two or all of the amino acids are replaced.

[0154] The humanized positions in FR2 can be selected from L34 or M34 (the first amino acid position in FR2), S35 or G35 (the second amino acid position in FR2), V37 or F37 (the fourth amino acid position in FR2), Q43 or K43 (the tenth amino acid position in FR2), G44 (the eleventh amino acid position in FR2), L45 (the twelfth amino acid position in FR2), E46 (the thirteenth amino acid position in FR2), W47 or A47 (the fourteenth amino acid position in FR2), S49 (the sixteenth amino acid position in FR2), and / or P50, A50, V50, or G50 (the seventeenth amino acid position in FR2). In one embodiment, any one of these humanized positions in FR2, or a combination of any two positions, is used to humanize FR2 until 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.

[0155] The humanized positions in FR3 can be selected from Y58 (the 1st amino acid position in FR3), S74 (the 17th amino acid position in FR3), K75 (the 18th amino acid position in FR3), N76 (the 19th amino acid position in FR3), L78 or I78 (the 21st amino acid position in FR3), N84, S84 or T84 (the 27th amino acid position in FR3), R86 (the 29th amino acid position in FR3), A87 (the 30th amino acid position in FR3), A96 (the 39th amino acid position in FR3) and / or R97 or A97 (the 40th amino acid position in FR3). In one embodiment, any one of these humanized positions in FR3, or a combination of any two positions, is used for humanizing FR3 until all these positions are humanized.

[0156] The humanized position in FR4 can be L106 or M106 (the sixth amino acid position in FR4).

[0157] I78 also has a stabilizing effect on VHH. Therefore, Ile at position 78 stabilizes VHH.

[0158] In one embodiment, the VHH antibody of the present invention binds to protein A. In a specific embodiment, the VHH antibody of the present invention binds to a protein A-based resin, such as a protein A chromatography resin, such as MabSelect PrismA TM resin.

[0159] Protein A is a 42 kDa surface protein originally present in the cell wall of Staphylococcus aureus. It is encoded by the spa gene. Due to its ability to bind immunoglobulins, it is used in biochemical research. It consists of five homologous Ig-binding domains, each domain folding into a three-helix bundle. Each domain is capable of binding proteins of many mammalian species, most notably IgG. It binds to the heavy chain in the Fc region of most immunoglobulins and also binds to the heavy chain in the Fab region in the case of the human VH3 family. Protein A generally does not bind to camelid VHH antibodies or binds only weakly to the antibodies.

[0160] Henry KA et al., A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies, PLoS One 11(9): e0163113 (2016) discloses how to make non-protein A-binding VHH antibodies capable of binding protein A, see Table 5.

[0161] 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.

[0162] QVQLQESGGGLVQA G G S L R LSCAAS KB_B01 FR1

[0163] ----------S--------------Protein A-binding FR1

[0164] In addition, 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 achieves protein A binding by replacing amino acid residue 83 from D with N. However, in an optional embodiment, the N at amino acid residue 84 can be replaced with S.

[0165] N Y ADSV KGR F T I S RDNA E GTVYL Q M DN LKPEDTAVYYC KB_B01 FR3

[0166] E----------------KN------NS-----------Protein A-binding FR3

[0167] 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.

[0168] In one embodiment, the VHH antibody of the present invention is an isolated VHH antibody.

[0169] The term "isolated", when used in connection with a VHH antibody, e.g., in the expressions "isolated VHH antibody" and like 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 having 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 be cross-reactive with other antigens (e.g., TfR1 molecules from other species such as cTfR1 and mTfR1). In addition, an isolated VHH antibody may be substantially free of other cellular materials and / or chemicals. For example, an isolated VHH antibody may be purified to a purity greater than 95% or 99%, as determined by, e.g., electrophoresis (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)). Those skilled in the art will understand that although the term "isolated" is not explicitly mentioned each time the term "VHH antibody" etc. is used, an isolated VHH antibody is meant herein.

[0170] A further aspect of the embodiments includes nucleic acid molecules encoding a VHH antibody according to the embodiments or a fusion molecule according to the embodiments, see below. As used herein, nucleic acid molecules include polynucleotides, oligonucleotides, and nucleic acid sequences, and generally refer to polymers of DNA or RNA, which may be single-stranded or double-stranded, which may contain natural, unnatural, or modified nucleotides, and which may contain natural, unnatural, or modified internucleotide linkages, such as phosphoramidate linkages or phosphorothioate linkages, rather than the phosphodiester found between nucleotides in an unmodified oligonucleotide. The term "nucleic acid molecule" also includes complementary DNA (cDNA) and messenger RNA (mRNA).

[0171] 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.

[0172] 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.

[0173] The nucleic acid molecule may also encode other molecules in addition to the VHH antibody of the embodiments, such as a VHH antibody that is genetically fused to another molecule, i.e., in the form of a VHH-containing fusion protein further described herein.

[0174] Another aspect of the embodiments relates to a vector comprising a nucleic acid molecule according to the embodiments.

[0175] The vector is preferably an expression vector, i.e., a vector comprising at least one nucleic acid molecule, said nucleic acid molecule comprising a coding sequence that can be expressed (such as transcribed and translated) in a host cell comprising said expression vector. Thus, the expression vector comprises a nucleic acid molecule according to the embodiments operably linked to a promoter.

[0176] As used herein, operably linked means that the nucleic acid molecule is in the correct functional position and / or orientation relative to the promoter such that the nucleic acid molecule can be expressed in a host cell, i.e., the promoter (constitutive or inducible) controls the transcription of the nucleic acid molecule.

[0177] In one embodiment, the expression vector is selected from DNA molecules, RNA molecules, plasmids, episomes, and viral vectors. In such embodiments, in addition to the nucleic acid sequence encoding the VHH antibody, the expression vector further comprises control sequences required to produce 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 contained in the expression vector. A promoter is a DNA sequence to which a protein binds to initiate the transcription of an RNA molecule of the downstream DNA (gene). The promoter is preferably selected based on the particular host cell in which the VHH antibody is to be expressed, such as the T5, T7, lac, BL21 promoters for expressing the VHH antibody in bacterial cells, the GAL1, MET25, CUP1, LAC4, ADH2, SUC2, GAPDH promoters for expressing the VHH antibody in yeast, and the EF1a, CMV, or CAG promoters for expressing 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 in all cases of the host cell. An inducible promoter, also known as an inducibly active promoter, is only regulated and becomes active in a host cell in response to a specific stimulus, such as a chemically inducible promoter, a temperature-inducible promoter, or a light-inducible promoter. The expression vector may optionally comprise other control sequences, such as enhancers. An enhancer is a short DNA region that can be bound by an activator, thereby increasing the likelihood of transcription of a particular gene occurring. An optional signal peptide can be provided at the N-terminus or C-terminus of the polypeptide encoded by the expression vector.

[0178] Another aspect of the embodiments relates to a host cell comprising a nucleic acid molecule or an expression vector according to the embodiments.

[0179] Then, the nucleic acid molecule or the expression vector can be transcribed in the host cell, thereby producing the VHH antibody in the cell.

[0180] In one embodiment, the cell is selected from the group consisting of bacterial cells, yeast cells, and mammalian cells.

[0181] Another aspect of the embodiment relates to a VHH antibody according to the invention linked to at least one molecule. Thus, this aspect of the 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 a VHH antibody and at least one molecule. In this case, the above nucleic acid molecule encodes not only the VHH antibody but also at least one other molecule. For example, the nucleic acid molecule may include, from the 5' end to the 3' end, a nucleic acid sequence encoding a VHH molecule and a nucleic acid sequence encoding at least one other molecule, or include a nucleic acid sequence encoding at least one other molecule and a nucleic acid sequence encoding a VHH molecule. Thus, the VHH molecule may be covalently linked to at least one other molecule at its N-terminus or C-terminus, see Figure 2 .

[0182] It is also possible to produce fusion molecules that contain more than one molecule in addition to the VHH antibody. For example, multiple different molecules or multiple copies of a single molecule may be linked or attached to the N-terminus of the VHH antibody, the C-terminus of the VHH antibody, or at least one molecule is linked or attached to the N-terminus of the VHH antibody and at least one molecule is linked or attached to the C-terminus of the VHH molecule.

[0183] In one embodiment, the fusion molecule contains at least one other molecule linked or attached to the C-terminus of the VHH antibody.

[0184] As described above, the VHH antibody is preferably linked to at least one other molecule by genetic fusion with at least one other molecule. In such embodiments, the nucleic acid molecule encodes a VHH antibody and at least one other molecule as a VHH-containing fusion protein. This means that the nucleic acid molecule contains a nucleic acid sequence encoding a VHH antibody that is linked or joined 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.

[0185] However, the VHH antibody can be linked, joined, attached, or conjugated to at least one molecule by means other than genetic fusion, such as by chemically linking the VHH antibody to at least one molecule. For example, the VHH antibody can be covalently linked to at least one molecule by the reaction of maleimide with an amine or a thiol, which is commonly referred to as thiol-maleimide or amine-maleimide click chemistry. In such embodiments, additional amino acid residues (such as cysteine, arginine, or lysine) can be added to the N-terminus and / or C-terminus of the VHH antibody to enable such reactions with maleimide.

[0186] It is also possible to enzymatically link the VHH antibody to at least one molecule, 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 such embodiments, additional amino acid residues (such as glutamine or lysine) can be added to the N-terminus and / or C-terminus of the VHH antibody to enable such enzymatic reactions.

[0187] In one embodiment, the VHH antibody according to the present invention is covalently linked to at least one molecule.

[0188] The VHH antibody can be directly linked (such as fused) to at least one molecule. Alternatively, the VHH antibody is covalently linked to at least one molecule through a linker. According to the embodiment, various such linkers can be used, especially peptide linkers, including but not limited to the G n linker (where n is an integer equal to or greater than 1 and usually equal to or less than 10), the S m linker (where m is an integer equal to or greater than 1 and usually equal to or less than 10), the A q linker (where q is an integer equal to or greater than 1 and usually equal to or less than 10), various GS linkers or GA linkers, that is, a combination of one or more Gs with one or more Ss or one or more As, 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 usually equal to or less than 10. For example, linkers such as G4A, G4S, G3S and their combinations (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), and the linkers listed in Table 1 on pages 156-157 are hereby incorporated by reference as illustrative but non-limiting examples of peptide linkers that can be used to covalently interconnect the VHH antibody and at least one molecule.

[0189] The fusion molecule or VHH antibody 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. Such tags may be present at the N-terminus and / or C-terminus of the fusion molecule or VHH antibody. The addition of such tags for purification may also be accompanied by protease cleavage sites, such as but not limited to TEV site, so as to subsequently and effectively remove the purification tags from the VHH antibody or fusion molecule.

[0190] In addition to comprising Figure 2 at least one molecule as shown, the fusion molecule may further comprise a plurality of VHH antibodies.

[0191] In one embodiment, the fusion molecule is a monovalent fusion molecule relative to the VHH antibody, that is, preferably it comprises only a single VHH antibody in addition to at least one molecule. There is in vitro and in vivo evidence indicating that the monovalent binding mode promotes transcellular transport, while the bivalent binding mode results 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)).

[0192] The at least one molecule may be any molecule, such as a medicament or drug, a diagnostic agent, an imaging agent, a tracer, a half-life extender, etc. Examples of such molecules include but are not limited to antibiotics, antiviral agents, immunomodulators, anti-tumor agents, anti-inflammatory agents, adjuvants, peptides, polypeptides, and proteins, such as enzymes, hormones, neurotrophic factors, neuropeptides, cytokines, apolipoproteins, growth factors, antigens, antibodies or parts of antibodies, adjuvants, etc., nucleic acids, such as RNA or DNA, including for example coding genes, inhibitory nucleic acids, such as ribozymes, antisense nucleic acids, interfering nucleic acids, whole genomes or parts thereof, plasmids, etc.

[0193] 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.

[0194] As used herein, an imaging agent refers to a reagent or molecule used to visualize the imaging agent during imaging, such as being taken up by cells when administered into the body of a patient. Examples of such imaging agents include medicaments or molecules containing radioactive atoms or isotopes, such as radioactive tracers for positron emission tomography (PET) imaging agents, such as 18 F-containing PET imaging agents, 11 C-containing PET imaging agents, 64 Cu-containing PET imaging agents, or single photon emission computed tomography (SPECT) imaging agents, such as99m Technetium (Tc) or 111 single photon emission computed tomography (SPECT) tracers of indium (In). Other examples of imaging agents include fluorescent probes, luminescent probes, probes containing metal complexes, near-infrared (NIR) fluorescent probes, and the like.

[0195] Generally speaking, there is an urgent need for diagnostic tools that can detect diseases or medical conditions (especially in the ventricles) at an early stage. Such early detection will contribute to effective disease-modifying therapies, especially for evaluating the therapeutic effects in preclinical and clinical trials of new drug candidates.

[0196] Classical positron emission tomography (PET) ligands or tracers based on small molecules that can cross the blood-brain barrier (BBB) are difficult to specifically target pathogenic proteins, including but not limited to amyloid-beta, tau, and alpha-synuclein, or false markers of diseases such as inflammation. PET ligands or tracers based on specific binding molecules (such as antibodies) are generally preferred. However, PET tracers based on monoclonal antibodies (mAbs) usually have too long a circulating half-life to be of any use as PET ligands or tracers. This means that the radioactivity from radiolabeled mAbs in the cerebral blood circulation will interfere with the specific signals at the target sites in the brain. Therefore, for use in PET applications, antibody-based PET ligands or tracers should have a rapid blood clearance rate and a high brain-blood ratio. Due to renal excretion, smaller antibody domains or fragments (average molecular weight equal to or less than 60 kDa), such as VHH antibodies, have a faster whole-body elimination compared to mAbs. Therefore, since VHH antibodies are smaller than mAbs, they are suitable for use as PET ligands or tracers ( et al., Abispecific 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)).

[0197] In these embodiments, the fusion molecule can be used as a diagnostic agent for diagnosing various diseases or disorders.

[0198] For example, the VHH antibodies of the embodiments can be linked to another antibody, an antigen-binding fragment or domain thereof, including another VHH antibody, which specifically binds to a disease-specific marker. In such cases, it is preferred to label the other antibody to detect its binding to the disease-specific marker after delivery, for example, by using the VHH antibody as a receptor-mediated transcytosis of the BBB transporter to be delivered to the cerebral ventricle.

[0199] Antigen-binding fragments or domains of antibodies as used herein can be selected from the group consisting of: single-chain antibodies, Fv fragments, scFv fragments, Fab fragments, F(ab’)2 fragments, Fab' fragments, Fd fragments, single-domain antibodies (sdAb), scFv-Fc fragments, di-scFv fragments, and groups of two or more CDR regions.

[0200] For therapeutic purposes, in addition to the therapeutic agent, the fusion molecule can also contain a half-life extender or group, also known as a stabilizer or group. Then, such a half-life extender is added to increase the half-life of the fusion molecule when administered to a patient. Any half-life extender that can be linked to the VHH antibody without adverse biological effects can be used, including but not limited to the Fc fragment of IgG, serum proteins (such as human serum albumin (HSA)), albumin-binding protein scaffolds, IgG, various polyethylene glycol (PEG) molecules, or unstructured polypeptides (such as XTEN) or PASylation as an alternative to PEGylation.

[0201] The present invention also relates to a pharmaceutical composition comprising a fusion molecule as defined above, wherein at least one molecule is a therapeutic agent. The pharmaceutical composition also contains a pharmaceutically acceptable vehicle or excipient.

[0202] A pharmaceutically acceptable excipient can be any pharmaceutically acceptable excipient or vehicle that is compatible with the other components of the pharmaceutical composition. The pharmaceutically acceptable vehicle can be selected from the conventionally used vehicles according to each mode of administration. The pharmaceutical composition can be a solid pharmaceutical composition, such as tablets, pills, powders, or granules; a semi-solid pharmaceutical composition, such as suppositories; or a liquid pharmaceutical composition, such as soft capsules or injection solutions.

[0203] Pharmaceutically acceptable vehicles or excipients include but are not limited to diluents, such as lactose, glucose, sucrose, mannitol, sorbitol, cellulose, and glycine; lubricants, such as silica, talc, stearic acid (including its salts), and polyethylene glycol; binders, such as magnesium silicate and aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone; disintegrants, such as starch, agar, alginic acid, and sodium alginate; absorbents; dyes; flavoring agents; sweetening agents; polypropylene glycol; liquid vehicles, such as water, physiological saline solution, aqueous glucose solution, glycerol, ethanol, and oils.

[0204] The embodiments also relate to the above fusion molecules for use as medicaments, wherein at least one molecule is a therapeutic molecule.

[0205] In an embodiment, the therapeutic agent is capable of treating a CNS disease or disorder.

[0206] In a specific embodiment, the above fusion molecule is used to treat a CNS disease or disorder. In such embodiments, at least one molecule is a therapeutic agent capable of treating a CNS disease or disorder.

[0207] A further embodiment relates to a method of treating a CNS disease or disorder in a patient. The method comprises administering to the patient an effective amount of the above fusion molecule or the above pharmaceutical composition. In such embodiments, at least one molecule is a therapeutic agent capable of treating a CNS disease or disorder.

[0208] Illustrative but non-limiting examples of CNS diseases or disorders 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 monomelic amyotrophy (MMA), multiple sclerosis (MS), neurofibromatosis, Parkinson's disease (PD), lysosomal storage diseases, neuronal lysosomal storage diseases, ischemic stroke, intracerebral hemorrhage, traumatic brain injury (TBI), vascular dementia, frontotemporal dementia, amyloidosis, tauopathies, Creutzfeldt-Jakob disease, neuroinflammation and neuropathic pain.

[0209] Malignant cells often overexpress TfR1, and this increased expression may be associated with poor prognosis in different types of cancers (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, and its level is usually several times higher than that in normal cells. In fact, TfR1 has been identified as a universal cancer biomarker. Increased TfR1 expression is associated with advanced and / or poor prognosis in multiple cancers, including solid cancers 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 nervous system cancers, as well as hematological malignancies such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and non-Hodgkin 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)).

[0210] The elevated expression level of TfR1 on malignant cells, combined with its extracellular accessibility, internalization ability, and central role in cancer cell pathology, makes this receptor an attractive target for antibody-mediated therapy. The VHH antibodies of the present invention can target TfR1 for cancer treatment by using VHH antibodies conjugated to anti-cancer agents that are internalized through receptor-mediated endocytosis.

[0211] In another embodiment, the therapeutic agent is capable of treating cancer.

[0212] In a specific embodiment, the above fusion molecule is used for treating cancer. In such embodiments, at least one molecule is a therapeutic agent, immunotherapeutic agent, and / or adjuvant therapeutic agent capable of treating cancer.

[0213] Further embodiments relate to a method of treating cancer in a patient. The method comprises administering to the patient an effective amount of the above fusion molecule or the above pharmaceutical composition. In such embodiments, at least one molecule is a therapeutic agent capable of treating cancer.

[0214] 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 oligonucleotide using FORCE TM conjugation in mdx mice, Nucleic Acids Research gka c641, 2022; Sugo et al., Development of antibody-siRNA conjugate targeted to cardiac and skeletal muscles, J Control Release 237:1-13 (2016)). Thus, the fusion molecules of the present invention can be used to treat various muscle diseases, particularly muscular dystrophies.

[0215] The fusion molecules can also be used to treat muscular dystrophies such as Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), myotonic 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.

[0216] In further embodiments, the therapeutic agent is capable of treating muscular dystrophy.

[0217] In specific embodiments, the above fusion molecules are used to treat muscular dystrophy. In such embodiments, at least one molecule is a therapeutic agent capable of treating muscular dystrophy.

[0218] Further embodiments relate to a method of treating a patient having muscular dystrophy. The method comprises administering to the patient an effective amount of the above-described fusion molecule or the above-described pharmaceutical composition. In such embodiments, at least one molecule is a therapeutic agent capable of treating muscular dystrophy.

[0219] As used herein, an effective amount means an amount effective at dosages and for periods of time necessary to achieve the desired result. An effective amount may vary according to factors such as disease state, age, sex, and weight of the patient. As used herein and well understood in the art, "treating" or "treatment" means a method for obtaining a beneficial or desired result, including a clinical result. A beneficial or desired clinical result can include, for example, alleviation or improvement of one or more symptoms or conditions, diminishment of the extent of disease, stabilization of a disease state (i.e., preventing worsening), prevention of the spread of disease, delay or slowing of disease progression, amelioration or palliation of a disease state, reduction of the recurrence of disease, and remission. "Treatment" can also extend survival as compared to expected survival if not receiving any treatment.

[0220] As used herein and well understood in the art, "preventing" or "prophylaxis" means a method of reducing or preventing the risk of developing a disease or disorder, including prolonging or delaying disease development. For example, patients who are predisposed to developing a disease (such as due to genetic or hereditary susceptibility) may benefit from a fusion molecule or pharmaceutical composition to prevent, reduce the risk of the disease, and delay and / or slow the development of the disease.

[0221] The patient is preferably a human patient. However, the embodiments can also be applied to veterinary applications, i.e., non-human patients, such as non-human mammals, including, for example, primates, monkeys, apes, cows, sheep, pigs, goats, horses, cats, dogs, mice, rats, and guinea pigs.

[0222] The fusion molecule or pharmaceutical composition according to the embodiments can be administered to the patient by various routes, including, for example, parenteral routes, such as by injection via subcutaneous, intravenous, intraperitoneal, or intramuscular routes; oral routes; rectal routes; topical routes; intranasal routes; transmucosal routes; or intraocular routes. In a specific embodiment, the route of administration is subcutaneous or intravenous administration.

[0223] Examples

[0224] Example I - Production of Proteins for Llama Immunization and Assays

[0225] The extracellular domains of hTfR1 (amino acids R121 - F760, SEQ ID NO: 48), mTfR1 (S122 - F763, SEQ ID NO: 49), and cTfR1 (R121 - F760, SEQ ID NO: 50) were cloned into the pCDNA3.4 - TOPO plasmid using the stop codon TGA, TGA, and Kozak sequences (generated using the Geneart tool (Thermo Fischer Scientific)). The plasmid has 5' cloning sites: HindIII, KpnI, NheI and 3' cloning sites: XhoI, PacI, and is optimized for expression in the grey hamster (Cricetulus griseus). Expression was carried out using an N - terminal signal peptide (MDWLRNLLFLMAAAQSINA, SEQ ID NO: 51) followed by a single amino acid linker "A", a histidine tag (6×His, SEQ ID NO: 56), and an SG linker (for hTfR1, cTfR1) or a G linker (for mTfR1). The plasmid was ordered from Geneart (Thermo Fischer Scientific, Germany). Transient transfection of Chinese hamster ovary (CHO) cells was carried out according to standard procedures. The cultures were harvested on day 6. The supernatant was filtered (0.45 μm syringe filter) and then purified using a HisTrapExcel 5 mL column (Cytiva) with immobilized metal ion chelation (IMAC). The buffers used were as follows: equilibration / wash buffer (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, NaCl 500 mM, imidazole, pH 8). The elution fractions were concentrated using an Amicon Ultra 10000 MWCO spin concentrator. The protein was dialyzed overnight at 4 °C in PBS containing 10% glycerol (Medicago, tablets, glycerol diluted from an 80% stock solution) (cassette slide - a - lyzer 3 ml) with a cut - off of 10 kDa (Pierce). The PBS was set to pH 8. The protein concentration was measured using the absorbance at 280 nm.

[0226] Example II - Generation and Selection of a VHH Library That Binds to Human TfR1

[0227] According to ModiPhage of Modiquest Research BV (the Netherlands) TMMethods were used to immunize llama animals (N = 2) to generate antibodies. Two llama animals were primed using an adaptive immunization protocol based on monitoring of the specific immune response to immunogens hTfR1 and mTfR1 (generated as described in Example I). As Figure 1A shown, immunizations were performed by intramuscular (i.m.) administration of a single dose of 500 μg protein on Day 1 (hTfR1), Day 21 (mTfR1), and Day 42 (hTfR1). On Day 54, blood was drawn to study the immune response by ELISA. Serum samples were serially diluted in 3-fold steps (from 1:100 to 1:72900) and screened against plate-fixed 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 using goat anti-rabbit horseradish peroxidase (HRP) and goat anti-mouse HRP, respectively, and detected at 480 nm absorbance using 3,3',5,5'-tetramethylbenzidine (TMB) substrate in a spectrophotometric plate reader. Two additional doses of 500 μg hTfR1 were injected on Days 86 and 107. On Day 117, blood was drawn and ELISA was performed as described above. One animal was selected for final harvest and received an additional booster dose on Day 120, a 1:1 mixture of hTfR1 and mTfR1 (250 μg + 250 μg). Four days later (Day 124), peripheral blood was drawn and PBLs were isolated by density gradient centrifugation using Ficoll-Paque TM PLUS ∼1.5×10 9 PBLs were isolated by density gradient centrifugation using Ficoll-Paque PLUS ∼1.5×10 9 RNA was extracted from approximately 0.7×10 9 PBLs using TRIzol (Thermo Fischer Scientific), and reverse transcription to cDNA was performed by oligo(dT)-primed reverse transcription of 400 μg freshly isolated RNA.

[0228] All subsequent steps for constructing the VHH phage display library were performed as described by 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 for a PCR reaction to amplify the IgG2 / 3 library. A first PCR reaction was performed to amplify all antibodies (IgG1 and IgG2 / 3), and then a second nested PCR reaction was performed to amplify and isolate the VHH library, including subcloning into the overhang site in the phagemid pADL10b using primers CALL001 (SEQ ID NO:52) and CALL002 (SEQ ID NO:53). 3 μg of the cDNA template was used with a High kit from ThermoFischer Scientific: 10 μl of 5× High Fidelity buffer, 1 μl of dNTP (dinucleotide triphosphate; 10 mM each, 0.2 mM final concentration), 25 pmol of each primer (0.5 μM final primer concentration), 0.5 μl polymerase (1 U / reaction), ddH2O (double-distilled water) to 50 μl, 1 - 2 μl of template, cycling program: 98 °C for 5 minutes, 98 °C for 30 seconds (30×), 72 °C for 30 seconds (30×), 72 °C for 10 minutes, store at 4 °C. A total of 36 wells were pooled, then purified using Genejet enzyme purification (Thermo Fischer, catalog number: K0832), and PCR dimers were removed. The mixture was purified through four purification columns and eluted in 4 × 20 μl of elution buffer. The eluate was loaded onto a 1% agarose gel, and SYBRSafe DNA Gel Stain (SigmaAldrich) was used for safe gel extraction, and the extracted product was used as a template in the subsequent PCR reaction. Next, amplification was performed using a nested PCR reaction, including the pADL10 cloning site using the following oligonucleotides VHH-R-SfiI (SEQ ID NO:54) and VHH-F-SfiI (SEQ ID NO:55). Two PCR mixtures were prepared separately, 0.5 μM of each primer (VHH-F-SfiI and VHH-R-SfiI) and 0.1 μM of each primer, and 1 μl of template was thermocycled as follows: 98 °C for 5 minutes, 98 °C for 30 seconds (20×), 72 °C for 1 minute (20×), 72 °C for 10 minutes, store at 4 °C. Approximately 50 ng of the amplified and gel-extracted heavy-chain antibody was used as a template in each well. After amplification, all wells were combined and purified using Genejet PCR purification according to the manufacturer's protocol, and eluted in elution buffer (10 mM Tris-HCl pH 8.5). The concentration of the PCR product was measured using Nanodrop (result: 160 ng / μl). The product was digested with SfiI restriction enzyme (Thermo Fischer Scientific). The 400 bp DNA fragment was extracted on a 2% agarose gel using Genejet gel extraction method.

[0229] Digest the pADL10b vector overnight at 37 °C using SfiI restriction enzyme (Thermo Fischer Scientific) according to the manufacturer's instructions. The digested vector was purified on a 1% agarose gel and visualized using Sybr Safe (Thermo Fischer Scientific). Repeat the process until the final concentration is 154.6 ng / μl.

[0230] After PCR amplification, digest the library at 50 °C (using Sfil) for 2 hours. Remove the enzyme using the Genejet PCR purification column described above. Elute the resulting material in 20 μl of elution buffer (10 mM Tris-HCl pH 8.5). The DNA concentration was measured to be 73 ng / μl. Ligate the mixture again. First, heat the material at 50 °C and slowly cool it back to room temperature (RT, 20 - 25 °C), then incubate it with shaking at 16 °C overnight. Purify as above and elute in 10 mM Tris-HCl pH 8.5. A total of 1600 ng of DNA was used. Electroporate 10 μl of TG1 Escherichia coli in a total reaction volume of 25 μl with 1 or 2 μl of the ligation mixture (5.48 ms 1.92 kV and 5.48 ms 1.92 kV, respectively). Remove the cells from the -70 °C freezer and use them immediately after thawing on ice. Distribute the transformants on large agar plates (20 cm) and let them grow under standard conditions until colonies appear and are ready for harvesting.

[0231] Harvest the cells by adding Luria Broth (LB) medium. Count the colonies and estimate the library size to be 3.2×10 8 . After overnight growth on agar plates, prepare a culture of the final library pool. Shake the culture at 200 revolutions per minute (rpm) at 37 °C for 2 hours. Add Hyperphage at a multiplicity of infection (MOI) of 2 without shaking at 37 °C for 30 minutes. Remove the medium by centrifuging at 4500 g for 5 minutes in a Falcon tube. Then resuspend the cells 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). Shake the culture at 200 rpm overnight at 30 °C.

[0232] Prepare 5 mL cultures from the selected clones' overnight cultures in 15 mL falcon tubes supplemented with 100 μg / mL ampicillin in LB, omitting glucose. Dilute the overnight cultures 1:100 in 5 mL LB and incubate at 150 rpm for 2 hours until the cultures become turbid. Add 1 μL of hyperphage helper phage (Hyperpha ge, titer: 1.4×10 12 cfu / ml, Progen). Allow the helper phage to infect for 30 minutes at 37 °C. Discard the LB containing the phage by centrifugation at 4500 g. Resuspend the bacterial pellet in 5 mL TB medium supplemented with 100 μg / mL ampicillin, 50 μg / mL kanamycin, and 1 mM IPTG. Shake the cultures overnight at 200 rpm and 30 °C. Centrifuge the cultures at 4500 g for 4 minutes to remove the bacteria, add 0.25×V of 20% PEG8000 / 2.5 M NaCl, and incubate on ice for 2 hours in the centrifuge tube. Pellet the phage / bacteria at 15000 g for 10 minutes and resuspend in 0.5 mL PBS. Transfer the phage-containing supernatant to a separate microcentrifuge tube pre-blocked with PBS / casein.

[0233] Perform panning with biotinylated TfR1

[0234] Biotinylate hTfR1, cTfR1, and mTfR1 according to the standard procedure using the biotinylation kit EZ-link Sulfo NHS PEG4 (Thermo FIscher Scientific) with a 1:1 molar ratio of 1 mg of target protein to 2 mM biotin at pH 8. Remove free biotin by dialysis in a Pierce 3 mL 10 kDa MWCO (molecular weight cut-off) dialysis device with 1 L of PBS buffer at pH 8 for 3×1 hour at 4 °C. Concentrate the protein to 850 μL using an Amicon spin concentrator with a 10 kDa MWCO. Pre-block the phage supernatant with PBS / casein and mix with 100 nM biotinylated hTfR1, cTfR1, or mTfR1 and incubate for 1 hour. Use 50 μL of Streptavidin (M280, Invitrogen, Thermo Fischer Scientific) for bead blocking and use containing 0.15% Wash with 20% PBS (PBST). Resuspend the beads in 1 ml of ELISA blocker (PBS / casein ELISA blocker (Thermo Scientific)) and incubate for 30 minutes at room temperature in a rotary mixer. Replace the blocker with the phage + target mixture and gently rotate at room temperature for 30 minutes to capture biotinylated TfR1 on the beads. Wash the beads 5 times with PBS. Elute both mTfR1 and hTfR1 captures in 400 μl of PBS containing 0.25 mg / ml trypsin.

[0235] Digest the phage with trypsin at room temperature for 30 minutes, then titrate and rescue. TG1 E. coli culture is started by inoculating 10 ml of culture into 10 ml of LB medium and growing a single TG1 colony on an M9 minimal agar plate. Incubate the culture at 37 °C at 200 rpm until the OD 600 reaches the logarithmic phase (about 4 hours, OD 600 is about 0.6). Keep the culture on ice, prepare dilutions (1 - 10 fold) and mix with 100 μl of TG1 and incubate for 5 minutes, then inoculate the mixture onto an LB Lennox agar plate (Merck - Sigma Aldrich) supplemented with 2% glucose and 100 μg / ml ampicillin. First, trypsinize the input (1 mg / ml trypsin) by mixing 75 μl of the phage stock solution with 25 μl and incubate at room temperature for 30 minutes, then dilute and titrate. Mix each of the remaining phage outputs with 2 ml of TG1 E. coli and incubate at room temperature for 5 minutes (without shaking). Inoculate each mixture onto two large LB Lennox agar plates and let them dry completely, then incubate in a plastic bag at 37 °C.

[0236] According to Table 1 below, repeat the procedure of selection (and subsequent rescue) using captured Dynabeads with 100 nM, 25 nM, 10 nM, and 5 nM of hTfR1, cTfR1, or mTfR1 (the latter two in the presence of 1 μM human transferrin (recombinant in rice, Sigma Aldrich, human transferrin)) in the following three rounds. After the 4th round of selection, randomly pick 25 colonies from the agar plates representing each track and elution method (trypsin or citrate / phosphate buffer at pH 5.5 containing 0.15% 20) and send them to the Gene Analysis Core Facility at Karolinska Institute (Sweden) for sequencing using the primer pADL10fwd.

[0237] Table 1 - Biopanning protocol for selection

[0238]

[0239]

[0240] Example III - Combining with ELISA to select VHHs that bind to hTfR1

[0241] Before performing ELISA, the selected phage samples were diluted 1:10 in blocking buffer Casein ELISA Blocking Reagent (ThermoFischer Scientific), 2.5 M NaCl containing 20% v / v PEG8000. The targets (hTfR1, cTfR1, mTfR1) were coated at 1 μg / ml in PBS pH 7.4 (100 μl) overnight at 4 °C in 96-well Greiner plates (untreated). The coating buffer was discarded and replaced with 100 μl of blocking solution. The plates were covered and incubated at 900 rpm at room temperature for 2 hours. Anti-human TfR1 antibody BA3 (MEM189, Sigma, 1 mg / ml) and anti-mTfR1 (8D3, Novusbiologicals, 1 mg / ml) were diluted in blocking buffer to a starting concentration of 100 nM and serially diluted 1:2 by transferring 100 μl into 100 μl of blocking buffer. The plates were incubated at room temperature, 900 rpm for 1 hour and the wells were washed 3×300 μl in PBST (PBS containing 0.1% 20). Anti-M13 antibody (Anti-PVIII GE Healthcare) was diluted 1:4000 in blocking buffer and 100 μl was added to the target-coated wells and incubated at 900 rpm at room temperature for 1 hour. Washing was performed with PBST. Anti-mouse secondary antibody (Sigma) was diluted 1:4000 in blocking buffer and incubated at 900 rpm at room temperature for 1 hour. After washing in PBST, development was carried out with TMB substrate. The reaction was stopped by adding 2 M H2SO4 to each well and absorbance readings at 450 nm were measured using a SpectraMax 3000 (Molecular Devices). One clone (KB_B01) used in the following examples was selected based on cross-reactive binding to both cTfR1 and hTfR1 in ELISA. The selection criteria were a reactivity to human and cynomolgus monkey receptors greater than 10-fold above the blank (negative control). The 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 brain delivery of therapeutic agents.

[0242] Example IV - Generation of free VHH or fusion variants

[0243] Selected VHH clone (KB_B01) was produced as a single VHH unit (12 - 14 kDa), see Figure 2 , which has a C-terminal 6×His tag (SEQ ID NO:56), followed by a "C tag" (C-terminal amino acids EPEA, SEQ ID NO:57), ordered from Genscript Inc. and codon-optimized using the expression vector pEt-22b(+). The protein was obtained from the periplasmic space of E. coli using standard methods and purified in one step on a Ni column.

[0244] The human Fc region was gene-fused to the VHH via a GG spacer, resulting in a bivalent TfR1-binding functional entity, see Figure 2 .

[0245] More specifically, the VHHFc fusion protein (SEQ ID NO:59) between human IgG1 Fc (SEQ ID NO:58) and KB_B01 (SEQ ID NO:30) was cloned together with the signal peptide from murine IgGκ light chain (SEQ ID NO:81) into the pcDNA3.4 backbone with codon-optimized sequences (performed by GeneArt, Thermo Fischer Scientific) and transiently expressed in Expi293 TM cells (Thermo Fischer Scientific). A 200 mL culture was grown under standard conditions and harvested 6 days after transfection. Purification of the medium was performed on MabSelect Sure TM and refined in HiLoad Superdex200, yielding a dimer VHH-Fc fusion fraction with >99% purity, as verified by analytical size exclusion chromatography (SEC). The same method was performed to generate the fusion protein of the benchmark VHH (SEQ ID NO:80) and human IgG1 Fc disclosed in WO 2020 / 144233. This benchmark VHH-containing fusion protein is referred to as BV herein.

[0246] In addition, 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) are based on the same human IgG1 Fc and GG linker sequences and signal peptide (SEQ ID NO:79) as described above. After sequence optimization and cloning into a proprietary plasmid at GenScript Biotech Corporation, they were produced in a 4 mL volume in the Turbo-CHO TM high-performance platform system, yielding the following Fc fusions: 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). The final samples were harvested on day 6 post-transfection, purified on a protein A-based system at Genscript, and verified by SDS-PAGE or analytical size exclusion chromatography (SEC) with a purity of 95-99%.

[0247] Functional fusion proteins are produced in a form fused with VHH and single-chain variable domain (scFv). Such fusion proteins have monomeric TfR1 binding, but due to the presence of the scFv domain capable of binding to a target different from TfR1 or a different epitope on the same target, they have the potential for bispecific binding. The amyloid-β binding murine antibody 3D6 was selected as a functional and representative scFv unit. The scFv of the 3D6 antibody consists of VH (SEQ ID NO: 68) and VL (SEQ ID NO: 69), which are fused to the N-terminus or C-terminus of KB_B01 and joined to BV via a 3×(G4S) linker (SEQ ID NO: 70), generating KB_B01-scFv (SEQ ID NO: 71) or scFv-BV (SEQ ID NO: 72), BV-scFv (SEQ ID NO: 73) or scFv-KB_B01 (SEQ ID NO: 74). GenScript Biotech Corporation optimized the sequences of the gene constructs, synthesized them and cloned them into custom plasmids, and expressed them in transiently transfected TurboCHO TM high-performance platform cells. According to standard procedures, purification was performed using HiTrap TM FF Crude (Cytiva), and then further purified using HiLoad TM 26 / 600 Superdex (Cytiva).

[0248] Using the vector pcDNA3.4, the reference antibody 128.1 (BA1) was expressed as full-length human IgG1 (SEQ ID NO: 75) in transiently transfected FreeStyle TM -293 and harvested from the culture medium, and purified in one step on protein A resin. The reference antibody JCR-IgG (BA2) (SEQ ID NO: 76) was designed based on the information in US 9,994,641 as an IgG1 antibody reactive to 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 and expressed in transiently transfected ExpiCHO TM cells, and harvested on day 8. The protein was purified by MabSelect Sure TM (Cytiva), subsequently purified by Superdex 16 / 60 (Cytiva), and analyzed according to standard procedures known to those skilled in the art.

[0249] Binding characteristics of Example V-KB_B01

[0250] During the phage display panning process for hTfR1 and cTfR1, the binding of the llama VHH KB_B01 to hTfR1 and cTfR1 was identified by ELISA. The binding kinetics were further analyzed by surface plasmon resonance (SPR) and biolayer interferometry (BLI). The binding affinity of VHH KB_B01 to the target human protein and the lack of interference of transferrin (Tf) with the affinity were tested. SPR analysis was performed on a Biacore T200 or Biacore 8K instrument (Cytiva, Sweden). SPR S series chips SA and S series CM5 chips or S series protein A chips (Cytiva, Uppsala Sweden) were used. Biotinylated transferrin was coupled to the SA chip.

[0251] Human TfR1, mTfR1, and cTfR1 were biotinylated using a biotinylation kit: EZ-Link NHS-PEG4-Biotin, No-Weigh type (Thermo Fisher Scientific) according to the manufacturer's instructions. The stock biotin solution vial was dissolved and stored in DMSO at -70 °C at a concentration of 20 nM. This tube was thawed and freshly diluted to 2 mM with ddH2O and mixed with the TfR1 protein in PBS containing a 2× molar excess of biotin, with a total volume of 200 μL. The mixture was incubated with shaking at 25 °C for 45 minutes and then incubated overnight 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 at pH 8 was used for buffer exchange. Protein concentration was determined using absorbance at 280, and the yield was >75%.

[0252] Using single-cycle kinetics, the binding characteristics of monomeric VHH KB_B01 to the reference full-length antibody BA1 were evaluated by SPR on a Biacore T200 instrument. Human Tf was amine-coupled (to approximately 3000 RU) on a CM5 chip, and then hTfR1 was captured on the immobilized human transferrin (hTf). Human TfR1 was loaded onto hTf by injection at 100 nM, and then analytes (VHH KB_B01 or BA1) with increasing concentrations in the range of 6.25 to 100 nM were injected in 2-fold increments. The contact time for each concentration was 150 seconds at 30 μL / s, and the final dissociation step was 500 seconds. At the end of each single-cycle run, the hTf surface was completely regenerated by injecting 10 mM glycine pH 2 for 30 seconds. Using the Biacore T200 software evaluation tool and the Langmuir 1:1 model, data on the association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (K D ) were evaluated.

[0253] The monomeric VHH KB_B01 and the reference antibody BA1 bind to hTfR1 (Table 2 and Figure 3A , 3B ), demonstrating human non-Tf interference binding. Since hTfR1 is immobilized by being loaded on hTf, the binding of the analyte means that neither KB_B01 nor BA1 competes with hTf for binding to hTfR1. In addition, comparing the results of KB_B01 and BA1 in terms of affinity and binding kinetics shows that due to the significant difference in the dissociation rate (kd), the affinity (K D ) of the monomeric KB_B01 for binding to pre-complexed hTfR1-Tf is 166-fold lower than that of the bivalent BA1 (K D = 0.0199 nM), being 3.31 nM. It has been shown that too high an affinity may cause the TfR1-binding protein and any protein fused thereto to be retained in the blood vessels and / or lead to 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 the following binding characteristics: being more suitable as a brain drug transporter due to its lower affinity for hTfR1 than BA1.

[0254] Table 2. Affinities of monomeric VHH and full-length reference antibody BA1 for hTfR1 measured by SPR

[0255] Clone TfR1 species <![CDATA[ka(M -1 s -1 )]]> <![CDATA[kd(s -1 )]]> <![CDATA[K D (M)]]> KB_B01 Human <![CDATA[5.97×10 5 > <![CDATA[1.98×10 -2 > <![CDATA[3.31×10 -9 > BA1 Human <![CDATA[2.82×10 6 > <![CDATA[5.63×10 -5 > <![CDATA[1.99×10 -11 >

[0256] Characterization of the Fc-fused VHH KB_B01 of Example VI

[0257] Affinity measurements were performed by SPR on a Biacore T200 (Cytiva), which was loaded with KB_B01-Fc, BV-Fc, and BA2 (Example IV) at a high level (100 nM for 60 seconds) onto a Protein A chip (Cytiva). Subsequently, these were exposed to 5-fold increments of increasing concentrations of hTfR1 in the range of 0.16 to 100 nM. The data are summarized in Table 3, and the sensorgrams are shown in Figure 4A-4I . The dimeric KB_B01-Fc binds to hTfR1 with a K D value of 0.176 nM (Table 3, Figure 4A ), i.e., the affinity is 19-fold higher than that of the free monomeric KB_B01 for binding to immobilized hTfR1 (KD = 3.31 nM, see Table 2). As a control, the reference BV-Fc and BA2 were run under the same conditions and showed 0.00228 nM ( Figure 4D ) and 0.0249 nM ( Figure 4G ) affinity (K D value), respectively. Cynomolgus and mouse TfR1 were also run under the same conditions, and the binding affinity of KB_B01-Fc to mTfR1 was 2.82 nM ( Figure 4C ). The reference BV-Fc bound to mTfR1 with a high affinity (K D ) of 0.444 nM ( Figure 4F ), which means that when both are in the Fc fusion form, the binding affinity of KB-B01 to mTfR1 is 6.4-fold lower than that of the reference BV. In contrast, the reference antibody BA2 did not bind to mTfR1 with a detectable signal ( Figure 4I ). This experiment demonstrated that KB_B01 has the desirable property of a low nM affinity range and an interesting K D profile in terms of release (OFF) to the receptor.

[0258] In addition, KB_B01 showed broad species cross-reactivity, which is a highly desirable feature from a translational perspective when predicting pharmacokinetics and pharmacodynamics from mouse to human.

[0259] Table 3 - Binding kinetics of KB_B01 and BV as dimeric Fc fusions to human, cynomolgus, and mouse TfR1 studied using SPR

[0260]

[0261] To further investigate the effect of soluble human transferrin on binding to human TfR1, the above 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 (4-fold increments) in the range of 0.156 to 40 nM of the receptor in the absence or presence of a saturating concentration of 250 nM hTf. In the presence of 250 nM hTf, the high affinity for hTfR1 was retained, indicating that KB_B01-Fc and the two reference antibodies BA1 and BA2 do not interfere with the binding of Tf to human TfR1 (Table 4, Figure 5A-5F ). This experiment demonstrated that KB_B01 has desirable and unique properties for using the TfR1 molecule in humans to increase the delivery of therapeutic or diagnostic proteins to the CNS.

[0262] An interesting difference between KB_B01 and the reference VHH BV is that the binding of BV-Fc to hTfR1 is more affected by the presence of hTf than the binding of KB_B01-Fc. In the presence of 250 nM hTf, the affinity of BV-Fc for hTfR1 decreased nearly 390-fold compared to the absence of hTf, but the affinity of KB_B01-Fc for hTfR1 decreased only 10-fold.

[0263] Table 4 - Effect of the presence of transferrin on binding to human TfR1

[0264]

[0265] Example VIII - Analysis of Bioinformatics and Additional Cloning

[0266] Using the Jalview multiple sequence alignment editor and analysis workbench version 2 (Barton group, University of Dundee) software, KB_B01 was aligned with all clones based on the preliminary screening (panning) sequencing of the clones. Based on sequence identity with KB_B01 and positive binding confirmed in ELISA, nine additional clones, KB_B02 to KB_B10 (see Figure 6 for the alignment), were selected and generated as fusions with human IgG1 Fc (Example IV). A clone known to have a high interference with Tf binding was selected as a reference, KB_BC.

[0267] Based on the above, KB_B02 to KB_B10 were generated as fusions with human IgG1 Fc (see Example IV), and their affinities for hTfR1 and mTfR1 were tested 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) at 5 μL / s, eight at a time, for 120 seconds, and then increasing concentrations of the receptor in the range of 0.8 to 500 nM were injected in five-fold increments (contact time 120 seconds, dissociation 600 seconds). After each complete single-cycle kinetics run, the surface was regenerated with 10 mM glycine, pH 1.5 at 30 μL / s for 30 seconds. Table 5 presents the kinetic data for KB_B02 to KB_B10 as Fc fusions. KB_B02 to KB_B10 all bound to hTfR1 with high affinity, which was defined by KD values in the range of less than 0.00309 nM to 0.451 nM (Table 5). The affinity for mTfR1 ranged from less than 0.0171 nM to 89.7 nM. D values defined (Table 5). The affinity for mTfR1 ranged from less than 0.0171 nM to 89.7 nM.

[0268] Table 5 - Binding kinetics of KB_B02 and KB_B10 as dimeric Fc fusions to human and murine TfR1 using SPR

[0269]

[0270]

[0271] Next, epitope binning was performed by pairwise testing the binders of hTfR1 in a combinatorial manner using SPR on a Biacore 8K. KB_B01, KB_B04, KB_B05, KB_B08, KB_B09, and KB_B10, together with the reference antibodies BA1 and BA2, were immobilized onto a CM5 chip series S (Cytiva) by amine coupling. Another VHH-Fc known to bind to a different epitope, KB_BC, was also immobilized. The proteins to be immobilized were diluted to 25 μg / mL in sodium acetate at pH 5.5, and the proteins were coupled with an SPR signal of approximately 6000 resonance units (RU). The first injection used 50 nM of the extracellular domain of hTfR1 (contact time 120 s, 10 μL / s), and the second injection used 100 nM of each analyte; KB_B01 to KB_B10, BA1, BA2, KB_BC, murine transferrin (mTf), and human transferrin (hTf), with a contact time of 150 s and 10 μL / s. After each cycle, the surface was regenerated with 10 mM glycine at pH 2.1 at 30 μL / s for 30 s. Analysis was performed using the Biacore Insight Evaluation package version 4.0.8.20368. The results showed that KB_B01 to KB_B10 all competed with each other for binding to hTfR1, thus forming distinct epitopes (Table 6). The reference antibody BA1 is 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)), competes with BA2, but not with KB_B01 to KB_B10. The control VHH-Fc molecule KB_BC neither blocked any of the other anti-TfR1 molecules nor BA1 and BA2, but completely prevented the binding of mTf and hTf to hTfR1. The KB_BC control indicated that none of the other controls competed with Tf, and thus KB_B01 to KB_B10 have desirable brain transport properties.

[0272] Table 6 - Epitope binning of dimeric VHH-Fc fusions and reference antibodies using SPR

[0273]

[0274] B = Block, - = Non-block, X = Autologous competition (against itself). Squares represent the KB_B01 to KB_B10 epitopes.

[0275] To demonstrate functionality when fused to non-Fc proteins, KB_B01 and the reference BV were produced as fusions with single-chain variable domains (VH-VL type scFv with the heavy chain at the amino terminus and the light chain at the carboxyl terminus) located at the amino terminus (scFv-KB_B01) or carboxyl terminus (KB_B01-scFv), see Examples IV and Figure 2 .. To verify that these fusion proteins still bind to hTfR1, the binding kinetics were studied by SPR using an 8K Biacore. Briefly, the receptor hTfR1 was immobilized onto a CM5 chip (Cytiva) by amine coupling and exposed to increasing concentrations of scFv-KB_B01 or KB_B01-scFv in the range of 0.25 to 64 nM in 4-fold increments using single-cycle kinetics. Similarly, the reference VHH BV was produced as a similar fusion protein; scFc-BV and BV-scFv, respectively, and tested in the same experiment but with a slightly wider concentration range from 0.08 to 50 nM in 5-fold increments. The kinetic binding data and sensorgrams are presented in Table 7 and Figure 7A-7D .. Thus, it can be concluded that KB_B01 can be conjugated to another protein at the amino terminus (scFv-KB_B01, K D 1.80 nM) or carboxyl terminus (KB_B01-scFv, K D 0.845 nM) without loss of affinity for hTfR1. In fact, compared to monomeric KB_B01 complexed with hTf (K D 3.31 nM, Table 2), the affinity is slightly higher, indicating that KB_B01 can be used as a BBB transport vehicle fused to therapeutic molecules without loss of function.

[0276] Table 7 - Binding kinetics of fusions of KB_B01 and reference VHH BV with scFv to hTfR1 determined using SPR

[0277] Construct <![CDATA[ka(M -1 s -1 )]]> <![CDATA[kd(s -1 )]]> <![CDATA[K D (M)]]> Rmax (RU) scFv-KB_B01 <![CDATA[1.44×10 5 > <![CDATA[2.59×10 -4 > <![CDATA[1.80×10 -9 > 16.2 KB_B01-scFv <![CDATA[5.35×10 5 > <![CDATA[4.50×10 -4 > <![CDATA[8.45×10 -10 > 21.0 scFv-BV <![CDATA[1.75×10 5 > <![CDATA[1.22×10 -4 > <![CDATA[6.98×10 -10 > 18.5 BV-scFv <![CDATA[6.17×10 5 > <![CDATA[1.17×10 -4 > <![CDATA[1.89×10 -10 > 24.9

[0278] Example VII - TfR1-mediated human cell uptake

[0279] Next, it was tested whether the KB_B01-Fc fusion protein could be internalized in cells expressing human TfR1. Well-known adherent HEK293T cells (ATCC) expressing TfR1 were grown in collagen-coated 96-well plates (ThermoFischer Scientific) at an inoculation density of 30,000 cells / well. The cells were grown in DMEM (Dulbecco's Modified Eagle Medium) for 3 - 5 days until approximately 80 - 90% confluence. KB_B01, as well as the benchmark BV-Fc fusion and the known reference binder BA1 and a negative control (a VHH-Fc-like that does not bind to hTfR1) were used in this assay.

[0280] The cell assay was established based on titrating the test items and the negative control in the range of 0.85 nM to 82.5 nM within 5 - 15 - 30 - 45 minutes at 37 °C and 5% CO2. To compare the cell fate of the added protein after long-term exposure (set here to 120 minutes), after 30 minutes of incubation, the medium was replaced with fresh DMEM and maintained for 90 minutes (incubated at 37 °C and 5% CO2), and then the medium was replaced a second time. After 120 minutes, the cells were brought to the bench and immediately washed with PBS (1×) and fixed with 4% paraformaldehyde for 10 - 15 minutes. After fixation, the cells were washed with PBS and permeabilized with PBS containing 0.1% Triton-X100 TM and then blocked with PBS containing 1% bovine serum albumin (BSA), and then the solution Alexa Fluor-488-anti-human IgG (Fc fragment specific, stock solution 0.75 mg / ml) (Jackson Immunoresearch) was added. During the last 20 minutes of the secondary antibody incubation, Texas Red-phalloidin 300 U (a 1:200 dilution of a 10 μg / mL stock solution) was added to the wells. Thereafter, the cells were washed with PBS at least 3 times and stored at 4 °C until imaging. The cells were imaged in an inverted Zeiss 710 laser scanning confocal microscope using 488, 555, and 647 lasers and a 20× air objective.

[0281] The results of these experiments showed that KB_B01, as well as BV and BA1, were readily taken up by HEK293T cells at various test concentrations (0.7 - 85 nM) and incubation times from 5 to 240 minutes. As Figure 8As shown, KB_B01-Fc was efficiently internalized by cells at 40 minutes and a concentration of 20 nM, similar to BV-Fc. At longer time points, a 120-minute incubation with a 30-minute wash to remove excess test protein from the medium, equal amounts of KB_B01-Fc, BV-Fc, and BA1 were found. The negative control (VHH-Fc, which does not bind to hTfR1) did not accumulate in the cells ( Figure 8 ), or show any presence at any time point or test concentration, indicating that the assay and uptake are dependent on TfR1.

[0282] Example VIII - In Vivo and In Vitro Evaluation of VHH-Fc and scFv Fusions

[0283] To evaluate the in vivo brain targeting properties of KB_B01, KB_B01-Fc was compared to an equivalent VHH-Fc (KB-neg ctrl) lacking mTfR1 binding in wild-type (WT) mice. The scFv-VHH fusions (described in Examples IV, VI, and Table 7) were then compared to the negative control based on the N-terminal vs. C-terminal orientation of the fusion partner. Using the method described below, all fusion proteins were radiolabeled with iodine-125 ( 125 I): Greenwood et al., The preparation of 131 I-labelled human growth hor mone of high specific radioactivity. Biochem J 89:114–123 (1963). An amount of 15 - 80 μg of protein was mixed with 260 ± 26 MBq / μg or 61 ± 2.4 kBq / μg stock 125 I (PerkinElmer Inc, Waltham, MA, USA) and 5 μg chloramine T (Sigma Aldrich) in PBS. The reaction mixture (110 μL) was incubated for 90 seconds and then quenched with 10 μg sodium metabisulfite (Sigma-Aldrich). The radiolabeled samples were immediately purified on a 7 kDa cut-off Zeba column (Thermo Fischer).

[0284] Retention binding to mTfR1 and hTfR1 was verified by ELISA. Briefly, high-binding half-area 96-well plates (Costar 3690, Merck) were coated with 1 - 4 μg / mL of the extracellular domains of hTfR1 and mTfR1 (in PBS pH 7.4) and left overnight at 4 °C. Using 1% BSA 0.05% The PBS from 20 (Merck) was used to block the plate for 1 hour. After washing (with PBS wash buffer), the radiolabeled sample (or the original sample) was incubated for 2 hours or overnight (at 4 °C) in a dilution buffer (PBS - 20 with 0.05%) at serial dilutions of 1:5, then washed 4 times and incubated with a secondary antibody for detecting VHH (anti-VHH secondary antibody (A01861, Genscript Biotech Corporation)) at a dilution of 1:6000. All reported experiments confirmed the binding to the targets (mTfR1, hTfR1).

[0285] In vivo experiments were conducted in 12-week-old C57BL / 6 mice. Additionally, to study the amyloid-β retention of the bispecific VHH-scFv fusion with amyloid-β-binding 3D6 scFv, an APP NL-GF AD model (Nilsson P et al., ACS Chem. Neurosci. 2014, 5, 7, 499–502) was used, and the mice were 13 - 15 months old. After radiolabeling, the VHH-Fc (5 nmol / kg; 3.4 MBq / nmol) or VHH-scFv (5 nmol / kg; 6.8 ± 0.5 MBq / nmol) fusion protein was immediately administered intravenously to the mice via the tail vein. Blood samples (8 μL) were obtained from the tail vein at 5 minutes, 0.5 hour, 1 hour, 2 hours, 4 hours, and 9 hours after injection. The mice were sacrificed by bleeding from the heart followed by a 2.5-minute cardiac perfusion with 40 mL of NaCl. The sacrifice time points after injection were as follows: for WT mice, 2.5 hours after injection of the VHH-Fc construct; and for WT and APP NL-G-F mice, 2, 6, or 24 hours after injection of the VHH-scFv fusion to study brain retention. Only KB_B01-scFv was used at the 6-hour time point. The brain and major organs were dissected. The blood was separated into plasma and blood cell pellet by centrifugation at 10 000×g for 5 minutes. The brain was divided into the left and right hemispheres. The cerebellum was excised from the left hemisphere, and the remaining tissue in the left hemisphere is hereinafter referred to as the "brain". Then, the radioactivity in the brain, blood fraction, and major organs was measured using a gamma counter (2480WizardTM, Wallac Oy PerkinElmer, Turku, Finland). As Figure 9A shown, at 2.5 hours after injection, compared with the negative control VHH-Fc fusion, the distribution of the dimeric VHH-Fc fusion KB_B01 in the brains of WT mice had a significantly enhanced SUV, indicating an in vivo functional brain targeting mechanism. The blood levels of the non-mTfR1-binding negative control were higher than those of VHH-Fc-fused KB_B01(Figure 9B )。These experiments showed that KB_B01, as an Fc fusion, targeted the ventricles depending on its mTfR1 binding property.

[0286] Next, the fate of the scFv-VHH fusions at 2 hours and 24 hours post-injection was explored using both N-terminal and C-terminal constructs. For KB_B01, N-terminal localization of VHH (KB_B01-scFv) showed good brain concentration at 2 hours (Table 8). Benchmark BV showed a similar relationship between brain concentration direction and total brain uptake at 2 hours (Table 8).

[0287] Table 8 - Brain concentration at 2 hours after injection of VHH-scFv3D6 fusion protein into WT mice

[0288] Protein <![CDATA[% ID / g 脑 > Protein <![CDATA[% ID / g 脑 <!-- 29 -->]]> <![CDATA 125 I]scFv-KB_B01]]> 0.15±0.01 <![CDATA 125 I]KB_B01-scFv]]> 0.52±0.05 <![CDATA 125 I]KB_negative control]]> 0.07±0.01 n / a n / a <![CDATA 125 I]scFv-BV]]> 0.47±0.04 <![CDATA 125 I]scFv-BV]]> 0.94±0.03

[0289] Mean ± SD.

[0290] Next, brain concentrations of WT and APP NL-GF mice were evaluated at 2 hours and 24 hours post-injection. Retention of the protein by binding to amyloid-β is crucial for both the diagnostic and therapeutic efficacy of the active moiety / conjugate. As Figure 10A shown, at 2 hours post-injection, the distribution of BV-scFv in the brain was higher than that of KB_B01-scFv. At 6 hours, only KB-B01 was studied and showed significant brain parenchymal retention in APP NL-GF mice ( Figure 10B ). At 24 hours, both KB_B01 and BV showed lower brain concentrations than at 2 hours ( Figure 10C ). Here, non-specific retention in the brain (not binding to amyloid-β protein) was favorable, which was reflected in the low levels in WT brains, especially in KB_B01-scFv. As shown in Table 9, in WT mouse brains, the detectable level of the radiolabeled fusion protein of KB_B01-scFv was the lowest 24 hours after injection. In addition, at 24 hours, the brain concentration ratio between APP NL-GF mice and WT mice was higher in KB_B01-scFv (17.2 versus 13.4, fold difference between WT and APP NL-GF mice, see Table 9). In addition, looking more closely at the peripheral pharmacokinetics, at 24 hours after injection of BV-scFv, the specific distribution in red blood cells (erythrocytes, i.e., the pellet) was higher than that of KB_B01-scFv ( Figure 10D ).

[0291] Table 9 - Brain concentration at 24 hours after injection of VHH-scFv3D6 fusion protein

[0292]

[0293]

[0294] Mean ± SD.

[0295] In vitro characterization assessment of the Example IX-VHH-scFv fusion

[0296] To study the VHH distribution between blood vessels and brain parenchyma, sagittal cryosections of 20 μm were fixed in 4% paraformaldehyde (PFA) washed with PBS for 10 minutes and blocked with 5% normal goat serum. The sections were incubated with PBS 0.1% 20 for 15 minutes and then incubated overnight at 4 °C with the antibodies rat anti-mouse CD31 (BD, #553370) and rabbit anti-Aβ42 (Agrisera). The next day, the sections were washed with PBS and incubated with goat anti-rat antibody (Alexa 647; Molecular Probes, Thermo Fischer Scientific) and goat anti-rabbit antibody (Alexa 488, MolecularProbes, Thermo Fischer Scientific). The sections were stored in PBS until the same day for the nuclear track emulsion (NTE) procedure (described below).

[0297] NTE was performed according to a previously published protocol (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)). Under darkroom conditions with a safelight, Ilford K5 emulsion (Ilford Photo, Harman Techologies) was melted in a 40 °C water bath and prepared as a 50% solution with MQ-H2O according to the manufacturer's instructions. Brain sections were immersed in the emulsion for 10 s and then air-dried at room temperature for 2 h. The sections were stored in a light-tight box at 4 °C for 4 weeks. Development was performed according to the manufacturer's instructions. After NTE, sections with immunostaining and developed emulsion were visualized, and systemic images were acquired as follows: using a Zeiss Observer Z.1 microscope with ZEN software (Carl Zeiss Microimaging GmbH, Jena, Germany), with wide-field illumination (bright field for NTE) or fluorescence (for CD31), using a filter block with an emission wavelength of 647. Subsequently, the images were overlaid (merged), and NTE spots within vascular structures or within the parenchymal space were quantified based on macroscopic image analysis. The relative parenchymal versus capillary signal (% parenchyma) in NTE images was analyzed for n = 20 images per animal from one animal per group at the median (i.e., most representative) total brain concentration. Quantification was performed using a standardized macro in Fiji (ImageJ) as previously described ( et al., Brain pharmacokinetics of two BBB-penetrating bispecific antibodies of different size, Fluids and Barriers of the CNS 18:26 (2021)).

[0298] As Figure 11A shown, KB_B01-scFv distributed to the brain parenchyma without retention in blood vessels 2 h after injection, superior to the reference BV-scFv. Figure 11A A representative vascular structure outlined with a thin white line is shown to demonstrate the vascular versus parenchymal localization of NTE spots (white dots). Quantification revealed that KB_B01-scFv was preferentially distributed in the brain parenchyma at 78 ± 6%; while BV-scFv was 52 ± 10% ( Figure 11B)。This means that each dose of KB_B01-scFv can reach the target site (epitope) in the brain in a more efficient manner and is most suitable for specifically imaging disease biomarkers in situ in the test constructs for diagnostic PET imaging. For imaging agents used in PET, non-specific retention in blood vessels is undesirable, and better parenchymal targeting is also a priority for therapeutic agents to reach their targets.

[0299] Example X - Generation of Protein A - Binding Antibodies

[0300] Llama VHHs generally do not bind to the protein A - based resin PrismA TM but by changing the amino acids in the framework sequence, PrismA TM binding and thus altering protein A binding, VHHs can be generated. To explore the possibility of enabling protein A to bind to KB_B01 and KB_B03 and fusion proteins containing KB_B01 and KB_B03, these two VHHs were mutated according to a common protein A - binding sequence, which is based on Henry et al., A Rational Engineering Strategy for Designing Protein A - Binding Camelid Single - Domain Antibodies, PLoS One 11(9):e0163113(2016). KB_B01 was mutated from glutamate to lysine at position 75, from aspartic acid to asparagine at position 83, and from 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), as free VHHs without the His6 tag. KB_B12 was also generated as a fusion protein with scFv (SEQ ID NO:85), and the scFv was fused to the His6 tag via a short Gly2 linker (GGHHHHHH, SEQ ID NO:86) so that in addition to purification based on PrismA TM purification, Ni - NTA - based purification can also be performed. A positive control known to bind to protein A (SEQ ID NO:87) was also included.

[0301] The protein was produced by ProteoGenix SAS, France in CHO cells. The cDNA encoding the VHH (and its fusions with scFv) was chemically synthesized, optimized for expression in CHO cells, and subcloned into a Proteogenix proprietary mammalian cell expression vector. The vector was transfected into XtenCHO cells by the XtenCHO transfection protocol. Culture medium with a total volume of 3.5 mL was collected 8 days after transfection and purified using Mabselect PrismA resin (Cytiva). The culture medium was clarified by filtration (0.22 μm) and loaded onto the pre-equilibrated (PBS pH 7.5) PrismA resin. According to the resin manufacturer's instructions, the bound VHH was eluted by changing the pH with citric acid, and the eluate was immediately neutralized by adding 1 M Tris-HCl pH 9.0. The protein concentration was determined by A280 spectrophotometry. Subsequently, the flow-through fraction of the scFv fusion construct was purified by Ni-NTA-based purification method 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 stepwise elution. The protein concentration was determined by A280 nm spectrophotometry and qualitatively and quantitatively analyzed by SDS-PAGE. TM After elution, the protein concentrations of KB_B11, KB_B12, and the scFv fusion KB_B12-scFv were generally low, approximately 5, 8, and 7 μg / mL culture, respectively. Subsequent purification of the scFv fusion protein by Ni-NTS IMAC revealed that there was no substance in the flow-through fraction, demonstrating the functionality of the PrismA resin-based purification for this protein construct. TM The KB_B011, KB_B12, and the fusion of scFv with KB_B12 were purified using PrismA resin. The binding of KB_B011, KB_B12, the fusion of scFv with KB_B12, and a ProA positive control (SEQ ID NO:90) to PrismA was also verified by SPR using a pre-coated chip (PrismA chip, Cytiva).

[0302] PrismA TM After elution, the protein concentrations of KB_B11, KB_B12, and the scFv fusion KB_B12-scFv were generally low, approximately 5, 8, and 7 μg / mL culture, respectively. Subsequent purification of the scFv fusion protein by Ni-NTS IMAC revealed that there was no substance in the flow-through fraction, demonstrating the functionality of the PrismA resin-based purification for this protein construct. TM The KB_B011, KB_B12, and the fusion of scFv with KB_B12 were purified using PrismA resin. The binding of KB_B011, KB_B12, the fusion of scFv with KB_B12, and a ProA positive control (SEQ ID NO:90) to PrismA was also verified by SPR using a pre-coated chip (PrismA chip, Cytiva).

[0303] PrismA TM After elution, the protein concentrations of KB_B11, KB_B12, and the scFv fusion KB_B12-scFv were generally low, approximately 5, 8, and 7 μg / mL culture, respectively. Subsequent purification of the scFv fusion protein by Ni-NTS IMAC revealed that there was no substance in the flow-through fraction, demonstrating the functionality of the PrismA resin-based purification for this protein construct. TMBinding of the resin. Also included is a negative control VHH with an unoptimized sequence against ProA. The positive and negative controls were produced in E. coli and contain a C-terminal His tag (SEQ ID NO:86). The binding signals of KB_B11, KB_B12, and the positive control reached 1232, 651, and 1193 RU, respectively, at the highest concentration (500 nM), while the binding signal of KB_B12-scFv reached 545 RU at the highest tested concentration (125 nM), confirming the functionality of VHH-mediated binding to PrismA TM resin binding (Table 10). For the negative control, no signal above baseline was recorded.

[0304] Table 10. PrismA TM Binding data validation function of supported VHH

[0305] VHH construct Mutation relative to the corresponding WT VHH <![CDATA[Prism A TM Combined]]> <![CDATA[KB_B11(KB_B01 PrismA TM )]]> E75K, D83N, N84S Yes <![CDATA[KB_B12(KB_B03 PrismA TM )]]> D83N Yes KB_B12-scFv D83N Yes

[0306] Example XI - In vivo and ex vivo evaluation of VHH-Fc of KB_B03 in human extracellular domain of transferrin receptor 1 humanized mice (hECD-TfR1-mice)

[0307] To evaluate the in vivo brain targeting properties of KB_B03, the distribution properties of KB_B03 were tested in homozygous, heterozygous, and wild-type genotype hECD-TfR1 mice. Homozygous mice express only a partially humanized TfR1, i.e., a human-mouse chimeric TfR1. Taconic Biosciences GmbH, Leverkusen, Germany generated humanized mice expressing a chimeric receptor that contains the extracellular domain of hTfR1 and the intracellular and transmembrane domains of mTfR1.

[0308] Taconic Biosciences GmbH, Leverkusen, Germany generated a genetically engineered mouse model in which the endogenous mouse Tfrc gene was partially humanized such that the engineered mice produce a chimeric TFRC protein containing the extracellular domain of human TFRC. Using methods known to those skilled in the art, a targeting vector was assembled using BAC-derived mouse and human genomic DNA fragments and including a standard selection cassette.

[0309] 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. Morphologically distinct ES cell colonies (ES clones) were isolated and analyzed by Southern blotting and / or PCR. Homologous recombination and single integration at the 3' and 5' sites were confirmed by PCR and Southern blotting using conventional methods known to those skilled in the art. The homologous recombinant ES cell clones were amplified and cryopreserved in liquid nitrogen.

[0310] Generation of chimeras and generation of heterozygotes: After hormone administration, superovulated BALB / c females were mated with BALB / c males. Blastocysts were isolated from the uterus at dpc 3.5 and placed in a drop of DMEM containing 15% FCS (fetal bovine serum) under mineral oil. 10 - 15 targeted ES cells were injected into each blastocyst using a microinjection pipette. After recovery, approximately 8 injected blastocysts were transferred to each uterine horn of pseudopregnant NMRI females. The degree of chimerism in the chimeras (F0) was evaluated by observing the contribution of ES cells to the coat color of the BALB / c host (black / white). In vitro fertilization was performed using oocytes from superovulated C57BL / 6NTac females and thawed sperm from previously cryopreserved sperm of male chimeras. The fertilized embryos were then transferred into pseudopregnant Swiss Webster recipient females. Germline transmission was first identified by the presence of black-furred offspring (strain C57BL / 6NTac) and confirmed by genotyping the black offspring by PCR. F1 mice heterozygous for the modified Tfrc allele were mated to subsequently generate homozygous (HOM), heterozygous (HET), and wild-type (WT) genotypes for the following studies.

[0311] As described in Example VIII, using the chloramine T method, with iodine-125 ( 125I) Radioactively label KB_B03-Fc. In vivo experiments were conducted in 6 - 10-week-old C57BL / 6 mice. After radioactive labeling, KB_B03-Fc (5 nmol / kg; 3.4 MBq / nmol) was immediately administered intravenously to the mice via the tail vein. Blood samples (8 μL) were obtained from the tail vein at 5 minutes, 0.5 hour, 1 hour, and finally at 2 hours after injection. At 2 hours after injection, the mice were euthanized by cardiac puncture followed by a 2.5-minute cardiac perfusion with 40 mL of NaCl. The brain and major organs were dissected. Blood was separated into plasma and blood cell pellet by centrifugation at 10,000×g for 5 minutes. The brain was divided into the left and right hemispheres. The cerebellum was excised from the left hemisphere, and the remaining tissue of the left hemisphere is hereinafter referred to as the "brain". Then, a g-counter (2480WizardTM, Wallac OyPerkinElmer, Turku, Finland) was used to measure the radioactivity in the brain, blood fractions, and major organs. As Figure 12A and 12B shown, the blood distribution profile of hECD-TfR1 mice showed that compared with HET mice and WT mice, the blood level of HOM mice decreased faster at 5 minutes to 30 minutes, while the blood levels were similar at 30 minutes to 120 minutes. Blood compartment analysis showed that due to the slightly higher affinity of KB_B_03 for hTfR1 than for mTfR1, the binding rate of HOM mice to the pellet was slightly higher (Example VIII, Table 5). Normalized brain uptake ( Figure 12C ) indicated that due to the cross-specificity property of KB_B03, the uptake rates of all genotypes were high, as expected. The results showed that KB_B03 has the ability to target the brain in functional hECD-TfR1 in vivo.

[0312] Table 11 - Sequence Information

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323] The above-described embodiments are to be understood as some illustrative embodiments of the present invention. Those skilled in the art should understand that various modifications, combinations, and changes can be made to the embodiments without departing from the scope of the present invention. Specifically, where technically possible, different partial solutions in different embodiments can be combined in other configurations. However, the scope of the present invention is defined by the appended claims.

Claims

1. A variable heavy chain only (VHH) antibody that specifically binds to transferrin receptor 1 (TfR1), the antibody comprising: Complementary determining region 1 (CDR1), which consists 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; CDR2, which consists of the amino acid sequence FTX6X7GX8T 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; and CDR3, which is composed of the amino acid sequence X9X 10 LX 11 X 12 and X9 is Y or H, X 10 is F or Y, X 11 is D or G, and X 12 is V, N or D.

2. The VHH antibody according to claim 1, wherein the CDR1 consists of the amino acid sequence GTX2FX3X4NY defined in SEQ ID NO:2, where X2 is P or D, X3 is S or T, and X4 is N, L or M; and The CDR3 consists of the amino acid sequence X9FLX 11 X 12 where X9 is Y or H, and X 11 is D or G, and X 12 is V, N or D.

3. The VHH antibody according to claim 2, wherein the CDR1 consists of the amino acid sequence GTDFSX4NY defined in SEQ ID NO:3, where X4 is L or M; the CDR2 consists of the amino acid sequence FTX6X7GST defined in SEQ ID NO:4, where X6 is S or A, and X7 is S or T; and the CDR3 consists of the amino acid sequence HFLGD defined in SEQ ID NO:

5.

4. The VHH antibody according to claim 3, wherein the CDR1 consists of the amino acid sequence GTDFSLNY defined in SEQ ID NO:6; the CDR2 consists of the amino acid sequence FTAX7GST defined in SEQ ID NO:7, where X7 is S or T; and the CDR3 consists of the amino acid sequence HFLGD defined in SEQ ID NO:

5.

5. The VHH antibody according to claim 4, wherein 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; and the CDR3 consists of the amino acid sequence HFLGD defined in SEQ ID NO:

5.

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

5.

7. The VHH antibody according to claim 3, wherein 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; and the CDR3 consists of the amino acid sequence HFLGD defined in SEQ ID NO:

5.

8. The VHH antibody according to claim 2, wherein the CDR1 consists of the amino acid sequence GTX2FX3X4NY as defined in SEQ ID NO:2, wherein X2 is P or D, X3 is S or T, and X4 is N, L or M; the CDR2 consists of the amino acid sequence FTX6X7GX8T as defined in SEQ ID NO:1, wherein X6 is S or T, X7 is G, A, H or S, and X8 is S, N or D; and The CDR3 consists of the amino acid sequence YFLDX defined in SEQ ID NO:12 12 wherein X 12 is V or N.

9. The VHH antibody according to claim 8, wherein the CDR1 consists of the amino acid sequence GTPFX3NNY as defined in SEQ ID NO:13, wherein X3 is S or T; the 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; and the CDR3 consists of the amino acid sequence YFLDV as defined in SEQ ID NO:

15.

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

15.

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

15.

12. The VHH antibody according to claim 8, wherein the CDR1 consists of the amino acid sequence GTDFSX4NY as defined in SEQ ID NO:3, wherein X4 is L or M; the CDR2 consists of the amino acid sequence FTX6X7GX8T as defined in SEQ ID NO:1, wherein X6 is S or T, X7 is H or S, and X8 is N or D; and the CDR3 consists of the amino acid sequence YFLDN as defined in SEQ ID NO:

20.

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

20.

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

20.

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

25.

16. The VHH antibody according to any one of claims 1 to 15, wherein the VHH antibody has the following 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, X 15 is L or V, X 16 is A or T, X 17 is A, T or V, and X 18 is A, L or V; FR2 has the amino acid sequence X defined in SEQ ID NO:27 19 RWYRQAPG X 20 QREX 21 VAG, where X 19 is M or L, X 20 is K or N, and X 21 is W or F; FR3 has the amino acid sequence NYX defined in SEQ ID NO:28 20 DSX 21 KG RFTIX 22 RDNAX 23 X 24 TVYLQMDX25LX 26 PEDTAVYX 27 C, where X 20 is A, P or G, X 21 is V or M, X 22 is S or G, X 23 is E or K, X 24 is G or N, X 25 is N or S, X 26 is K, I or T, and X 27 is Y or F; and FR4 has the amino acid sequence WGQGTQVTVSS defined in SEQ ID NO:

29.

17. The VHH antibody according to claim 1, wherein the VHH antibody has an amino acid sequence selected from the group consisting of SEQ ID NO:30 to 37.

18. A heavy chain variable domain only VHH antibody that specifically binds to transferrin receptor 1 TfR1, the antibody comprising: Complementary determining region 1 (CDR1), which consists of the amino acid sequence DSAFX defined in SEQ ID NO:38 28 composed of MNT, where X 28 is S or N; CDR2, which consists of the amino acid sequence IVSDDNT defined in SEQ ID NO:39; and CDR3, which consists of the amino acid sequence KGDVV defined in SEQ ID NO:

40.

19. The VHH antibody according to claim 18, wherein 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; and the CDR3 consists of the amino acid sequence KGDVV defined in SEQ ID NO:

40.

20. The VHH antibody according to claim 18, wherein 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; and the CDR3 consists of the amino acid sequence KGDVV defined in SEQ ID NO:

40.

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

29.

22. The VHH antibody according to claim 18, wherein the VHH antibody has an amino acid sequence selected from the group consisting of SEQ ID NO:46 to 47.

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

24. The VHH antibody according to any one of claims 1 to 23, wherein the VHH antibody specifically binds to murine TfR1.

25. The VHH antibody according to any one of claims 1 to 24, wherein the VHH antibody 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 camelid 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 the VHH antibody according to any one of claims 1 to 28 linked to at least one molecule.

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

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

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

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

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

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

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

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

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

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