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] This invention relates to heavy chain-only variable domain (VHH) antibodies capable of binding to transferrin receptors, and the use of such VHH antibodies to transport molecules across the blood-brain barrier to relevant targets in the brain. Background Technology

[0002] Drug exposure to the brain for central nervous system (CNS) diseases is inherently difficult 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 within the brain. Some small molecules are also excluded from the ventricles due to their properties, such as lack of lipophilicity or substrates for efflux pumps. Generally, compounds with a molecular weight less than 600 Daltons (Da) can cross the BBB without the repulsion of other forces. Larger molecules (such as proteins) cannot easily enter the brain in large quantities without specific active transport aids. For therapeutic antibodies and protein-based drugs, it is estimated that less than 0.1% of systemically injected therapeutic antibodies reach the ventricles. Several strategies to overcome this formidable barrier have been tested and evaluated.

[0003] The brain's endothelial cell block (BBB), composed of brain endothelial cells (BEC), is the first barrier to entry into the brain. Other cells in the so-called neurovascular unit (NVU) are also important for the transport and interaction of target cells within the brain parenchyma. In the human brain, the total span of cerebral blood vessels is approximately 20 meters. 2 This represents a large surface area, which can provide opportunities for brain exposure to circulating therapeutic agents.

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

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

[0006] Receptor-mediated transcytosis is a natural mechanism that utilizes endogenous receptors expressed on the surface of the BBB lumen. It has been reported to successfully increase brain exposure to therapeutic molecules and has demonstrated clinical efficacy and safety.

[0007] WO 2020 / 144233 discloses a camelid heavy chain variable domain (VHH) molecule that binds to the transferrin receptor (TfR), and its use in delivering molecules of 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 gastrointestinal tract. These TfR-specific binding moieties include single-domain nurse shark neoantigen receptor variable domain (VNAR) antibodies that bind to TfR.

[0009] WO 2016 / 081643 relates to anti-transferrin receptor antibodies and their methods of use.

[0010] Effective therapeutic molecular transporters are still needed to reach the brain. These transporters should be able to conjugate or fuse with therapeutic or diagnostic molecules in a manner that does not affect the transporter's target binding or the therapeutic efficacy of the therapeutic molecule. Summary of the Invention

[0011] The overall goal is to provide VHH molecules that are specific to transferrin receptor 1 and can efficiently transport cargo to the desired compartment via receptor-mediated transcytosis.

[0012] The embodiments of the present invention satisfy these and other objectives.

[0013] This invention is defined in the independent technical solution. Other embodiments of this invention are defined in the dependent technical solutions.

[0014] 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 a complementarity-determining region 1 (CDR1) having an amino acid sequence selected from the group consisting of GSIFGSKR as defined in SEQ ID NO:1 and GSIFGFNA as defined in SEQ ID NO:2. The VHH antibody further comprises a CDR2 having an amino acid sequence selected from the group consisting of ITYRGTT as defined in SEQ ID NO:3 and IAVAGST as defined in SEQ ID NO:4. The VHH antibody further comprises a CDR3 having an amino acid sequence selected from the group consisting of WMFTTDNY as defined in SEQ ID NO:5 and WMYATANY as defined in SEQ ID NO:6. If CDR1 has an amino acid sequence as defined in SEQ ID NO:2, then CDR3 has an amino acid sequence as defined in SEQ ID NO:6. VHH antibodies do not contain CDR1 having the amino acid sequence as defined in SEQ ID NO:2, CDR2 having the amino acid sequence as defined in SEQ ID NO:4, or CDR3 having the amino acid sequence as defined in SEQ ID NO:6.

[0015] Another aspect of the present invention relates to a VHH antibody that specifically binds to TfR1. The VHH antibody comprises CDR1 consisting of the amino acid sequence X1X2IX3GSKR as defined in SEQ ID NO:7, wherein X1 is G or E, X2 is S, D, or I, and X3 is F or N. The VHH antibody further comprises CDR2 consisting of the amino acid sequence ITX4X5GTT as defined in SEQ ID NO:8, wherein X4 is Y or V, and X5 is R, H, or G. The VHH antibody further comprises CDR3 consisting of the amino acid sequence WMFTTX6NY as defined in SEQ ID NO:9, wherein X6 is D, T, or N.

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

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

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

[0019] Other aspects of the present invention relate to nucleic acid molecules encoding the above-mentioned VHH antibody or fusion molecule, expression vectors containing the above-mentioned nucleic acid molecules operatively linked to a promoter, and host cells containing the above-mentioned nucleic acid molecules or expression vectors.

[0020] The VHH antibody of this 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 optimally suited for transcytosis by endothelial cells at the BBB. Therefore, the VHH antibody can serve as a transporter for various molecules (including therapeutic or diagnostic imaging agents) to enter the ventricles during systemic administration. Attached Figure Description

[0021] The following description, together with the accompanying drawings, best illustrates the implementation scheme and its additional objectives and advantages:

[0022] Figure 1. Production strategy of TfR1 combined with VHH

[0023] A graphic illustration of immunization of llamas with an immunogen (1A). Based on ModiPhage, Modiquest Research BV (the Netherlands). TMMethods: Llamas (N=2) were immunized. Primary immunization was performed on day 1 using the extracellular domain of human transferrin receptor 1 (hTfR1) (500 μg protein + complete Freund's adjuvant (CFA), intramuscularly). A booster dose of mouse TfR1 (mTfR1) (500 μg protein + incomplete Freund's adjuvant (IFA), intramuscularly) was administered on day 21, and a booster dose of hTfR1 was administered on day 42. Peripheral blood was collected on day 54, and the immune response was studied using enzyme-linked immunosorbent assay (ELISA) to test the presence of immunoglobulin G1 (IgG1) and IgG2 / 3 antibodies and their responsiveness to mTfR1 and hTfR1. Repeat immunizations were performed on day 86 (hTfR1 + IFA) and day 107 (hTfR1 + IFA). Fresh peripheral blood samples were analyzed on day 117. An additional pre-harvest booster dose of a 1:1 mixture of hTfR1 and mTfR1 (250 μg + 250 μg + IFA) was given on day 120, followed by harvesting peripheral blood lymphocytes (PBL) on day 124.

[0024] A graphical overview of the phage library construction (1B). This was achieved using Ficoll-Paque. TM PLUS ~ 1.5 x 10 9 PBLs were isolated by density gradient centrifugation. RNA was extracted and then reverse transcribed into complementary deoxyribonucleic acid (cDNA). The cDNA was then 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), followed by a second nested PCR reaction to amplify and isolate the VHH library (as described in Pardon et al., A general protocol for the generation of Nanobodies for structural biology. Nat Protoc 9:674–693 (2014)). After vector digestion, DNA amplification was performed as described above, using a total of 1600 ng of DNA to electroporate TG1 E. coli, yielding an estimated size of 3.2 × 10⁻⁶. 8 The library.

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

[0026] Figure 2This diagram illustrates VHH monomers and various VHH-containing fusion proteins. Free VHH binds as a monomer to a binding site on TfR1. Bivalent VHH fusion proteins are formed between two VHHs and a crystallizable (Fc) region of a human fragment. Functional monovalent fusion proteins are generated between a VHH and a single-chain variable fragment (scFv) or between a VHH and a non-antibody-derived molecule (X). Figure 2 A bivalent VHH fusion protein with two non-antibody-derived molecules (X) was also shown.

[0027] Figure 3. Affinity of monomer VHH to hTfR1 measured using SPR.

[0028] This figure shows representative sensory mappings of KB_A01 (3A) binding to hTfR1 loaded on human transferrin (hTf) at progressively increasing concentrations from 6.25 to 100 nM, the transferrin being immobilized via amine coupling on a dextran-coated gold (CM5) chip (Cytiva). As a control, the full-length monoclonal antibody BA1 (3B) was run at the same concentration.

[0029] Figure 4. Affinity of the dimer VHH-Fc protein to hTfR1 measured using SPR.

[0030] This figure shows representative sensory maps of the binding of hTfR1 at concentrations ranging from 0.16 to 100 nM to the Fc fusion KB_A01 (4A) loaded on a protein A-coated chip (Cytiva). As controls, the full-length monoclonal antibody BA2 (4B) and the baseline VHH-Fc BV (4C) were also tested. KB_A01, BA2, and BV were also tested for mTfR1, with only BV binding with detectable affinity (4D).

[0031] Figure 5. Affinity measurements of dimer KB_A01-Fc with hTfR1 and cTfR1 in the absence and presence of hTf.

[0032] This figure shows representative sensory maps of the KB_A01-Fc fusion protein binding to hTfR1 (5A-5B) or cynomolgus monkey TfR1 (cTfR1) (5C-5D) in the absence of (5A and 5C) and in the presence of (5B and 5D) excess concentrations of 250 nM hTf.

[0033] Figure 6. Measurement of the affinity of the benchmark antibody to hTfR1 in the absence and presence of hTf.

[0034] This figure shows representative sensor plots of the binding of baseline antibody BA2 (6A-6B) or baseline antibody BA1 (6C-6D) to hTfR1 in the absence of (6A and 6C) and in the presence of (6B and 6D) excess concentrations of 250 nM hTf.

[0035] Figure 7 Sequence alignment of sequences closely related to KB_A01

[0036] The figure displays the amino acid sequences of KB_A01 (SEQ ID NO:23) and three closely related clones KB_A09 to KB_A11 using single-letter codes. Dots indicate amino acids identical to the reference sequence (KB_A01), and boxes indicate the three complementarity-determining regions (CDRs).

[0037] Figure 8. Binding to the apex and extracellular domain of hTfR1 measured by ELISA.

[0038] The apical domain of hTfR1 was expressed using M13 phage as a stable unit and was used to determine apical binding of KB_A01, the baseline antibody BA2, and the baseline VHH-Fc BV, as well as the absence of BV-Fc (8A). The system also assayed variants of KB_A01, with representative clones demonstrating binding to the apical domain (8B). Validation ELISA using the soluble extracellular domain (without M13 phage) was performed on representative clones or the baseline antibody, showing full-length IgG at 2.5 μg / mL and VHH-Fcs at 1.25 μg / mL (8C).

[0039] Figure 9. Affinity of VHH and scFv fusion proteins to hTfR1 measured using SPR.

[0040] Human TfR1 was coupled to an amine on the CM5 chip surface and exposed to KB_A01 fused to the C-terminal (VHH-scFv, 9A) or N-terminal (scFv-VHH, 9B) scFv gene. Exposure was increased in 1:4 increments over a concentration range of 0.25 to 64 nM.

[0041] Figure 10. HEK293T cells undergoing cell uptake via TfR1

[0042] HEK293T cells were cultured in 96-well plates until confluence (4–5 days). Test compounds (KB_A01-Fc, BA1, BA2, and negative unbound VHH-Fc, negative control) were diluted at 3.3–20 nM in DMEM and incubated for 15 or 45 minutes, washed with PBS, and fixed in 4% PFA (10A, 10B). For longer incubation times (10C), the medium was changed twice with fresh DMEM; once at t=30 minutes and again at t=120 minutes. After 15, 40–45, or 240 minutes of incubation, cells were immunostained and analyzed using confocal microscopy. Representative images of cell uptake after 40 minutes of incubation at 20 nM for KB_A01-Fc, BA1, and negative control are shown here (10A), or representative images of KB_01-Fc and BA1 after 15 and 45 minutes of incubation at 3.3 nM (10B). The cultures of KB_A01-Fc, BA1, BA2, and the negative control were displayed in reverse grayscale (10C) at 240 minutes. The results at t=240 minutes showed that BA1 had significantly less signal (intracellular presence) at this time point compared to BA2 and KB_A01-Fc, indicating a higher degree of intracellular degradation. Figure 10A and 10C No signal was observed when using a non-hTfR1-bound VHH-Fc negative control. Images are displayed as 8-bit grayscale (10A, 10B) or inverted grayscale (10C). Scale bar 50 μM.

[0043] Figure 11 Transcytosis of brain-like endothelial cells in an in vitro BBB model

[0044] like As described in *Transport study of interleukin-1 inhibitors using a human in vitro model of the blood-brain barrier*, *Brain Behaviour*, *Immunity Health* 16:100307 (2021), in vitro BBB transwell assays were performed using brain-like endothelial cells. The test compound (500 nM in physiological buffer) was added to donor wells, and compartments (n = 3 independent wells per sample) were harvested at 180 minutes. Cells were lysed, and the contents of all compartments were analyzed using an ELISA reactive to human Fc. The control IgG used was a known anti-IL1β antibody. All assays were reactive to human Fc and analyzed against their own standard curves and IgG standards on the same sample plate. KB_A01-Fc showed higher transcytosis capacity than the control IgG antibody and the baseline antibody BA2.

[0045] Figure 12. PrismA TM Cause KB_A01 to mutate

[0046] The figure shows that KB_A01, KB_A12, and positive control 1 all carry His tags produced in E. coli, purified using IMAC (Ni-NTA), and run on SDS-PAGE (12A). Subsequently, protein A-based resin (PrismA) was used. TM IMAC purification material was purified using resin. The acid elution fraction (12B) and flow fraction (12C) were analyzed by SDS-PAGE. The comparison of KB_A01 (12D) and KB_A12 (12E) with PrismA on a pre-fixed chip (Cytiva) was also performed by SPR in the concentration range of 1.95 to 500 nM. TM The combination of.

[0047] Figure 13. Brain and blood distribution after systemic administration of VHH-Fc fusion compound KB_A01 to humanized mice with the extracellular domain of TfR1.

[0048] As described in Example XI, the radioactively labeled [ 125 I]VHH-Fc fusion protein was injected into human extracellular domain chimeric TfR1 (hECD-TfR1) mice with homozygous (HOM), heterozygous (HET), and wild-type (WT) genotypes. Blood concentrations (13A) are shown in blood samples collected at t=5 min, t=30 min, t=1 h, and finally (2.0 h). Two hours after injection, animals were euthanized, perfused with NaCl via the heart, and the brain was removed for radioactive analysis, showing blood chamber and brain concentrations at 2.0 h (13B, 13C). Blood concentrations are expressed as (% of injected dose, radioactivity), while brain uptake is expressed as normalized uptake values ​​SUV (% of injected dose corrected for animal body weight). Detailed Implementation

[0049] This invention relates to heavy chain variable domain (VHH) antibodies, also known as single-domain antibodies, particularly VHH antibodies capable of binding to transferrin (Tf) receptor 1 (TfR1), and the use of such VHH antibodies to transport molecules across the blood-brain barrier (BBB).

[0050] Drug exposure to the brain is inherently challenging for drugs targeting central nervous system (CNS) diseases because the brain's blood-brain barrier (BBB) ​​protects it from unwanted substances present in the peripheral circulation, including antibodies, proteins, and other molecules that may have therapeutic effects within the brain. Receptor-mediated transcytosis, a natural mechanism utilizing endogenous receptors expressed on the surface of the BBB lumen, is thought to increase drug exposure in the BBB. One endogenous receptor that can be used to achieve receptor-mediated transcytosis is the transferrin receptor.

[0051] The transferrin receptor is the carrier protein of transferrin, a glycoprotein that binds to plasma and mediates the transport of iron (Fe) in plasma. The transferrin receptor internalizes the transferrin-iron complex via receptor-mediated endocytosis, thereby introducing iron. In humans and other mammals, there are two types of transferrin receptors: transferrin receptor 1 (TfR1) and transferrin receptor 2 (TfR2). TfR1 is a high-affinity, universally expressed receptor, while TfR2 expression is limited to certain cell types and is unaffected by intracellular iron concentration. TfR2 has a 25-30 fold lower affinity for transferrin than TfR1. The transferrin receptor used in this article refers to the TfR1 homolog, also known as differentiation cluster 71 (CD71), which is encoded by the human TFRC gene.

[0052] The extracellular domains of human TfR1 (hTfR1) and mouse TfR1 (mTfR1) were used to immunize llama animals and create a clonal library expressing VHH antibodies. These VHH antibodies were screened for binding to hTfR1, and a key VHH antibody KB_A01 with the desired binding properties was selected. Further VHH antibodies were generated by modifying selected CDRs and identifying VHH antibodies with sequences similar to KB_A01. The VHH antibodies of this invention specifically bind to hTfR1 and also to cynomolgus monkey TfR1 (cTfR1), and their binding characteristics are suitable for transcytosis, including binding affinity, non-interference with transferrin binding sites, and binding to the apical domain of hTfR1. As shown in the experimental section, these VHH antibodies retain their TfR1 binding characteristics when presented as VHH-containing fusion proteins, including bivalent VHH fusion proteins formed between two VHH antibodies and the crystallizable region (Fc) of a human constant antibody fragment, as well as functional fusion proteins fused with drug molecules, such as single-chain variable fragments (scFv). Experimental data further demonstrate that VHH antibodies (including VHH-containing fusion proteins) are taken up by human cells expressing hTfR1 and transcytose on a monolayer of human brain-like endothelial cells used as an in vitro BBB model.

[0053] Compared to the VHH antibodies disclosed in WO 2020 / 144233, the VHH antibody of the present invention has further advantages. First, compared to the prior art VHH antibodies disclosed in Example VIII, the VHH antibody of the present invention binds to a different epitope on hTfR1. The epitopes bound by the prior art VHH antibodies are not within the so-called apical domain of hTfR1 bound by the VHH antibody of the present invention. Binding to the apical domain of the extracellular domain of TfR1 is more advantageous for BBB delivery and receptor-mediated transcytosis than binding to helical or protease-like domains, because these domains do not interfere with transferrin binding (Daniels et al., The transferrin receptor part I: Biology and targeting with cytotoxic antibodies for the treatment of cancer. Clin Immunol. 2006; 121:144–158 (2006); WO 2016 / 081643). Furthermore, the binding of the VHH antibody of the present invention to the apical domain of TfR1 does not interfere with the binding of all-transferrin to TfR1, see Figure 5 and Table 5. Another significant advantage of the VHH antibody of the present invention compared to the VHH antibody disclosed in WO 2020 / 144233 is that the VHH antibody of the present invention has binding properties in terms of both the range of affinity for hTfR1 and its release from hTfR1, thereby allowing efficient passage across the BBB and a high relative uptake rate in the brain. The VHH antibodies disclosed in WO 2020 / 144233 have a significantly higher affinity for hTfR1, which makes them more susceptible to lysosomal degradation during endocytosis within BBB endothelial cells rather than crossing the BBB. Therefore, the VHH antibody of WO 2020 / 144233 may be trapped in endothelial cells and eventually degraded therein. Another significant advantage of the VHH antibody of the present invention compared to the VHH antibody disclosed in WO 2020 / 144233 is that the affinity of the VHH antibody of the present invention for hTfR1 does not change significantly in the presence or absence of human transferrin (hTf). However, as shown in Table 5, the affinity of the VHH antibody disclosed in WO 2020 / 144233 for hTfR1 is significantly reduced in the presence of hTf compared to the absence of hTf. This dependence of the affinity for hTfR1 on hTf concentration makes it difficult to select the appropriate amount of VHH antibody required to achieve the desired receptor-mediated transBBB transcytosis.

[0054] Therefore, 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 a complementarity-determining region 1 (CDR1) having an amino acid sequence selected from the group consisting of GSIFGSKR as defined in SEQ ID NO:1 and GSIFGFNA as defined in SEQ ID NO:2. The VHH antibody further comprises a CDR2 having an amino acid sequence selected from the group consisting of ITYRGTT as defined in SEQ ID NO:3 and IAVAGST as defined in SEQ ID NO:4. The VHH antibody further comprises a CDR3 having an amino acid sequence selected from the group consisting of WMFTTDNY as defined in SEQ ID NO:5 and WMYATANY as defined in SEQ ID NO:6. According to the present invention, if CDR1 has an amino acid sequence as defined in SEQ ID NO:2, then CDR3 has an amino acid sequence as defined in SEQ ID NO:6. However, the VHH antibody does not contain CDR1 having the amino acid sequence as defined in SEQ ID NO:2, CDR2 having the amino acid sequence as defined in SEQ ID NO:4, or CDR3 having the amino acid sequence as defined in SEQ ID NO:6.

[0055] The VHH antibodies in this regard are generated based on CDR shuffling between KB_A01 and VHH antibodies in the CDR containing several alanine mutations compared to KB_A01. The strategy involves treating the VHH antibody containing the alanine mutation in the CDR (referred to herein as KB_ref) as a naturally occurring alanine scan, but replacing the entire CDR with individual amino acid residues from KB_A01. In ELISA analysis, KB_ref was initially identified as an hTfR1 binder. However, when this VHH antibody KB_ref was immobilized with protein A and analyzed using surface plasmon resonance (SPR), it unexpectedly failed to bind to hTfR1. However, several VHH antibodies KB_A03, KB_A04, KB_A06, and KB_A07, obtained through selected CDR shuffling, did indeed bind to VHH with the desired binding properties. This was quite unexpected, because the VHH antibody KB_ref (some of the CDRs in KB_A03, KB_A04, KB_A06 and KB_A07 are derived from the antibody) cannot bind to hTfR1.

[0056] More specifically, the scaffold VHH antibody lacking an alanine residue in the CDR, namely KB_A01, has CDR1 as defined in SEQ ID NO:1, CDR2 as defined in SEQ ID NO:3, and CDR3 as defined in SEQ ID NO:5, while the VHH antibody containing an alanine mutation in the CDR, namely KB_ref, has CDR1 as defined in SEQ ID NO:2, CDR2 as defined in SEQ ID NO:4, and CDR3 as defined in SEQ ID NO:6.

[0057] As shown in Table 7, replacing all CDRs of KB_A01 with the CDR of KB_ref results in the loss of binding to hTfR1 and KB_A08. Therefore, VHH antibodies in this respect do not contain CDR1 having the amino acid sequence defined in SEQ ID NO:2, CDR2 having the amino acid sequence defined in SEQ ID NO:4, and CDR3 having the amino acid sequence defined in SEQ ID NO:6. Furthermore, when the CDR1 of KB_A01 is replaced with the CDR1 of KB_ref, hTfR1 binding is lost unless the CDR3 of KB_A01 is also replaced with the CDR3 of KB_ref, see KB_A02 and KB_A05 in Table 5. Therefore, if the CDR1 of the VHH antibody in this respect has the amino acid sequence defined in SEQ ID NO:2, then the CDR3 has the amino acid sequence defined in SEQ ID NO:6.

[0058] Table 6 summarizes the CDRs of VHH antibodies KB_A01 to KB_A08, while Table 7 shows the binding kinetics of these VHH antibodies in the form of the dimer VHH-Fc fusion protein with hTfR1.

[0059] In one embodiment, the VHH antibody does not contain CDR1 having the amino acid sequence defined in SEQ ID NO:2, CDR2 having the amino acid sequence defined in SEQ ID NO:3 or 4, or CDR3 having the amino acid sequence defined in SEQ ID NO:6. In this embodiment, the VHH antibody does not contain CDR1 having the amino acid sequence defined in SEQ ID NO:2, CDR2 having the amino acid sequence defined in SEQ ID NO:3, or CDR3 having the amino acid sequence defined in SEQ ID NO:6. Furthermore, in this particular embodiment, the VHH antibody does not contain CDR1 having the amino acid sequence defined in SEQ ID NO:2, CDR2 having the amino acid sequence defined in SEQ ID NO:4, or CDR3 having the amino acid sequence defined in SEQ ID NO:6.

[0060] In one embodiment, CDR1 has the amino acid sequence defined in SEQ ID NO:1, CDR2 has the amino acid sequence selected from the group consisting of SEQ ID NO:3 and 4, and CDR3 has the amino acid sequence defined in SEQ ID NO:5.

[0061] In one specific embodiment, CDR1 has the amino acid sequence defined in SEQ ID NO:1, CDR2 has the amino acid sequence defined in SEQ ID NO:4, and CDR3 has the amino acid sequence defined in SEQ ID NO:5.

[0062] In another specific embodiment, CDR1 has the amino acid sequence defined in SEQ ID NO:1, CDR2 has the amino acid sequence defined in SEQ ID NO:3, and CDR3 has the amino acid sequence defined in SEQ ID NO:5.

[0063] In another embodiment, CDR1 has an amino acid sequence selected from the group consisting of SEQ ID NO:1 and 2, CDR2 has an amino acid sequence defined in SEQ ID NO:3, and CDR3 has an amino acid sequence defined in SEQ ID NO:6.

[0064] In one specific embodiment, CDR1 has the amino acid sequence defined in SEQ ID NO:1, CDR2 has the amino acid sequence defined in SEQ ID NO:3, and CDR3 has the amino acid sequence defined in SEQ ID NO:6.

[0065] In another specific embodiment, CDR1 has the amino acid sequence defined in SEQ ID NO:2, CDR2 has the amino acid sequence defined in SEQ ID NO:3, and CDR3 has the amino acid sequence defined in SEQ ID NO:6.

[0066] In another specific embodiment, CDR1 has the amino acid sequence defined in SEQ ID NO:1, CDR2 has the amino acid sequence defined in SEQ ID NO:4, and CDR3 has the amino acid sequence defined in SEQ ID NO:6.

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

[0068] In one embodiment, FR1 has the amino acid sequence QVQLQESGGGSVQAGGSLSLSCAAS as defined in SEQ ID NO:57.

[0069] In one embodiment, FR2 has the amino acid sequence MGWFRQAPGEQRDVVAT as defined in SEQ ID NO:58.

[0070] In one embodiment, FR3 has the amino acid sequence EYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYC as defined in SEQ ID NO:59.

[0071] In one embodiment, FR4 has the amino acid sequence WGQGTQVTVSS as defined in SEQ ID NO:22.

[0072] In one specific embodiment, the VHH antibody has the amino acid sequences of FR1, FR2, FR3 and FR4 as defined in SEQ ID NO:57, 58, 59 and 22.

[0073] In one embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO:23. This type of VHH antibody is referred to herein as KB_A01.

[0074] In another embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO:24. This type of VHH antibody is referred to herein as KB_A03.

[0075] In another embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO:25. This type of VHH antibody is referred to herein as KB_A04.

[0076] In another embodiment, the VHH has the amino acid sequence defined in SEQ ID NO:44. This type of VHH antibody is referred to herein as KB_A06.

[0077] In another embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO:26. This type of VHH antibody is referred to herein as KB_A07.

[0078] In one embodiment, the VHH antibody has an amino acid sequence selected from the group consisting of SEQ ID NO:23 to 26, 44, preferably an amino acid sequence selected from the group consisting of SEQ ID NO:23 to 26.

[0079] Another aspect of the present invention relates to a VHH antibody that specifically binds to TfR1. The VHH antibody comprises CDR1 consisting of the amino acid sequence X1X2IX3GSKR as defined in SEQ ID NO:7, wherein X1 is G or E, X2 is S, D, or I, and X3 is F or N. The VHH antibody further comprises CDR2 consisting of the amino acid sequence ITX4X5GTT as defined in SEQ ID NO:8, wherein X4 is Y or V, and X5 is R, H, or G. The VHH antibody further comprises CDR3 consisting of the amino acid sequence WMFTTX6NY as defined in SEQ ID NO:9, wherein X6 is D, T, or N.

[0080] This aspect of the invention relates to a family of VHH antibodies that have closely related CDR regions and all bind to hTfR1 with high affinity.

[0081] In one embodiment, CDR1 consists of the amino acid sequence X1X2IX3GSKR as defined in SEQ ID NO:7, wherein X1 is G or E, X2 is D or I, and X3 is F or N. In this embodiment, CDR2 consists of the amino acid sequence ITX4X5GTT as defined in SEQ ID NO:8, wherein X4 is Y or V, and X5 is R, H, or G. Furthermore, CDR3 consists of the amino acid sequence WMFTTX6NY as defined in SEQ ID NO:9, wherein X6 is D, T, or N.

[0082] In one specific embodiment, CDR1 consists of the amino acid sequence GDIX3GSKR as defined in SEQ ID NO:11, where X3 is F or N. In this embodiment, CDR2 consists of the amino acid sequence ITVX5GTT as defined in SEQ ID NO:12, where X5 is R or G. Furthermore, CDR3 consists of the amino acid sequence WMFTTX6NY as defined in SEQ ID NO:9, where X6 is T or N.

[0083] In a preferred embodiment, CDR1 consists of the amino acid sequence GDINGSKR as defined in SEQ ID NO:13, CDR2 consists of the amino acid sequence ITVRGTT as defined in SEQ ID NO:14, and CDR3 consists of the amino acid sequence WMFTTTNY as defined in SEQ ID NO:10.

[0084] In another preferred embodiment, CDR1 consists of the amino acid sequence GDIFGSKR as defined in SEQ ID NO:15, CDR2 consists of the amino acid sequence ITVGGTT as defined in SEQ ID NO:16, and CDR3 consists of the amino acid sequence WMFTTNNY as defined in SEQ ID NO:55.

[0085] In another preferred embodiment, CDR1 consists of the amino acid sequence EIINFGSKR defined in SEQ ID NO:17, CDR2 consists of the amino acid sequence ITYHGTT defined in SEQ ID NO:18, and CDR3 consists of the amino acid sequence WMFTTDNY defined in SEQ ID NO:5.

[0086] In one embodiment, the VHH antibody has the following formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0087] In one embodiment, FR1 has the amino acid sequence QVQLQESGGGX7VQAGGSLX8LSCAAS as defined in SEQ ID NO:19, where X7 is S or L and X8 is S or R.

[0088] In one embodiment, FR2 has the amino acid sequence MGWFRQAPGX9X as defined in SEQ ID NO:20. 10 RDX 11 VAT, where X9 is E, K, or Q, X 10 It is either Q or A, and X 11 It can be V or L.

[0089] In one embodiment, FR3 has the amino acid sequence X as defined in SEQ ID NO:21. 12 YX 13 DSVKGRFTISRDNAX 14 NTVYLQMNX 15 LKPEDTAX 16 YYC, where X 12 For E or K, X 13 For A or E, X 14 For K or N, X 15 It is N or S, and X 16 It can be V or F.

[0090] In one embodiment, FR4 has the amino acid sequence WGQGTQVTVSS as defined in SEQ ID NO:22.

[0091] In one specific embodiment, the VHH antibody has the amino acid sequences of FR1, FR2, FR3 and FR4 as defined in SEQ ID NO:19, 20, 21 and 22.

[0092] The present invention also covers VHH antibodies having FR1 amino acid sequences comprising or consisting of SEQ ID NO:19 or 57, 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:19 or 57.

[0093] The present invention also covers VHH antibodies having a FR2 amino acid sequence comprising or consisting of SEQ ID NO:20 or 58, or variants thereof having at least 82% sequence identity with SEQ ID NO:20 or 58, preferably at least 88% sequence identity and more preferably at least 94% sequence identity.

[0094] The present invention also covers VHH antibodies having FR3 amino acid sequences comprising or consisting of SEQ ID NO:21 or 59, 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:21 or 59.

[0095] The present invention also covers VHH antibodies having an FR4 amino acid sequence comprising or consisting of SEQ ID NO:22, 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:22.

[0096] 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: The query sequence is aligned to the target sequence using the CLUSTAL W algorithm. A window corresponding to the shortest sequence in the aligned sequence is compared. In some cases, the shortest sequence in the aligned sequence may be the target sequence. In other cases, the query sequence may constitute the shortest sequence in the aligned sequence. Amino acid residues at each position are compared, and the percentage of positions in the query sequence that have the same correspondence with the target sequence is reported as sequence identity % (%).

[0097] An amino acid sequence having a defined sequence identity percentage of a reference amino acid sequence is preferably obtained through amino acid substitution, such as through conserved amino acid replacement. Conserved amino acid replacement, also known as conserved amino acid substitution or mutation, is an amino acid substitution in an amino acid sequence that changes a given amino acid to a different amino acid with similar biochemical, structural, and / or chemical properties.

[0098] For example, based on the structure and general chemical characteristics of amino acids' side chains (R groups), amino acids can be divided into six major classes:

[0099] • Aliphatic: Isoleucine (I), Leucine (L), Glycine (G), Alanine (A), Valine (V);

[0100] • Containing hydroxyl groups or sulfur / selenium: serine (S), cysteine ​​(C), threonine (T), methionine (M);

[0101] • Cyclic: Proline (P)

[0102] • Aromatic compounds: phenylalanine (F), tyrosine (Y), tryptophan (W);

[0103] • Basic: Histidine (H), Lysine (K), Arginine (R); and

[0104] • Acids and their amides: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q).

[0105] This means that an amino acid sequence having a defined sequence identity % of the reference amino acid sequence is preferably obtained by replacing one or more amino acid residues in the reference amino acid sequence with one or more conserved amino acid substitutions using the corresponding amino acid from the R group listed above that is the same as the given amino acid residue.

[0106] In one embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO:27. This type of VHH antibody is referred to herein as KB A09.

[0107] In another embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO:28. This type of VHH antibody is referred to herein as KB_A10.

[0108] In another embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO:29. This type of VHH antibody is referred to herein as KB_A11.

[0109] In another embodiment, the VHH antibody has the amino acid sequence defined in SEQ ID NO:23. This type of VHH antibody is referred to herein as KB_A01.

[0110] Figure 7 Sequence alignments of closely related VHH antibodies KB_A01, KB_A09, KB_A10, and KB_A11 are shown. Table 7 illustrates the binding kinetics of the VHH antibodies KB_A09, KB_A10, and KB_A11 in the form of the dimer VHH-Fc fusion protein with hTfR1.

[0111] In one embodiment, the VHH antibody has an amino acid sequence selected from the group consisting of SEQ ID NO:23, 27 to 29, preferably an amino acid sequence selected from the group consisting of SEQ ID NO:27 to 29.

[0112] In one embodiment, the VHH antibody has an amino acid sequence selected from the group consisting of SEQ ID NO:23-29 and 44. In a preferred embodiment, the VHH has an amino acid sequence selected from the group consisting of SEQ ID NO:23-29.

[0113] The VHH antibody of this invention specifically binds to TfR1.

[0114] "Specific binding" and similar expressions mean that the molecule in question (e.g., a 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 cohesion. It is determined by the equilibrium dissociation constant (K0) between the antigen and antibody. D Affinity, represented by K, is a measure of the binding strength between an antigenic determinant (i.e., an epitope) and an antigen-binding site on an antibody. D The lower the 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 ), that is, 1 / K D As technicians will know, affinity can be determined in a manner known per se based on the target-specific antigen.

[0115] Typically, antibodies will be in 10 -5 Up to 10 -12 mol / L (M) or lower, and preferably 10 -7 Up to 10 -12 M or lower, and more preferably 10 -8 Up to 10 -12 The equilibrium dissociation constant of M (K) D ), that is, with 10 5 Up to 10 12 M -1 Or larger, and preferably 10 7 Up to 10 12 M -1 or larger and more preferably 10 8 Up to 10 12 M -1 Affinity constant (K) A ) binds to its antigen. Generally speaking, greater than 10 -4 M any K D Value (or below 10) 4 M -1 Any K A Values ​​are considered to indicate nonspecific binding.

[0116] The specific binding of an antibody to an antigen or antigenic determinant can be determined by any suitable method known per se, including, for example, Scatcherd analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competitive assay, surface plasmon resonance (SPR), biolayer interferometry (BLI), and various variations thereof known per se in the art.

[0117] The affinity of VHH antibodies for TfR1 should be tuned to a specific range to achieve efficient BBB crossing and high relative uptake in the brain. Generally, if the antibody's affinity for TfR1 is too low, such as K... D If the antibody affinity is greater than approximately 1 μM, its affinity 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. Conversely, if the antibody affinity is too high, such as K... D At concentrations significantly below 1 nM (<< 1 nM), antibodies are endocytosed but instead undergo lysosomal degradation in BBB endothelial cells. This means that to achieve optimal receptor-mediated endocytosis (i.e., receptor-mediated cellular uptake) and transcytosis (i.e., transcellular transport), VHH antibodies should have an affinity (K0) 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).

[0118] As shown in Table 2, the affinity (K) of the VHH antibody KB_A01 of the present invention in monomeric form is... D The affinity is 4.1 nM, which is within the nM range. Furthermore, when included in VHH-containing fusion proteins, the affinity remains within the desired low nM to nM range (see [link to relevant documentation]). Figure 2 ), for example K D The dimerized-VHH Fc fusion protein is 0.18 nM (see Table 4), or K D The fusion protein with scFv has a concentration of 3.2-3.4 nM (see Table 9). Furthermore, other VHH antibodies of the present invention have an affinity for TfR1 in the low nM to nM range, thus making them suitable for BBB crossing via receptor-mediated endocytosis and transcytosis (see Table 7), wherein the K of the dimer-VHH Fc fusion protein is... D The range is from 0.15 to 5.1 nM.

[0119] In one embodiment, the VHH antibody of the present invention specifically binds to human TfR1 (hTfR1).

[0120] In one embodiment, the VHH antibody of the present invention has an affinity (K0.1) selected in the range of 0.1 to 150 nM, preferably in the range of 0.1 to 100 nM. D ) Specifically binds to hTfR1. In a specific embodiment, the monovalent form of the VHH antibody of the present invention specifically binds to hTfR1, wherein K D Selected from the range of 1 to 150 nM, preferably from the range of 1 to 100 nM, and more preferably from the range of 1 to 50 nM.

[0121] 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 not only specifically binds to hTfR1, but also binds to cTfR1, as shown in Table 5.

[0122] In one embodiment, the VHH antibody of the present invention does not specifically bind to mTfR1.

[0123] Structurally, hTfR1 is a dimeric transmembrane glycoprotein with a large extracellular domain (residues 90-760), an intramembrane region (residues 62-89), and the remaining 61 residues in the cytoplasm. The extracellular domain has three domains: a helical domain (residues 606-760), a protease-like domain (residues 121-183, 384-605), and a apical domain (residues 184-383). The helical domain is responsible for receptor dimerization. Transferrin binds to the helical domain and the protease-like domain.

[0124] In one embodiment, the VHH antibody of the present invention specifically binds to the apical domain of TfR1, such as hTfR1. Binding to the apical domain of the extracellular domain of TfR1 is more advantageous for BBB delivery and receptor-mediated transcytosis than binding to helical or protease-like domains because these domains do not interfere with transferrin binding (Daniels et al., The transferrin receptor part I: Biology and targeting with cytotoxic antibodies for the treatment of cancer. Clin Immunol. 2006; 121:144–158(2006); WO 2016 / 081643). Furthermore, the binding of the VHH antibody of the present invention to the apical domain of TfR1 does not interfere with the binding of holotransferrin to TfR1, see Figure 5 and Table 5. Therefore, the VHH antibody of the present invention does not interfere with the use of TfR1 for transferrin binding to achieve cellular iron uptake. Therefore, in terms of transcytosis capability, binding of the VHH antibody to the apical domain of TfR1 is preferred.

[0125] In one embodiment, the VHH antibody of the present invention is a camel family VHH antibody.

[0126] In another embodiment, the VHH antibody of the present invention is a humanized VHH antibody. For example, the CDR region of the VHH antibody can be transplanted onto the human skeleton. Soler et al., Effect of Humanizing Mutations on the Stability of the Llama Single-Domain Variable Region, Biomolecules 11(2):163(2021), identified several amino acid positions and the N-terminal Gln as promising candidates for converting Camelidae VHH into a human common germline sequence.

[0127] The following shows the sequence alignments between the KB_A01 framework region, the reference human VH (sVH), the universal VHH (uVHH), and the humanized VHH sequence (hVHH), as disclosed in the aforementioned Biomolecules article.

[0128] QVQLQESGGGSVQAGGSLSLSCAAS KB_A01 FR1

[0129] ----VQ----L——-P----R------sVH FR1

[0130] ----V--------P----R---T--uVHH FR1

[0131] ----V-----L——-P----R------hVHH FR1

[0132] MGWFRQAPGEQRDVVAT KB_A01 FR2

[0133] -SV-----KGLEW-SP sVH FR2

[0134] L--------QE-EA—-AuVHH FR2

[0135] L--------QGLEA—-AhVHH FR2

[0136] EYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYC KB_A01 FR3

[0137] Y--------------S---L-----T-RA--------sVH FR3

[0138] Y------------------------T----------------I---uVHH FR3

[0139] Y--------------S---L-----S-RA--------hVHH FR2

[0140] WGQGTQVTVSS KB_A01 FR4

[0141] -----M-----sVH FR4

[0142] -----------UVHH FR4

[0143] -----L-----sVH FR4

[0144] The VHH antibody of the present invention can be humanized by modification of FR1, FR2, FR3 and / or FR4, such as amino acid substitution. The amino acid positions in FR1-FR4 referred to herein are... Figure 7 The amino acid positions in KB_A01 (SEQ ID NO:23) shown.

[0145] For example, the humanization position 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 humanization positions in FR1, or any combination of any two positions, is used to humanize FR1 until all of these positions are humanized. For example, humanized FR1 may be based on SEQ ID NO:57, but Q1 is replaced by E, Q5 by V, S19 by R, or two or all of these amino acids are replaced.

[0146] The humanization position 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 humanization positions in FR2, or any combination of two positions, is used to humanize FR2 until all of these positions are humanized. As an illustrative example, the humanized positions in FR2 could be V37, G44, L45, and W47 or F37, G44, L45, and A47.

[0147] The humanization position in FR3 can be selected from Y58 (the first 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 humanization positions in FR3, or any combination of two positions, is used to humanize FR3 until all of these positions are humanized.

[0148] The humanization site in FR4 can be L109 or M109 (the sixth amino acid position in FR4).

[0149] I78 also has a stabilizing effect on VHH. Therefore, Ile at position 78 stabilizes VHH.

[0150] Example XIV of this article describes the generation and testing of various humanized VHH antibodies based on the FR region of the VHH antibody KB_A01. The positions marked X below can be successfully mutated to humanize the VHH antibody KB_A01 while still maintaining binding to hTfR1.

[0151] FR1 (SEQ ID NO:78):

[0152] X1VQLX2ESGGGX3VQX4GGSLX5LSCAAS

[0153] FR2 (SEQ ID NO:79):

[0154] MGWFRQAPGX6X7X8X9VVAT

[0155] FR3 (SEQ ID NO:80):

[0156] EYADSVKGRFTISRDNX 10 KNTX 11 YLQMNX 12 LX 13 PEDTAVYYC

[0157] FR4 (SEQ ID NO:81):

[0158] WGQGTX 14 VTVSS

[0159] The wild-type FR region of KB_A01 has X1=Q, X2=Q, X3=S, X4=A, X5=S, X6=E, X7=Q, X8=R, X9=D, X 10 =A, X 11 =V,X 12 =N, X 13 =K

[0160] and X 14 =Q.

[0161] Examples of humanized VHH antibodies of the present invention include VHH antibodies having the FR1-FR4 regions according to the above (SEQ ID NO:78-81), including those having the X1-X regions according to the following. 14 VHH antibody of at least one of them and X1-X having the wild-type FR region according to KB_A01 listed above. 14 VHH antibodies in any remaining unmutated individuals:

[0162] X1=E, X2=V, X3=L, X4=P, X5=R, X6=K, X7=G, X8=L, X9=E, X 10 =S,X 11 =I,X 12 =S,X 13 =R and X 14 =L.

[0163] 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, for example, MabSelect PrismA. TM Resin.

[0164] Protein A is a 42 kDa surface protein originally found 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 folded into a triple-helix bundle. Each domain is capable of binding proteins from many mammalian species, most notably IgG. It binds to the heavy chain within the Fc region of most immunoglobulins, and in the case of the human VH3 family, also to the heavy chain within the Fab region. Protein A typically does not bind to camel VHH antibodies or only binds weakly to them.

[0165] Henry KA et al., A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies, PLoS One 11(9):e0163113 (2016), disclosed how to enable non-protein A binding VHH antibodies to bind protein A, see Table 5.

[0166] 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. If X8 is R, then the common FR1 sequence in SEQ ID NO:19 is the protein A binding FR1 region. In a preferred embodiment, the FR1 region of KB_A01 is modified by replacing S with R on amino acid residue 19 to obtain the protein A binding FR1 sequence shown below and in SEQ ID NO:82.

[0167] QVQLQESGGGSVQA G G S L S LSCAAS KB_A01 FR1

[0168] QVQLQESGGGXVQA G G S L X LSCAAS Common FR1

[0169] ------------------R------Protein A binds to FR1

[0170] Furthermore, amino acid residue 59 should be Y, amino acid residue 64 should be K or E, amino acid residue 65 should be G, amino acid residue 66 should be R, amino acid residue 68 should be T or A, amino acid residue 70 should be S, amino acid residue 75 should be A, E, K, Q or R, amino acid residue 81 should be Q, amino acid residue 83 should be N, and amino acid residue 84 should be S, N or G. The FR3 region of KB_A01 is the protein A binding region without any amino acid substitutions. However, in an optional embodiment, N at amino acid residue 84 can be replaced by S. If X 14 The inequalities are A, E, K, Q, or R, and X is... 15 If the sequence is S, N, or G, then the common FR3 sequence in SEQ ID NO:21 is the protein A binding FR3 region.

[0171] E Y ADSV KGR F T I S rDNA K NTVYL Q M NN LKPEDTAVYYCKB_A01 FR3

[0172] X Y XDSV KGR F T I S rDNA X NTVYL Q M NX LKPEDTAXYYC common FR3

[0173] -----------------K--------S-----------Protein A binds to FR3

[0174] In the implementation scheme, the FR1 region of KB_A01 is modified by replacing S with R at amino acid residue 19, and the FR3 region of KB_A01 is modified by replacing N with S at amino acid residue 84.

[0175] In one embodiment, the VHH antibody has the FR1 region of QVQLQESGGGSVQAGGSLRLSCAAS as defined in SEQ ID NO:82 and the FR3 region of EYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYC as defined in SEQ ID NO:83.

[0176] In one embodiment, the VHH antibody of the present invention is an isolated VHH antibody.

[0177] The term "isolated" when used in conjunction with VHH antibodies, such as in the expression "isolated VHH antibody," indicates that the VHH antibody has been purified and removed from its original environment. As used herein, isolated VHH antibody is intended to refer to a VHH antibody that is substantially free of other antibodies with different antigen specificities. For example, isolated VHH antibodies that specifically bind to TfR1, particularly human TfR1 (hTfR1), are substantially free of antibodies that specifically bind to antigens other than TfR1. However, isolated VHH antibodies that specifically bind to hTfR1 may be cross-reactive with other antigens, such as TfR1 molecules from other species, like cTfR1. Furthermore, isolated VHH antibodies may be substantially free of other cellular material and / or chemicals. For example, isolated VHH antibodies can be purified to a purity greater than 95% or 99%, as determined by methods such as electrophoresis (e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF), capillary electrophoresis) or chromatographic methods (e.g., ion exchange or reversed-phase high-performance liquid chromatography (HPLC)). Those skilled in the art will understand that although the word "isolated" is not explicitly mentioned each time the term "VHH antibody" and similar expressions are used, it refers to isolated VHH antibodies herein.

[0178] Further aspects of the implementation plan include nucleic acid molecules encoding VHH antibodies or fusion molecules according to the implementation plan, see below. As used herein, nucleic acid molecules include polynucleotides, oligonucleotides, and nucleic acid sequences, and generally refer to polymers of DNA or RNA that may be single-stranded or double-stranded, may contain natural, non-natural, or modified nucleotides, and may contain natural, non-natural, or modified internucleotide bonds, such as aminophosphate bonds or thiophosphate bonds, rather than phosphodiesters found between nucleotides of unmodified oligonucleotides. The term “nucleic acid molecule” also includes complementary DNA (cDNA) and messenger RNA (mRNA).

[0179] In one embodiment, the nucleic acid molecule is an isolated nucleic acid molecule encoding a VHH antibody or a fusion molecule according to the embodiment.

[0180] The nucleic acid molecule may encode a single VHH antibody according to the implementation scheme, multiple copies of a single VHH antibody according to the implementation scheme, or one or more copies of different VHH antibodies according to the implementation scheme.

[0181] Nucleic acid molecules may also encode other molecules besides the VHH antibody of the implementation scheme, such as a VHH antibody fused to another molecular gene, i.e., in the form of a VHH-containing fusion protein as further described herein.

[0182] Another aspect of the implementation plan involves a vector containing nucleic acid molecules according to the implementation plan.

[0183] The vector is preferably an expression vector, i.e., a vector containing at least one nucleic acid molecule that contains a coding sequence that can be expressed (such as transcribed and translated) in a host cell containing the expression vector. Therefore, the expression vector contains a nucleic acid molecule operatively linked to a promoter according to an embodiment.

[0184] The term "operable linkage" as used in this article refers to a nucleic acid molecule being in the correct functional position and / or orientation relative to a promoter so that the nucleic acid molecule can be expressed in a host cell; that is, the promoter (constitutive or inducible) controls the transcription of the nucleic acid molecule.

[0185] In one embodiment, the expression vector is selected from DNA molecules, RNA molecules, plasmids, free plasmids, and viral vectors. In such embodiments, in addition to the nucleic acid sequence encoding the VHH antibody, the expression vector also contains control sequences required for the production of the VHH antibody in the host cell. For example, the nucleic acid sequence encoding the VHH antibody is under transcriptional control by 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 its downstream DNA (gene). The promoter is preferably selected based on the specific host cell to which the VHH antibody is to be expressed, such as the T5, T7, lac, and BL21 promoters for expressing VHH antibodies in bacterial cells, the GAL1, MET25, CUP1, LAC4, ADH2, SUC2, and GAPDH promoters for expressing VHH antibodies in yeast, and the EF1a, CMV, or CAG promoters for expression in mammalian cells (e.g., human cells). The promoter can be a constitutive promoter or an inducible promoter. Constitutive promoters, also known as constitutively active promoters, are active in all cases of the host cell. Inducible promoters, also known as inducible promoters, are regulated and activated in the host cell only in response to specific stimuli; examples include chemically induced promoters, temperature-induced promoters, or light-induced promoters. Expression vectors may optionally contain 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 specific gene. Optional signal peptides may be provided at the N-terminus or C-terminus of the polypeptide encoded by the expression vector.

[0186] Another aspect of the implementation plan involves a host cell containing a nucleic acid molecule or expression vector according to the implementation plan.

[0187] Then, nucleic acid molecules or expression vectors can be transcribed in host cells, thereby producing VHH antibodies in the cells.

[0188] In one implementation, the cells are selected from the group consisting of bacterial cells, yeast cells, and mammalian cells.

[0189] Another aspect of the embodiments relates to a VHH antibody according to the invention linked to at least one molecule. Therefore, 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 preferably takes the form of a gene fusion between a VHH antibody and at least one molecule. In this case, the aforementioned nucleic acid molecule encodes not only a VHH antibody but also at least one other molecule. For example, the nucleic acid molecule may include, from its 5' end to its 3' end, a nucleic acid sequence encoding a VHH molecule and a nucleic acid sequence encoding at least one other molecule, or may include a nucleic acid sequence encoding at least one other molecule and a nucleic acid sequence encoding a VHH molecule. Therefore, the VHH molecule may be covalently linked to at least one other molecule at its N-terminus or C-terminus, see [link to relevant documentation]. Figure 2 .

[0190] It is also possible to generate 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 to or attached to the N-terminus of the VHH antibody, the C-terminus of the VHH antibody, or at least one molecule may be linked to or attached to the N-terminus of the VHH antibody and at least one molecule may be linked to or attached to the C-terminus of the VHH molecule.

[0191] In one embodiment, the fusion molecule comprises at least one other molecule linked to or attached to the C-terminus of the VHH antibody.

[0192] As described above, the VHH antibody is preferably linked to at least one other molecule by fusion with at least one other molecular gene. In such embodiments, the nucleic acid molecule encodes the VHH antibody and at least one other molecule as a VHH-containing fusion protein. This means that the nucleic acid molecule contains a nucleic acid sequence encoding the VHH antibody, which is linked to or connected 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.

[0193] However, VHH antibodies can be linked, attached, or conjugated to at least one molecule in ways other than gene fusion, such as by chemically linking the VHH antibody to at least one molecule. For example, a VHH antibody can be covalently linked to at least one molecule via a reaction of maleimide with an amine or thiol, commonly referred to as thiol-maleimide or amine-maleimide click chemistry. In such embodiments, additional amino acid residues (e.g., cysteine, arginine, or lysine) can be added to the N-terminus and / or C-terminus of the VHH antibody to enable such a reaction with maleimide.

[0194] It is also possible to link VHH antibodies to at least one molecule enzymatically, for example, via transglutaminase (TGase), which catalyzes the formation of an isopeptide bond between the g-carboxamide group (-(C=O)NH2) of the glutamine residue side chain and the e-amino group (-NH2) of the lysine residue side chain, subsequently releasing ammonia (NH3). In such embodiments, additional amino acid residues (e.g., glutamine or lysine) can be added to the N-terminus and / or C-terminus of the VHH antibody to enable such enzymatic reactions. Site-specific linker sites can also be prepared via sortase-mediated reactions (Guimaraes et al., Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions, Nate Protoc ols 8:1787-1799 (2013)).

[0195] In one embodiment, the VHH antibody according to the invention is covalently linked to at least one molecule.

[0196] VHH antibodies can be directly linked to at least one molecule (e.g., through fusion). Alternatively, VHH antibodies can be covalently linked to at least one molecule via a linker. Depending on the implementation, various such linkers can be used, particularly peptide linkers, including but not limited to G… n Connector (where n is an integer equal to or greater than 1, and usually equal to or less than 10), S m Connector (where m is an integer equal to or greater than 1, and usually equal to or less than 10), A q Connectors (where q is an integer equal to or greater than 1, and typically equal to or less than 10), various GS connectors or GA connectors, i.e., combinations 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 typically equal to or less than 10. For example, linkers such as G4A, G4S, G3S, and combinations thereof (e.g., 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), pages 156-157. The linkers listed in Table 1 are hereby incorporated by reference as illustrative but not limiting examples of peptide linkers that can be used to covalently link VHH antibodies and at least one molecule.

[0197] The fusion molecules or VHH antibodies of the present invention may contain one or more tags, including but not limited to affinity purification tags, such as His tags, C tags, Q tags, and / or myc tags. 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 to achieve purification may also be accompanied by protease sites, such as, but not limited to, TEV sites, to allow for the subsequent efficient removal of the purification tags from the VHH antibody or fusion molecule.

[0198] In addition to containing Figure 2 In addition to at least one molecule shown, it may also contain multiple VHH antibodies.

[0199] In one embodiment, the fusion molecule is a monovalent fusion molecule relative to the VHH antibody, i.e., preferably containing only a single VHH antibody in addition to at least one molecule. There is in vitro and in vivo evidence that the monovalent binding mode promotes transcellular transport, while the bivalent binding mode leads to 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)).

[0200] The at least one molecule can be any molecule, such as a pharmaceutical agent 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, antitumor agents, anti-inflammatory agents, adjuvants, peptides, polypeptides, and proteins, such as enzymes, hormones, neurotrophic factors, neuropeptides, cytokines, apolipoproteins, growth factors, antigens, antibodies or portions of antibodies, adjuvants, etc., nucleic acids, such as RNA or DNA, including, for example, gene-coding nucleic acids, repressive nucleic acids, such as ribozymes, antisense nucleic acids, interfering nucleic acids, whole genomes or portions thereof, plasmids, etc.

[0201] 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.

[0202] As used in this article, imaging agents refer to reagents or molecules used to visualize imaging agents during the imaging process, such as those taken up by cells when administered to a patient. Examples of such imaging agents include agents or molecules containing radioactive atoms or isotopes, such as radioactive tracers used in positron emission tomography (PET) tracers, for example, those containing... 18 F-type PET tracers, containing 11 C-type PET tracers, containing 64 Cu-containing PET tracers or single-photon emission computed tomography (SPECT) tracers, for example, those containing... 99m Tc or 111 In SPECT tracers. Other examples of imaging agents include fluorescent probes, luminescent probes, probes containing metal complexes, near-infrared (NIR) fluorescent probes, etc.

[0203] Overall, there is an urgent need for diagnostic tools capable of early detection of diseases or medical conditions, particularly in the ventricles of the brain. Such early detection would contribute to effective disease-modifying therapies, especially in evaluating the therapeutic efficacy of new drug candidates in preclinical and clinical trials.

[0204] Classical PET ligands or tracers based on small molecules capable of crossing the BBB have difficulty specifically targeting pathogenic proteins, including but not limited to amyloid β, τ, and α-synuclein, or pseudomarkers of diseases such as inflammation. PET ligands or tracers based on specific binding molecules (e.g., antibodies or antibody domains) are generally preferred. However, monoclonal antibody (mAb)-based PET tracers often have such long circulating half-lives that they are of no use as PET ligands or tracers. This means that the radioactivity of radiolabeled mAbs derived from the cerebral bloodstream interferes with specific signals at the target site in the brain. Therefore, to be effective in PET applications, antibody-based PET ligands or tracers should have rapid blood clearance and a high cerebral-to-blood ratio. Smaller antibody domains or fragments (with a mean molecular weight equal to or less than 60 kDa), such as VHH antibodies, have faster systemic elimination compared to mAbs due to renal excretion. Therefore, VHH antibodies are suitable as PET ligands or tracers because they are smaller than mAbs. et al., A bispecific Tribody PET ra dioligand for visualization of amyloid-beta protofibrils-a new conc ept forneuroimaging. Neuroimage 148:55–63. (2017); Vandesquille et al., Chemically-definedcamelid antibody bioconjugate for the mag netic resonance imaging of Alzheimer's disease. mAbs 9:1016–1027(2017)).

[0205] In these implementations, the fusion molecule can be used as a diagnostic agent to diagnose various diseases or conditions.

[0206] For example, the VHH antibody of the embodiment may be linked to another antibody, its antigen-binding fragment or domain, including another VHH antibody that specifically binds to a disease-specific biomarker. In this case, the other antibody is preferably labeled to detect its binding to the disease-specific biomarker after delivery, for example, by delivering it to the ventricle via transcytosis mediated by using the VHH antibody as a receptor for the BBB transporter.

[0207] The antigen-binding fragments or domains of the antibodies used in this article may 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 two or more CDRs.

[0208] For therapeutic purposes, in addition to the therapeutic agent, the fusion molecule may also contain a half-life extender or group, also known as a stabilizer or group. This half-life extender is then added to increase the half-life of the fusion molecule when administered to the patient. Any half-life extender that can be linked to a VHH antibody but has no adverse biological effects can be used, including but not limited to the Fc fragment of IgG, serum proteins (e.g., human serum albumin (HSA)), albumin-binding protein scaffolds, IgG, various polyethylene glycol (PEG) molecules or unstructured peptides (e.g., XTEN), or PAS-based alternatives to PEGylation.

[0209] The present invention also relates to pharmaceutical compositions comprising fusion molecules as defined above, wherein at least one molecule is a therapeutic agent. The pharmaceutical compositions further comprise pharmaceutically acceptable mediators or excipients.

[0210] Pharmaceutically acceptable excipients can be any pharmaceutically acceptable excipient or carrier that is compatible with the other components of the pharmaceutical composition. Pharmaceutically acceptable carriers can be selected from conventionally used carriers for each method 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 injectable solutions.

[0211] Pharmaceutically acceptable mediators 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 gum, methylcellulose, sodium carboxymethyl cellulose, and polyvinylpyrrolidone; disintegrants such as starch, agar, alginate, and sodium alginate; absorbents; dyes; flavorings; sweeteners; polypropylene glycol; and liquid mediators such as water, saline solution, glucose solution, glycerin, ethanol, and oil.

[0212] The implementation plan also involves the aforementioned fusion molecules used as pharmaceutical agents, wherein at least one molecule is a therapeutic molecule.

[0213] In the implementation plan, the therapeutic agent is capable of treating CNS diseases or conditions.

[0214] In specific embodiments, the aforementioned fusion molecule is used to treat CNS diseases or conditions. In such embodiments, at least one molecule is a therapeutic agent capable of treating CNS diseases or conditions.

[0215] A further embodiment relates to a method of treating a patient with a CNS disease or condition. The method includes administering an effective amount of the aforementioned fusion molecule or the aforementioned pharmaceutical composition to the patient. In such embodiments, at least one molecule is a therapeutic agent capable of treating a CNS disease or condition.

[0216] Illustrative but non-limiting examples of CNS diseases or conditions 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 monosomy muscular atrophy (MMA), multiple sclerosis (MS), neurofibromatosis, Parkinson's disease (PD), lysosomal storage diseases, neurolysosomal storage diseases, ischemic stroke, cerebral hemorrhage, traumatic brain injury (TBI), vascular dementia, frontotemporal dementia, amyloidosis, tau proteinosis, Creutzfeldt-Jakob disease, neuroinflammation, and neuropathic pain.

[0217] Malignant cells often overexpress TfR1, and this increased expression may be associated with poor prognosis in different types of cancer (Candelaria et al., Antibodies Targeting the TfR1 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 levels are often several times higher than 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 a variety of cancers, including solid cancers such as esophageal squamous cell carcinoma, breast cancer, ovarian cancer, lung cancer, cervical cancer, bladder cancer, osteosarcoma, pancreatic cancer, bile duct cancer, renal cell carcinoma, hepatocellular carcinoma, adrenocortical carcinoma, glioblastoma multiforme (GBM), and nervous system cancers, including brain metastases from peripheral cancers, as well as hematopoietic malignancies such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin's lymphoma (NHL) (Candelaria et al., Antibodies Targeting the Transferrin Receptor 1 (TfR1) as Direct Anti-cancer Agents. Front. Immunol. 12:607692 (2021); Ramalho et al., Transferrin Receptor-Targeted Nanocarriers: Overcoming Barriers to Treat Glioblastoma. Pharmaceutics). 14(2):279(2022)).

[0218] Elevated TfR1 expression levels on malignant cells, coupled with its extracellular accessibility, internalization capacity, and central role in cancer cell pathology, make this receptor an attractive target for antibody-mediated therapy. The VHH antibody of this invention targets TfR1 for cancer treatment by using a VHH antibody conjugated to an anticancer agent, immunotherapeutic agent, and / or adjuvant therapy agent internalized via receptor-mediated endocytosis.

[0219] In another implementation, the therapeutic agent is capable of treating cancer.

[0220] In a specific implementation, the aforementioned fusion molecule is used to treat cancer. In such implementations, at least one molecule is a therapeutic agent capable of treating cancer.

[0221] A further embodiment relates to a method of treating a patient with cancer. The method includes administering an effective amount of the aforementioned fusion molecule or the aforementioned pharmaceutical composition to the patient. In such embodiments, at least one molecule is a therapeutic agent capable of treating cancer.

[0222] hTfR1 is also a target for delivering therapeutic agents (including oligonucleotides) to muscles (Desjardins et al., Enhanced exon skipping and prolonged dystrophin restoration achieved by TfR1-targeted delivery of antisense oligonucleotides using FORCE). TM Conjugation in MDX mice, Nucleic Acids Research gk ac641, 2022; Sugo et al., Development of antibody-siRNA conjugate targeted to cardiac and skeletal muscles, J Control Release 237:1-13 (2016). Therefore, the fusion molecule of the present invention can be used to treat various muscle diseases, especially muscular dystrophy.

[0223] Fusion molecules can also be used to treat muscular dystrophy, 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), especially DMD, FSHD, or DM.

[0224] In a further implementation, the therapeutic agent is capable of treating muscular dystrophy.

[0225] In a specific implementation, the aforementioned fusion molecule is used to treat muscular dystrophy. In such implementations, at least one molecule is a therapeutic agent capable of treating muscular dystrophy.

[0226] A further embodiment relates to a method of treating muscular dystrophy in a patient. The method includes administering an effective amount of the aforementioned fusion molecule or the aforementioned pharmaceutical composition to the patient. In such embodiments, at least one molecule is a therapeutic agent capable of treating muscular dystrophy.

[0227] As used herein, an effective dose refers to the dose and time period necessary to achieve the desired outcome. Effective doses can vary depending on factors such as disease state, age, sex, and patient weight. As used herein and as well understood in the art, “treating” or “treatment” means a method used to obtain a beneficial or desired outcome (including clinical outcomes). Beneficial or desired clinical outcomes can include, for example, the reduction or improvement of one or more symptoms or symptom, a reduction in the extent of disease, a stable disease state (i.e., prevention of exacerbation), prevention of disease spread, a delay or slowing of disease progression, an improvement or mitigation of the disease state, a reduction in disease relapse, and remission. “Treatment” can also mean prolonged survival compared to expected survival without any treatment.

[0228] As used herein and as well understood in the art, prevention or prophylaxis means a method of reducing or preventing the risk of developing a disease or condition, including prolonging or delaying the development of the disease. For example, patients with a predisposition to developing a disease (such as due to a genetic or hereditary susceptibility) may benefit from fusion molecules or pharmaceutical compositions to prevent, reduce the risk of, delay, and / or slow the development of the disease.

[0229] The preferred patient is a human patient. However, the implementation scheme can also be applied to veterinary applications, i.e., non-human patients, such as non-human mammals, including, for example, primates, monkeys, apes, cattle, sheep, pigs, goats, horses, cats, dogs, mice, rats, and guinea pigs.

[0230] The fusion molecule or pharmaceutical composition according to the implementation plan can be administered to patients via various routes, including, for example, parenteral routes, such as subcutaneous, intravenous, intraperitoneal, or intramuscular injection; oral routes; rectal routes; local routes; intranasal routes; translingual routes; or intraocular routes. In one specific implementation plan, the route of administration is subcutaneous or intravenous.

[0231] Example

[0232] Example I - Production of proteins for llama immunization and assay

[0233] The extracellular domains of hTfR1 (amino acids R121-F760, SEQ ID NO:30), mTfR1 (S122-F763, SEQ ID NO:31), and cTfR1 (R121-F760, SEQ ID NO:32) were cloned into the pCDNA3.4-TOPO plasmid using the stop codons TGA, TGA, and Kozak sequences (generated using Geneart tools (Thermo Fischer Scientific)). The plasmid has 5' cloning sites: HindIII, KpnI, NheI and 3' cloning sites: XhoI, PacI, and was optimized for expression in gray hamsters (Cricetulus griseus). Expression was performed using an N-terminal signal peptide (MDWLRNLLFLMAAAQSINA, SEQ ID NO:33) followed by a single amino acid linker “A,” a histidine tag (6×His, SEQ ID NO:38), and either an SG linker (hTfR1, cTfR1) or a G linker (mTfR1). The plasmid was ordered from Geneart (Thermo Fischer Scientific, Germany). Transient transfection of Chinese hamster ovary (CHO) cells was performed according to standard procedures. Cultures were harvested on day 6. The supernatant was filtered (using a 0.45 μm syringe filter) and then purified using a HisTrapExcel 5 mL column (Cytiva) with an immobilized metal ion chelate (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 fraction was concentrated using an Amicon Ultra 10000MWCO rotary condenser. Protein was dialyzed overnight at 4°C in PBS containing 10% glycerol (Medicago tablets, glycerol diluted from 80% stock solution) using a 3 ml cassette slide-a-lyzer, with a cutoff of 10 kDa (Pierce). The PBS was set to pH 8. Protein concentration was measured using absorbance at 280 nm.

[0234] Example II - Generation and Selection of VHH Libraries Incorporating Human TfR1

[0235] According to Modiquest Research BV (the Netherlands) and ModiPhage TMMethods: Llama animals (N=2) were immunized to generate antibodies. Two llamas were initially immunized using an adaptive immunization protocol based on monitoring specific immune responses to immunogens hTfR1 and mTfR1 (generated as described in Example I). As shown in Figure 1, immunizations were performed on day 1 (hTfR1), day 21 (mTfR1), and day 42 (hTfR1) by intramuscular (im) administration of a single dose of 500 μg protein. On day 54, blood samples were drawn to investigate the immune response using ELISA. Serum samples were serially diluted 3-fold (from 1:100 to 1:72900) and screened for plate-fixed hTfR1, mTfR1, and the control protein bovine serum albumin (BSA), all at 1 μg / ml. The specific binding of anti-lamb IgG1 and IgG2 / IgG3 antibodies was detected using goat anti-rabbit horseradish peroxidase (HRP) and goat anti-mouse HRP, respectively, in a spectrophotometric plate reader with 3,3',5,5'-tetramethylbenzidine (TMB) substrate at an absorbance of 480 nm. Two additional doses of 500 μg hTfR1 were administered 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, namely a 1:1 mixture of hTfR1 and mTfR1 (250 μg + 250 μg). Peripheral blood was drawn four days later (day 124) and analyzed using Ficoll-Paque. TM PLUS ~ 1.5 x 10 9 PBLs were separated by density gradient centrifugation. TRIzol (Thermo Fischer Scientific) was used to separate the PBLs from approximately 0.7 × 10⁻⁶ PBLs. 9 RNA was extracted from PBL. 400 μg of freshly isolated RNA was reverse transcribed into cDNA via oligomeric (dT)-induced reverse transcription.

[0236] All subsequent steps in constructing the VHH phage display library were performed as described in Pardon et al., A general protocol for the generation of Nanobodies for structural biology. Nat Protoc 9:674–693 (2014), see [link to article]. Figure 1BMore specifically, the cDNA described above 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), followed by a second nested PCR reaction to amplify and isolate the VHH library, including subcloning into the dangling site in phagemid pADL10b using primers CALL001 (SEQ ID NO:34) and CALL002 (SEQ ID NO:35). A 3 μg amount of cDNA template was mixed with High-performance liquid from ThermoFischer Scientific. Use the kit together with: 10 μl 5× High Fidelity buffer, 1 μl dNTP (dinucleotide triphosphate; 10 mM each, 0.2 mM final concentration), 25 pmol of each primer (0.5 μM final primer concentration), and 0.5 μl Polymerase (1 U / reaction), ddH2O (double-distilled water) to 50 μl, 1–2 μl template, cycling program: 98℃ for 5 min, 98℃ for 30 s (30×), 72℃ for 30 s (30×), 72℃ for 10 min, 4℃ storage. A total of 36 wells were collected and purified using Genejet enzyme purification (Thermo Fischer, catalog number: K0832) to remove PCR dimers. The mixture was purified using four purification columns and eluted in 4×20 μl elution buffer. The eluent was loaded onto a 1% agarose gel, and gel extraction using SYBRSafeDNA GelStain (SigmaAldrich) was safe, and the extracted product was used as a template in subsequent PCR reactions. Next, amplification was performed using nested PCR reactions, including the use of the pADL10 cloning sites of the following oligonucleotides: VHH-R-SfiI (SEQ ID NO: 36) and VHH-F-SfiI (SEQ ID NO: 37). Two PCR mixtures were prepared separately, with 0.5 μM and 0.1 μM of each primer (VHH-F-SfiI and VHH-R-SfiI), and 1 μl of template was thermally cycled as follows: 98 °C for 5 min, 98 °C for 30 s (20×), 72 °C for 1 min (20×), 72 °C for 10 min, and stored at 4 °C. Approximately 50 ng of amplified and gel-extracted heavy chain antibody was used as template in each well. After amplification, all wells were pooled and purified using Genejet PCR purification according to the manufacturer's protocol, and eluted in elution buffer (10 mM Tris-HCl pH 8.5). The PCR product concentration was measured using Nanodrop (result: 160 ng / μl). The product was digested with SfiI restriction enzyme (Thermo Fischer Scientific). A 400 bp DNA fragment was extracted onto a 2% agarose gel using the Genejet gel extraction method.

[0237] Following the manufacturer's instructions, the pADL10b vector was digested overnight at 37°C using the SfiI restriction enzyme (Thermo Fischer Scientific). The digested vector was purified on a 1% agarose gel and visualized using Sybr Safe (Thermo Fischer Scientific). The procedure was repeated until a final concentration of 154.6 ng / μl was achieved.

[0238] After PCR amplification, the library was digested at 50°C (using Sfil) for 2 hours. Enzymes were removed using the Genejet PCR purification column described above. The resulting material was eluted in 20 μl of elution buffer (10 mM Tris-HCl, pH 8.5). The DNA concentration was measured to be 73 ng / μl. The mixture was ligated again. First, the material was heated at 50°C and slowly cooled to room temperature (RT, 20–25°C), then incubated overnight at 16°C with shaking. Purification was performed as above, followed by elution in 10 mM Tris-HCl, pH 8.5. A total of 1600 ng of DNA was used. 1 or 2 μl of the ligation mixture was used to electroporate 10 μl of TG1 E. coli in a 25 μl total reaction volume (5.48 ms 1.92 kV and 5.48 ms 1.92 kV, respectively). The cells were removed from the -70°C freezer and used immediately after thawing on ice. The transformants were distributed on large agar plates (20 cm) and allowed to grow under standard conditions until colonies appeared and the plates were ready for harvest.

[0239] Cells were harvested by adding Luria Broth (LB) medium. Colonies were counted, and the estimated library size was 3.2 × 10⁻⁶. 8 After overnight growth on agar plates, cultures for the final library pool were prepared. The cultures were incubated at 37°C with shaking at 200 rpm for 2 hours. Hyperphage was then added at 37°C without shaking for 30 minutes at a multiplicity of infection (MOI) of 2. The culture medium was removed by centrifugation at 4500g for 5 minutes in Falcon tubes. The cells were then resuspended in an equal volume of Terrific Broth (TB) medium supplemented with 100 μg / ml ampicillin, 50 μg / ml kanamycin, and 1 mM isopropyl β-thiogalactopyranoside (IPTG). The cultures were incubated overnight at 30°C with shaking at 200 rpm.

[0240] Prepare 5 mL cultures in LB broth from overnight cultures of selected clones in 15 mL Falcon tubes supplemented with 100 μg / mL ampicillin, omitting glucose. Dilute the overnight cultures 1:100 in 5 mL LB broth and incubate at 150 rpm for 2 hours until the cultures become turbid. Add 1 μl of hyperphage helper phage (Hyperphage, titer: 1.4 × 10⁻⁶) to each culture. 12 (cfu / ml, Progen). Infect with helper phages at 37°C for 30 minutes. Discard the LB containing the phage by centrifugation at 4500g. Resuspend the bacterial pellet in 5ml TB medium supplemented with 100μg / ml ampicillin, 50μg / ml kanamycin, and 1mM IPTG. Incubate the culture overnight at 200rpm and 30°C with shaking. Centrifuge the culture at 4500g for 4 minutes to remove bacteria, add 0.25×V of 20% PEG8000 / 2.5M NaCl, and incubate on ice for 2 hours in centrifuge tubes. Pelletize the phage / bacteria at 15000g for 10 minutes and resuspend in 0.5ml PBS. Transfer the phage-containing supernatant to separate microcentrifuge tubes pre-blocked with PBS / casein.

[0241] Using biotinylated TfR1 Select

[0242] Following standard procedures, hTfR1, cTfR1, and mTfR1 were biotinylated using a biotinylation kit EZ-link Sulfo NHS PEG4 (Thermo FIscher Scientific) at pH 8 with a 1:1 molar ratio of 1 mg target protein to 2 mM biotin. Free biotin was removed by dialyzing with 1 L PBS buffer at pH 8 for 3 × 1 h in a Pierce 3 mL 10 kDa MWCO (molecular weight cutoff) dialysis unit at 4 °C. The protein was concentrated to 850 μl using an Amicon rotary concentrater with 10 kDa MWCO. The phage supernatant was pre-blocked with PBS / casein and mixed with 100 nM biotinylated hTfR1, cTfR1, or mTfR1, and incubated for 1 h. 50 μl of Streptavidin containing hTfR1 (1.1 mg / mL) and mTfR1 (1 mg / mL) was then used. (M280, Invitrogen, Thermo Fischer Scientific) was used for bead blocking, and a solution containing 0.15% Wash with 20 μL of PBS (PBST). Resuspend the beads in 1 mL of ELISA blocking agent (PBS / casein ELISA blocking agent (Thermo Fischer Scientific)) and incubate for 30 min at room temperature in a rotary mixer. Replace the blocking agent with a phage + target mixture and gently rotate and incubate for 30 min at room temperature to capture biotinylated TfR1 on the beads. Wash the beads 5 times with PBS. Both mTfR1 and hTfR1 captures were eluted in 400 μL of PBS containing 0.25 mg / mL trypsin.

[0243] The phages were digested with trypsin at room temperature for 30 minutes, followed by titration and rescue. TG1 E. coli culture was initiated by inoculating 10 ml of culture into 10 ml of LB medium and growing a single TG1 colony on an M9 basic agar plate. The culture was incubated at 37°C and 200 rpm until OD was reached. 600 Reaching the logarithmic phase (approximately 4 hours, OD) 600 (Approximately 0.6). The culture was kept on ice. A dilution (1-10 times) was prepared and mixed with 100 μl TG1, and incubated for 5 minutes. The mixture was then inoculated onto LB Lennox agar plates (Merck-Sigma-Aldrich) supplemented with 2% glucose and 100 μg / ml ampicillin. The input was first trypsinized (1 mg / ml trypsin) by mixing 75 μl of the phage stock solution with 25 μl and incubating at room temperature for 30 minutes, followed by dilution and titration. All remaining phage output was individually mixed with 2 ml TG1 E. coli and incubated at room temperature for 5 minutes (without shaking). The mixture was then inoculated onto two large LB Lennox agar plates and allowed to dry completely before incubating in plastic bags at 37°C.

[0244] According to Table 1 below, hTfR1, cTfR1, or mTfR1 (the latter two were performed in the presence of 1 μM human transferrin (recombinantly prepared in rice, Sigma Aldrich, human transferrin)) were repeatedly used in the subsequent three rounds for capture. The selection (and subsequent rescue) procedure was carried out. After the fourth round of selection, samples were taken from each trajectory and elution method (trypsin or citrate / phosphate buffer at pH 5.5, containing 0.15%). Twenty-five colonies were randomly selected from the 20) agar plates and sent to the Karolinska Institute (Sweden) Genetic Analysis Core Facility for sequencing using primer pADL10fwd.

[0245] Table 1 - Biopharmaceutical screening schemes used for selection

[0246]

[0247] Example III - Combining with ELISA to select VHH that binds hTfR1 and cTfR1

[0248] Prior to ELISA, selected phage samples were diluted 1:10 in blocking buffer, casein ELISA blocking agent (ThermoFischer Scientific), and 2.5M NaCl containing 20% ​​v / v PEG8000. Targets (hTfR1, cTfR1, mTfR1) were coated overnight at 1 μg / mL in untreated PBS pH 7.4 in 96-well Greiner plates at 4°C (100 μL). The coating buffer was discarded and replaced with 100 μL of blocking solution. The plates were capped and incubated at 900 rpm for 2 hours at room temperature. Anti-human TfR1 antibody BA3 (MEM189, Sigma, 1 mg / mL) and anti-mTfR1 (8D3, ​​Novusbiologicals, 1 mg / mL) were diluted to an initial concentration of 100 nM in blocking buffer, and serially diluted 1:2 by transferring 100 μL to 100 μL of blocking buffer. The plate was incubated at room temperature and 900 rpm for 1 hour, and then washed with PBST (containing 0.1% PBST) using a 50TS microplate washer (Biotek). Wash wells with 3 × 300 μl of PBS (20%). Dilute anti-M13 antibody (Anti-PVIII GE Healthcare) 1:4000 in blocking buffer, add 100 μl to the target-coated wells, and incubate at 900 rpm for 1 hour at room temperature. Wash with PBST. Dilute anti-mouse secondary antibody (Sigma) 1:4000 in blocking buffer and incubate at 900 rpm for 1 hour at room temperature. After washing with PBST, develop with TMB substrate. Stop the reaction by adding 2 M H2SO4 to each well, and measure absorbance at 450 nm using a SpectraMax 3000 (Molecular Devices). One clone (KB_A01) used in the following examples was selected based on its cross-reactivity with both cTfR1 and hTfR1 in ELISA and its non-reactivity with mTfR1. The selection criterion was a reactivity to human and cynomolgus monkey receptors >10-fold higher than the blank (negative control). The selection criteria in this workflow are also based on non-competitive binding sites with Tf to Tf (see Example II), which are non-preferred binding sites for therapeutic agent brain transport.

[0249] Example IV - Generation of Free VHH or Fusion Variants

[0250] The selected VHH clone (KB_A01, SEQ ID NO:23) was generated as a single VHH unit (12-14 kDa), see [link to documentation]. Figure 2 The protein, which has a C-terminal 6×His tag (SEQ ID NO:38) followed by a "C tag" (C-terminal amino acid EPEA, SEQ ID NO:39), was ordered from Genscript Inc. and codon-optimized using the expression vector pET-22b(+). The protein was obtained from the periplasmic space of *E. coli* and purified in one step on a Ni column.

[0251] The human Fc region fuses with VHH via the GG spacer region to produce a bivalent TfR1-binding functional entity. See [link to relevant documentation]. Figure 2 More specifically, the VHH Fc fusion protein (SEQ ID NO:41) between human IgG1 Fc (SEQ ID NO:40) and KB_A01 (SEQ ID NO:23) and the signal peptide from the mouse IgGκ light chain (SEQ ID NO:61) were cloned into the vector pcDNA3.4 backbone with codon-optimized sequence (performed by GeneArt, Thermo Fischer Scientific) and in Expi293. TM Transient expression was performed in Thermo Fischer Scientific cells, with 200 mL cultures grown under standard conditions and harvested 6 days post-transfection. The culture medium was purified using MabSelect Sure. TM The fraction was purified in a HiLoad Superdex 200 and yielded a dimeric VHH-Fc fusion fraction with >99% purity, as verified by size exclusion chromatography (SEC). The same method was performed to produce a fusion protein of the baseline VHH (SEQ ID NO:60) and human IgG1 Fc disclosed in WO 2020 / 144233. This baseline VHH-containing fusion protein is referred to herein as BV.

[0252] Following bioinformatics evaluation, other VHH-Fc fusions were performed between human IgG1 Fc and KB_A02 (SEQ ID NO:42), KB_A03 (SEQ ID NO:24), KB_A04 (SEQ ID NO:25), KB_A05 (SEQ ID NO:43), KB_A06 (SEQ ID NO:44), KB_A07 (SEQ ID NO:26), KB_A08 (SEQ ID NO:45), KB_A09 (SEQ ID NO:27), KB_A10 (SEQ ID NO:28), and KB_A11 (SEQ ID NO:29). Based on the same human IgG1 Fc and GG adapter sequence and signal peptide (SEQ ID NO:56) as described above, after sequence optimization and cloning into a proprietary plasmid of GenScript Biotech Corporation, VHH-Fc fusions were performed at Turbo-CHO. TM These other VHH-Fc fusions were generated in 4 mL volumes using a high-performance platform system. The final samples were also harvested on day 6 post-transfection, purified on a Genscript protein A-based system, and validated by SDS-PAGE or analytical size exclusion chromatography (SEC) to a purity of 95-99%.

[0253] Functional fusion proteins are generated by fusing VHH and a single-chain variable domain (scFv). These fusion proteins have monomeric TfR1 binding, but possess the potential for bispecific binding due to the presence of scFv domains that can bind to targets different from TfR1 or to different epitopes on the same target. The amyloid β-binding antibody 3D6 was selected as the functional and representative scFv unit. The scFv of the 3D6 antibody consists of VH (SEQ ID NO:46) and VL (SEQ ID NO:47), which are fused to the N-terminus or C-terminus of KB_A01 and linked via a 3×(G4S) linker (SEQ ID NO:48) to generate KB_A01-scFv (SEQ ID NO:49) or scFv-KB_A01 (SEQ ID NO:50), see [link to 3DFv]. Figure 2 The gene construct optimized for expression in CHO by GenScript Biotech Corporation was transiently transfected into TurboCHO. TM It is expressed in high-performance platform cells.

[0254] FreeStyle DNA was transiently transfected using the vector pcDNA3.4. TMIn 293, the baseline antibody 128.1 (BA1) was expressed as full-length human IgG1 (SEQ ID NO:51) and harvested from the culture medium, and purified in one step on protein A resin. This baseline antibody BA1 binds to the extracellular region of hTfR1 and has the ability to cross the BBB (WO 93 / 10819; Friden PM et al., Characterization, receptor mapping and blood-brain barrier transcytosis of antibodies to the human transferrin receptor. J Pharmacol Exp Ther 278:1491–1498(1996)).

[0255] The baseline antibody, JCR-IgG (BA2), is a clinically validated TfR1-binding antibody targeting the CNS. Designed based on information in US 9,994,641, it is an IgG1 antibody reactive to hTfR1 via Fab'2 (SEQ ID NO:51). The heavy chain (HC) and light chain (LC) sequences were optimized, cloned into separate plasmids, and transiently transfected with ExpiC HO using standard reagents. TM Transfection and expression were performed in cells, and the protein was harvested on day 8. The protein was processed using MabSelect Sure. TM (Cytiva) purified, then purified by Superdex 16 / 60 (Cytiva) and analyzed according to standard procedures known to those skilled in the art.

[0256] Binding characteristics of Example V-KB_A01

[0257] During phage display and panning for hTfR1 and cTfR1, the binding of VHH KB_A01 to hTfR1 and cTfR1 was identified, and the reactivity of the clone was confirmed by ELISA. Binding kinetics were further analyzed using surface plasmon resonance (SPR) and biolayer interferometry (BLI). The binding affinity of VHH KB_A01 to TfR1 and the lack of interference from transferrin (Tf) 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 conjugated to the SA chip.

[0258] The binding characteristics of monomer VHH KB_A01 to the baseline antibody BA1 were evaluated using single-cycle kinetics via SPR on a Biacore T200 instrument. Human Tf was amine-conjugated on a CM5 chip (to approximately 3000 RU), followed by the capture of hTfR1 on immobilized human transferrin. Human TfR1 was loaded onto hTf by injection at 100 nM, followed by analyte (VHH KB_A01 or BA1) in 2-fold increments from 6.25 to 100 nM. The contact time for each concentration was 150 seconds at 30 μL / s, with a final dissociation step of 500 seconds. At the end of each single-cycle run, the hTf surface was completely regenerated by injection of 10 mM glycine at pH 2 for 30 seconds. Similarly, mTfR1 was amine-coupled to approximately 2200 RU on a CM5 chip and analytes (VHH KB_A01 or BA1) in the same concentration range as human TfR1 (6.25 to 100 nM in 2-fold increments) were applied to these surfaces, and the same SPR cycle as described above was applied, but without the loading step. At the end of each single-cycle run, the mTfR1 surface was completely regenerated by injecting 10 mM NaOH for 30 seconds. A blank run was performed after each single-cycle kinetic run for baseline correction. The association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (K) were evaluated using the Biacore T200 software tool with the Langmuir 1:1 model. D The data was evaluated.

[0259] The monomer KB_A01 and the benchmark antibody BA1 bound to hTfR1 (Table 2 and Figure 3), while neither antibody bound to mTfR1. Since hTfR1 is immobilized by loading onto hTf, the binding of the analyte implies that neither KB_A01 nor BA1 competes with hTf for binding to hTfR1. Furthermore, comparisons of the affinity and binding kinetics of KB_A01 and BA1 indicate that the affinity (Kd) of monomeric (free) KB_A01 for binding to pre-complexed hTfR1-Tf is significantly different due to the substantial difference in dissociation rate (kd). D )Compared to the divalent BA1(K) D=0.0199 nM) is 206 times lower, at 4.1 nM. It has been shown that excessive affinity may lead to the retention of VHH and any proteins fused with it in the blood vessels and / or to the 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_A01 has the following binding characteristics: due to its lower affinity for hTfR1 than BA1, it is more suitable as a drug transporter than BA1.

[0260] Table 2. Affinities of monomeric VHH and full-length baseline antibody BA1 to hTfR1 and mTfR1 as measured by SPR

[0261]

[0262] Example VI - Localization of the monomeric KB_A01 epitope by binning experiments using BLI targeting known hTfR1 binding antibodies BA1, BA2, and BA3

[0263] Recombinant KB_A01 was biotinylated using the EZ-Link NHS-PEG4-Biotin Biotinylation Kit (ThermoFisher) according to the manufacturer's instructions. Stock vials of biotin solution were dissolved at -70°C and stored in DMSO to a concentration of 20 nM. The solution was thawed and freshly diluted to 2 mM in ddH2O and mixed with VHH protein (12 nmol-57 nmol protein) at an equimolar ratio with biotin in PBS pH 7.4. PBS was added to a total volume of 0.5 or 1 mL. The mixture was incubated in a rotary mixer at room temperature for 30 minutes, followed by overnight incubation on a shaker at 4°C. The sample was concentrated using a 500 μL rotary concentrator (3000 MWCO, 13000 rpm for 5 minutes). This process was repeated for some samples until the concentration was adequate. The samples were then desalted using a NAP-5 column equilibrated with PBS pH 7.4 (Medicago) according to the manufacturer's instructions. In short, the sample was loaded into the resin, and 400 μL of PBS was added. Proteins were eluted by adding 500 μL of PBS. Protein concentration was estimated using absorbance at (λ)280. Sample yields were calculated, ranging from 24% to 46%.

[0264] hTfR1 binding and competition between KB_A01 and reference antibodies BA1, BA2, and BA3 were analyzed using BLI on an OctetRED96 instrument (ForteBio / Pall Life Sciences). Samples were loaded onto black polypropylene 96-well microplates (Greiner bio catalog number 655209). First, biotinylated KB_A01 (as described above, in the form of free VHH protein) was immobilized at 2 μM on Streptavidin Dip and Read plates for kinetic analysis. TM On a biosensor (Pall Lifesciences, ForteBio). 50 nM of hTfR1 protein was added to each sensor, followed by regeneration with glycine at pH 2 (5 seconds, then regeneration four times for 5 seconds each in run buffer (Kinetics Buffer 10X (ForteBio) diluted in PBS pH 7.4), repeated for four cycles. In the last cycle, competitive protein probes KB_A01 (control), BA1, and BA3 (purchased from Sigma Aldrich / Merck) were added at 10 nM, and binding was recorded. Kinetic measurements were performed as follows: baseline 100 seconds, loading 300 seconds, quenching 1200 seconds, association 300 seconds, and dissociation 200 seconds. Data were recorded and analyzed using Octet System Data Acquisition version 10.0 (ForteBio, Pall Life Sciences) and Octet System Data Analysis version 10.0 - Kinetics module (ForteBio, Pall Life Sciences). The binding results of the biotinylated monomer KB_A01 are shown in the first row of Table 3.

[0265] Next, full-length baseline antibodies BA1, BA2, and BA3 were immobilized at 1 μM onto a protein A (ProA) Dip and Read biosensor (Pall Life Sciences, Fortebio) for kinetics. hTfR1 (50 nM) was added to each biosensor following the same principle as described above, followed by the addition of either the competitive probe VHH KB_A01 (unbiotinylated) or either antibody BA1 or BA3 (both 10 nM). Due to the loading setup on protein A, the monoclonal antibodies did not antagonize each other (marked as nd = incomplete in Table 3). As shown in Table 3 (rows 2-4), KB_A01 prevented BA1 from binding to hTfR1. In contrast, KB_A01 and baseline antibody BA2, as well as KB_A01 and BA3, could bind to hTfR1 simultaneously. Therefore, KB_A01 binds to a specific epitope that overlaps with but is not identical to the epitope of BA1. It is known that BA1 and antibody BA3 can bind to overlapping epitopes in the apical domain, thereby competing for binding with hTfR1 (Sade et al., A Human Blood-Brain Barrier Transcytosis Assay Reveals Antibody Transcytosis Influenced by pH-Dependent Receptor Binding, PLOS one4(1):e936340(2014)). Therefore, we can further conclude that KB_A01 also binds to the apical domain.

[0266] Table 3 - Epitope binning of monomer VHH KB_A01 and reference antibody using BLI

[0267] Immobilized ligands KB_A01 BA1 BA3 KB_A01 X B - Antibody BA1 B X nd Antibody BA2 - nd nd Antibody BA3 - nd*(B) X

[0268] B = Blocked, - = Not blocked, nd = Undetermined, X = Self-competition (against itself). *(B) = Literature report.

[0269] Example VII: Characterization of Fc-Fc fusion with VHH

[0270] Affinity measurements were performed using SPR on a Biacore T200 (Cytiva) chip by loading the KB_A01-Fc fusion, BV (Fc fusion), and BA2 (Example IV) at a high level (100 nM for 60 seconds) onto a protein A chip (Cytiva). Subsequently, it was exposed to 5-fold incremental concentrations of hTfR1 ranging from 0.16 to 100 nM. The data are summarized in Table 4, and the sensing plot is shown in Figure 4. The dimer KB_A01-Fc was at 0.177 nM K DThe affinity for hTfR1 is 23 times higher than that for the free monomer KB_A01 bound to immobilized hTfR1 (Table 4). D =4.1 nM (see Table 2). As a control, baseline BV (VHH-Fc) and BA2 (IgG1) were run under the same conditions and showed an affinity of 0.00228 and 0.0249 nM, respectively (K0). D The mouse TfR1 antibody was also tested under the same conditions, and neither KB_A01-Fc nor the baseline antibody BA2 bound to mTfR1 with a detectable signal. In contrast, the baseline VHH-Fc (BV) showed a high affinity of 0.444 nM (K). D ) combined with mTfR1 ( Figure 4D These experiments demonstrate that KB_A01 possesses binding characteristics within an ideal nM affinity range and exhibits interesting K-type release (OFF) to the receptor. D Overview. Compared to BV, KB_A01 binds only hTfR1, but not mTfR1.

[0271] Table 4 - Binding kinetics of KB_A01 as a dimeric Fc fusion compound and control with human or mouse TfR1 determined using SPR

[0272]

[0273] To further investigate the effect of soluble human transferrin on binding to human and cynomolgus monkey TfR1, the above-mentioned SPR was performed in the presence and absence of human Tf. Mouse TfR1 was also included as a control. KB_A01-Fc, BV-Fc, BA1, and BA2 were immobilized on protein A sensor (Cytiva) and then exposed to escalating concentrations (4-fold increments) of the receptor in the absence or presence of a saturation concentration of 250 nM hTf. There was little difference in affinity when comparing the absence or presence of 250 nM hTf, indicating that KB_A01 and the two benchmark antibodies BA1 and BA2 do not interfere with the binding of Tf to human or cynomolgus monkey TfR1 (Table 5). Figures 5A-6D And 6A-6D). However, in the presence or absence of hTf, the affinity of BV-Fc for human TfR1 (K D Significant differences were observed. Furthermore, the data indicated high affinity for cynomolgus monkey TfR1, while binding with mouse TfR1 was not recorded (Table 5). This experiment demonstrates that KB_A01 possesses ideal and unique binding characteristics, making it suitable for use in non-human primates and humans to increase TfR1 delivery to the CNS.

[0274] Table 5 - Effect of transferrin presence on binding to TfR1 in humans and cynomolgus monkeys

[0275]

[0276]

[0277] Example VIII - Analysis of Bioinformatics and Additional Clones

[0278] Using the Jalview Multiple Sequence Alignment Editor and Analysis Workbench version 2 (Barton group, University of Dundee) software, KB_A01 was aligned with all clones sequenced based on preliminary clonal screening (panning). Based on sequence identity with KB_A01 and positive binding confirmed in ELISA, three additional clones, KB_A09, KB_A10, and KB_A11 (see [link to ELISA]), were selected. Figure 7 (Comparison of the results), and generated as a fusion with human IgG1 Fc (Example IV). Next, CDR combinations were designed in which CDRs from a sequencing clone (KB_ref, SEQ ID NO:53) were transplanted into the frame of KB_A01, generating KB_A03, KB_A04, KB_A06, and KB_A07, where CDR2 (KB_A03), CDR3 (KB_A04), CDR1 and CDR3 (KB_A06), and CDR2 and CDR3 (KB_A07) had been replaced in KB_A01. As shown in Table 7, replacing CDR1 (KB_A02) alone in KB_A01 or in combination with CDR2 (KB_A05) or CDR2 and CDR3 (KB_A08) resulted in VHH being unable to bind to hTfR1.

[0279] Table 6 - CDRs for KB_A01 and KB_A02 to KB_A08

[0280] VHH CDR1 CDR2 CDR3 KB_A01 <![CDATA[GSIFGSKR 1 ]]> <![CDATA[ITYRGTT 3 ]]> <![CDATA[WMFTTDNY 5 ]]> KB_A02 <![CDATA[GSIFGFNA 2 ]]> ITYRGTT WMFTTDNY KB_A03 GSIFGSKR <![CDATA[IAVAGST 4 ]]> WMFTTDNY KB_A04 GSIFGSKR ITYRGTT <![CDATA[WMYATANY 6 ]]> KB_A05 GSIFGFNA IAVAGST WMFTTDNY KB_A06 GSIFGFNA ITYRGTT WMYATANY KBA07 GSIFGSKR IAVAGST WMYATANY KB_A08 GSIFGFNA IAVAGST WMYATANY

[0281] 1 –SEQ ID NO:1 4 –SEQ ID NO:4

[0282] 2 –SEQ ID NO:2 5 –SEQ ID NO:5

[0283] 3 –SEQ ID NO:3 6 –SEQ ID NO:

[0284] Based on the above, KB_A02-KB_A11 were generated as fusions with human IgG1 Fc (see Example IV, SEQ ID NO: 62-71), and their affinity for hTfR1 and mTfR1 was tested using SPR. The VHH fused with the dimer Fc was then immobilized on a protein A chip as described above. In short, using 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, followed by five-fold incremental injections of receptors in the range of 0.8 to 500 nM (contact time 120 seconds, dissociation 600 seconds). After each complete single-cycle kinetic cycle, the surface was regenerated for 30 seconds with 10 mM glycine, pH 1.5, at 30 μL / s. Table 7 shows the kinetic data for KB_A02 to KB_A11 as Fc fusions. KB_A03, KB_A04, KB_A06, KB_A07, KB_A09, KB_A10, and KB_A11 all have K values ​​ranging from 0.150 to 5.12 nM. D Values ​​showed binding to hTfR1 (Table 7). In contrast, when KB_0A2, KB_A05, and KB_A08 were immobilized on protein A, they did not bind to hTfR1. No binding to hTfR1 was observed for KB_ref fused to human IgG1 Fc (SEQ ID NO:85).

[0285] Table 7 - Binding kinetics of KB_A02 to KB_A11 dimer Fc fusion protein to human TfR1 determined by SPR

[0286]

[0287] Next, epitope binning was performed by using SPR on a Biacore 8K to test hTfR1 binding agents in a combined manner. KB_A01 and KB_A09 were immobilized on a CM5 series S (Cytiva) chip with reference antibodies BA1, BA2 and reference VHH-Fc (BV) via amine coupling. The proteins to be immobilized were diluted to 25 μg / mL in sodium acetate at pH 5.5 and then bound to an SPR signal of approximately 6000 resonance units (RU). The first injection used 50 nM hTfR1 extracellular domain (contact time 120 s, 10 μL / s), and the second injection used 100 nM of each analyte: KB_A01, KB_A03, KB_A04, KB_A09, KB_A10, KB_A11, BA1, BA2, BV, mouse transferrin (mTf, purified, Jackson Immunoresearch Europe Ltd), and human transferrin (hTf, purified, Jackson Immunoresearch Europe Ltd), with a contact time of 150 s, 10 μL / s. After each cycle, the surface was regenerated for 30 s with 10 mM glycine at pH 2.1 at 30 μL / s. Analysis was performed using BiacoreInsight Evaluation package version 4.0.8.20368. The results showed that KB_A03, KB_A09, KB_A10, and KB_A11 all competed with KB_A01, KB_A09, and antibody BA1 (Table 8), but none competed with BA2 or BV. 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 allnew world hemorrhagic Fever arenaviruses. J Virol. 86(7):4024-4028(2012)), competing with BA2 and the aforementioned VHH-Fc fusion construct, but not with BV. Surprisingly, KB_A04 did not compete with BA1 (Table 8). Therefore, KB_A01, KB_A03, KB_A09, KB_A10, and KB_A11 form a separate bin that partially overlaps with, but does not completely overlap with, the bins of KB_A04 and BA1. The control VHH-Fc molecule BV does not block any other anti-TfR1 molecules, nor does it block the binding of mTf and hTf to hTfR1.Therefore, the VHH antibody of BV binds to epitopes that are completely different from KB_A01, KB_A03, KB_A09, KB_A10 and KB_A11.

[0288] Table 8 - Epitope binning of dimeric VHH-Fc fusion compound and benchmark antibody using SPR

[0289]

[0290]

[0291] B = blocking, - = non-blocking, X = self-competition (targeting itself).

[0292] To demonstrate its functionality when fused with non-Fc proteins, KB_A01 was also generated as a fusion with a single-stranded variable domain (scFv) located at the N-terminus (scFv-KB_A01) or C-terminus (KB_A01-scFv). See Example IV for details. Figure 2 To verify that these fusion proteins still bind to hTfR1, binding kinetics were investigated using 8K Biacore via SPR. Briefly, the receptor hTfR1 was immobilized to a CM5 chip (Cytiva) via amine coupling, and single-cycle kinetics were used to expose the protein to scFv-KB_A01 or KB_A01-scFv at 4-fold increments ranging from 0.25 to 64 nM. Kinetic binding data and sensor plots are presented in Table 9 and Figure 8.

[0293] Table 9. Binding kinetics of KB_A01 fusion protein with scFv human TfR1 using SPR

[0294] Construct <![CDATA[ka(M -1 s -1 )]]> <![CDATA[kd(s -1 )]]> <![CDATA[K D (M)]]> Rmax(RU) scFv-KB_A01 <![CDATA[1.70×10 6 ]]> <![CDATA[5.39×10 -3 ]]> <![CDATA[3.16×10 -9 ]]> 56.1 KB_A01-scFv <![CDATA[2.18×10 6 ]]> <![CDATA[7.50×10 -3 ]]> <![CDATA[3.44×10 -9 ]]> 51.8

[0295] Verification of apical domain binding of KB_A01 family binders using ELISA

[0296] To further evaluate whether KB_A01 and the associated anti-hTfR1 VHH bind to the apical domain of hTfR1, we expressed the apical domain by displaying it on M13 phage. Apical domains expressed as a single soluble domain are known to be unstable, which is why this approach was adopted. Genscript Biotech Corp. provides a synthetic gene (SEQ ID NO:54) encoding a recombinant variant of the hTfR1 apical domain (covering the Q191-F297 and S326-V380 regions of the full-length hTfR, corresponding to the Q80-F186 and S215-V269 regions of the hTfR extracellular domain, SEQ ID NO:30), in which mutations were introduced at Y222S and L329S to increase solubility; the amino acid sequence is shown in SEQ ID NO:72. The gene is flanked by restriction enzyme sites AgeI and NotI. The AP1 gene was cloned into phage particle pTG3 using restriction digestion and T4 ligation (New England Biolabs). The resulting phage particle pTG3-AP1 was transformed into *E. coli* XL1-blue via heat shock and inoculated onto LA-ampicillin (100 μg / ml) plates. The transformants were grown in 2xYT medium supplemented with ampicillin (100 μg / ml) and phage mini-prep kit (Monarch Plasmid Mini Prep Kit, New England Biolabs). The sequence of the pTG3-AP1 phage particle preparation was confirmed using Sanger sequencing (Eurofins Genomics GmbH).

[0297] E. coli XL1-blue transformed with pTG3-AP1 was cultured at 37°C in 30 ml of 2xYT medium supplemented with 5 μg / ml tetracycline and 100 μg / ml ampicillin until OD. 600 The concentration reached 0.7. Add 0.5 ml (3.1 × 10⁻⁶) of helper phage M13K07 to the culture. 11 Add kanamycin 50 μg / ml and IPTG 0.25 mM to the culture, and incubate at 37 °C for 1 h. Add kanamycin 50 μg / ml and IPTG 0.25 mM to the culture, and incubate overnight at 30 °C. Centrifuge the culture and transfer the supernatant to a new tube. Add 7.5 ml of NaCl-PEG (20% PEG-8000 / 2.5 M NaCl) to the supernatant, and incubate on ice for 30 min. Centrifuge the supernatant at 4 °C for 30 min. Discard the supernatant and dissolve the phage precipitate in 2 ml of 1% BSA PBS. Briefly centrifuge the resuspended phage preparation to remove larger aggregates, and then filter through a 0.2 μm filter.

[0298] Immunoassay plates (Nalge Nunc International) were coated with constructs KB_A01-Fc and KB_A02 to KB_A11 (including the baseline antibodies BA2 and BV-Fc) at a concentration of 10 μg / ml (5 μg / ml for Fc fusions) in 0.05 M sodium carbonate buffer, pH 9.4. The plates were incubated overnight at 4°C. The plates were washed twice in PBS-tween. Blocking buffer was added to PBS containing 1% BSA, and the plates were incubated at room temperature for 1 hour, followed by washing twice in PBST. Starting at a 1:1 PBST dilution, M13 phage displaying the hTfR1-top (SEQ ID NO:54) was added serially. For the extracellular domain of hTfR1 (SEQ ID NO:30), the plates were coated with 10 μg / ml hTfR1, and antibodies were added serially according to the dilution series described above. The plates were incubated at room temperature for 2 hours, followed by washing four times with PBST. Add anti-M13-HRP antibody (#11973-MM05T-H, Sino Biological) or anti-Fc HRP (Pierce #31423, 1 mg / mL) diluted 1:5000 in PBS-Tween, and then incubate at room temperature for 1 hour. Wash the plate four times with PBST, and then add 1-Step... TM Ultra TMB-ELISA substrate solution (ThermoFisher Scientific) was prepared and developed at room temperature for 30 minutes. The reaction was terminated with 2M H2SO4, and the absorbance was measured at 450 nm. These experiments showed that KB_A01, KB_A04, KB_A09, KB_A11, and BA2 bind to the apical domain but not to BV. Figure 8A and Figure 8B In the control experiments using the above ELISA method, KB_A01, BA2, and BV were all confirmed to bind to the extracellular domain of hTfR1 using the same method. Figure 8C This was also confirmed in all the SPR experiments mentioned above.

[0299] Based on the binning-related experiments described above, we can conclude that KB_A01, KB_A03, KB_A04, KB_A07, KB_A09, and KB_A11 bind to the same limited region of the hTfR1 apical domain. Although not tested in this study, the likelihood that KB_A07, which has sequence homology with other apical-binding VHH clones and blocks these clones in binning experiments, will not bind to the apical domain is extremely low. Furthermore, it was found that the baseline VHH antibody in BV cannot bind to the apical domain of hTfR1.

[0300] Example IX: Human Cell Uptake mediated by TfR1

[0301] Next, the internalization of the KB_A01 VHH-Fc fusion protein in TfR1-expressing cells was tested. Well-known TfR1-expressing adherent HEK293T cells (ATCC) were grown in collagen-coated 96-well plates (Thermo Fischer Scientific) at a seeding density of 30,000 cells / well. Cells were grown in DMEM (Dupuyet modified E. E.) medium for 3–5 days until approximately 80–90% confluence. KB_A01-Fc was used in this assay along with known reference binders BA1 and BA2, and a negative control (a VHH-Fc-like protein known not to bind hTfR1).

[0302] Cell assays were established based on titrations of test items KB_A01, BA1, BA2, and a negative control within the range of 0.85 nM to 82.5 nM over 5-15-30-45 minutes at 37°C and 5% CO2. To compare cell fate after prolonged exposure to added proteins, cells were incubated for 30 minutes, then replaced with fresh DMEM and maintained for 90 minutes (at 37°C and 5% CO2), followed by a second medium replacement. After 120 minutes, cells were transferred to the lab bench and immediately washed with PBS (1×) and fixed with 4% paraformaldehyde for 10-15 minutes. After fixation, cells were washed with PBS and treated with a solution containing 0.1% Triton-X 100. TM Cells were permeabilized with PBS, then blocked with PBS containing 1% bovine serum albumin (BSA), and then 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 secondary antibody incubation, 300 U of Texas Red-phalloidin (1:200 dilution of 10 μg / mL stock solution) was added to the wells. Cells were then washed with PBS at least three times and stored at 4°C until imaging. Cells were imaged using an inverted Zeiss 710 laser scanning confocal microscope with 488, 555, and 647 lasers and a 20× air objective.

[0303] The results of these experiments indicate that KB_A01, as well as BA1 and BA2, are readily taken up by HEK293T cells at various test concentrations (0.7-85 nM) and incubation times ranging from 5 to 240 minutes. Figure 10A As shown, KB_A01-Fc was efficiently taken up by cells similarly to BA1. At shorter time points (15-45 minutes), the amount was equivalent to that of KB_A01-Fc and BA1. At longer incubation time points, the intracellular presence of BA1 was significantly reduced compared to both KB_A01 and BA2. Figure 10CThe decrease in BA1 concentration in cells after 240 minutes indicates lysosomal degradation. The negative control (VHH-Fc, which does not bind to hTfR1) did not accumulate in cells and showed no presence at any time point or test concentration, indicating that assay and uptake are TfR1 dependent.

[0304] Example X: Assessment of transcytosis potential

[0305] Experiments were performed to assess the transcytosis potential of KB_A01 and compare it with selected reference IgG and baseline TfR1 binding to BA2. In vitro BBB Transwell assays of brain-like endothelial cells were performed in a tight monolayer configuration on a Transwell system, as shown below. The study, described in *Transport study of interleukin-1 inhibitors using a human in vitro model of the blood-brain barrier*, *Brain Behaviour*, *Immunity Health* 16:100307 (2021), was conducted at the Laboratoire de la barrière Hématoencéphalique (LBHE), Université d'Artois, France. To assess the transcytosis potential of the VHH-Fc fusion, three copies of the test compound (VHH-Fc or full-length antibody) were added to donor wells and incubated at 37°C for three hours. At the end of the experiment, all compartments were harvested. Cells were lysed, and the contents of all compartments (donor, cell, and recipient compartments) were analyzed using a human Ig (Fc-specific) ELISA from Mabtech, Swede, en. Plates were coated with anti-human IgG mAbMT145 (Fc-specific) and blocked with PBST containing 0.1% BSA. Add samples and standard diluents. Then, add the assay reagents in two steps: first, add biotinylated anti-human IgG mAb MT78 (Fc specific), then add HRP-conjugated streptavidin. Wash with PBST between each step. Add an equal volume of TMB, and finally terminate the reaction with 2M HCl and read the plate number. The substrate incubation time is set to 10 minutes. As a reference, a control monoclonal antibody lacking active transcytosis activity (responsive to interleukin 1-β) is used. This control monoclonal antibody does not bind to TfR1 and has a low BBB pass rate. Additionally, the clinically validated transporter BA2 (Example IV) is used. The method is validated by internal control of all wells to control monolayer compactness through co-incubation and measurement of human serum albumin (HSA) conjugated to fluorescent Alexa-647 and sodium fluorescein (NaF). All wells included in the comparison are compact and show similar HSA distribution in polarized cell monolayers.

[0306] like Figure 11 As shown, KB_A01 VHH-Fc exhibited significantly superior transcytosis capacity and remained within the near-compartment of the polarized cell monolayer. This experiment demonstrates that KB_A01, as a VHH-Fc fusion, possesses the ability to perform directed transcytosis across the human polarized brain-like endothelial cell monolayer.

[0307] Example XI - In vivo and in vitro evaluation of VHH-Fc of KB_A01 in humanized TfR1 extracellular domain mice (hECD-TfR1-mice)

[0308] To evaluate the in vivo brain-targeting properties of KB_A01, the distribution characteristics of KB_A01 were tested in homozygous, heterozygous, and wild-type hECD-TfR1 mice. Homozygous mice expressed only partially humanized TfR1, i.e., human-mouse chimeric TfR1. Taconic Biosciences GmbH, Leverkusen, Germany generated humanized mice expressing a chimeric receptor comprising the extracellular domain of hTfR1 and the intracellular and transmembrane domains of mTfR1.

[0309] Taconic Biosciences GmbH, Leverkusen, Germany, has developed a genetically engineered mouse model in which the endogenous mouse Tfrc gene has been partially humanized, resulting in engineered mice that produce chimeric TFRC proteins containing the human TFRC extracellular domain. The targeting vector was assembled using BAC-derived mouse and human genomic DNA fragments, including a standard selection cassette, using methods known to those skilled in the art.

[0310] Embryonic stem cells (ES) derived from C57BL / 6NTac mice were grown in ES cell culture medium containing leukemia inhibitory factor and fetal bovine serum on a mitotically inactivated feeder cell layer composed of mouse embryonic fibroblasts. The cells were electroporated with a linearized DNA-targeting vector according to methods known to those skilled in the art. Homologous recombination 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. Homologous recombination ES cell clones were amplified and frozen in liquid nitrogen.

[0311] Chimera and Heterozygote Generation: Following 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 a pseudopregnant NMRI female. Chimerism in the F0 was assessed by observing the contribution of ES cells to the coat color (black / white) of the BALB / c host. In vitro fertilization was performed using oocytes from superovulated C57BL / 6NTac females and thawed sperm from previously cryopreserved sperm from male chimeras. The fertilized embryos were then transferred into pseudopregnant Swiss Webster recipient females. Germplasm transmission was first identified by the presence of black-fured offspring (strain C57BL / 6NTac), and confirmed by genotyping of the black offspring using PCR. F1 mice heterozygous for the modified Tfrc allele were mated to generate homozygous (HOM), heterozygous (HET), and wild-type (WT) genotypes for the following studies.

[0312] Use the method described below with iodine-125 ( 125 I) Radiolabeling of KB_A01-Fc: Greenwood et al., The preparation of 131 I-labeled human growth hormone of high specific radioactivity. Biochem J 89:114–123 (1963). Stock up on 15–80 μg of protein with 260 ± 26 MBq / μg or 61 ± 2.4 kBq / μg. 125I (PerkinElmer Inc, Waltham, MA, USA) and 5 μg chloramine T (Sigma-Aldrich) were mixed in PBS. The reaction mixture (110 μL) was incubated for 90 seconds and then quenched with 10 μg sodium metabisulfite (Sigma-Aldrich). The radiolabeled sample was immediately purified on a Zeba column (ThermoFischer) with a 7 kDA cutoff. In vivo experiments were performed in 6–10 week old C57BL / 6 mice. Immediately after radiolabeling, KB_A01-Fc (5 nmol / kg; 3.4 MBq / nmol) was administered intravenously via the tail vein. Blood samples (8 μL) were obtained from the tail vein at 5 min, 0.5 h, 1 h, and finally 2 h post-injection. Two hours post-injection, mice were euthanized by blood collection from the heart followed by 2.5 min of transcardiac perfusion with 40 mL NaCl. The brain and major organs were dissected. The blood was separated into plasma and blood cell sediment by centrifugation at 10,000 × g for 5 minutes. The brain was divided into left and right hemispheres. The cerebellum was removed from the left hemisphere, and the remaining tissue in the left hemisphere is referred to as the "brain" below. Radioactivity in the brain, blood fraction, and major organs was then measured using a g-counter (2480 Wizard™, Wallac Oy PerkinElmer, Turku, Finland). Figure 13A and 13B As shown, pharmacokinetic blood profiles revealed the highest blood clearance in HOM mice, followed by HET mice, while WT mice exhibited the lowest blood clearance. Furthermore, the distribution among whole blood, sediment, and plasma showed the highest presence in the blood sediment of HOM mice (after rotating whole blood as described above), followed by HET mice, and the lowest presence in WT mice, indicating binding to hTfR1 on erythrocytes in vivo. Figure 13C The normalized brain uptake (SUV) was shown to be significantly higher in HOM mice than in WT mice, while the SUV of HET mice was nearly 50% lower than that of HOM mice. This clearly demonstrates the specific brain-targeting ability of KB_A01 in functional hECD-TfR1 in vivo.

[0313] Example XII - Epitope Characterization

[0314] The binding sites of KB_A01, KB_A03, and KB_A09, the Fc fusion forms described in Example IV, and two benchmark antibodies, BA1 and BA2, were investigated to determine the exact contribution of amino acids in the apical domain of human TfR1. Epitope localization was performed using Seqitope. TM (AAX Biotech AB, Sweden) This was based on massively parallel mutagenesis scanning. Seqitope TMThe binding ratio (BR) value for each amino acid residue of the target antigen is output, and this value is related to the importance of that amino acid residue in antibody binding. A higher BR value (>2.5) indicates that the amino acid residue contributes significantly to antibody binding, although this value appears to vary depending on the affinity of the antibody studied. A BR value of 1.0 indicates that the amino acid residue does not contribute to antibody binding.

[0315] The target antigen is the apical domain of hTfR1 expressed on M13 phage as described in Example VIII.

[0316] KB_A01, KB_A03, and KB_A09, as well as the tested baseline antibodies BA1 and BA2, bound to the apical domain expressed on M13 phage, while the baseline BV did not.

[0317] This embodiment resolved the epitopes of the baseline antibodies BA2 (D204, K205, N206, R208, V210, E369) and BA1 (D242, L246, P249, E369, V366, G351, R364) on hTfR1, as well as the antibodies KB_A01 (P354, L246, D245, G351, D242, R364, S355, D356), KB_A03 (D356, P354, D242, D245, G351, L246, S355) and KB_A09 (D356, D245, P354, G351, D242, L246, S355) (see Table 10). Antibodies in the KB_A01 group (KB_A01, KB_A03, and KB_A09) bind to similar epitopes in the apical domain.

[0318] Seqitope TM Detailed epitope analysis revealed that the BA1-binding epitope partially overlaps with the BA2-binding epitope (E369), but also overlaps with the antibody-binding epitopes (D242, L246, and G351) in the KB_A01 group. The conclusion is that the KB_A01 group's binding epitopes differ from, but partially overlap with, the BA1 epitopes, and do not overlap at all with the BA2 epitopes. This is consistent with the binning data in Example VI II, where KB_A01, KB_A03, KB_A09, and BA1 compete with each other, BA1 and BA2 compete with each other, while BA2 does not compete with members of the KB_A01 group. However, Seqitope TM The data provides further details and shows the differences between the current antibody and the benchmark antibody. The benchmark VHH BV was not included because it has been shown not to bind to the apical domain (see [link to data]). Figure 8AThe epitope of BA1 has been located in a apical domain between Ser324 and Ser368, where three identified amino acids are located (Helguer G. et al., An antibody recognizing the apical domain of human trans antrösferrin receptor 1 efficiently inhibits the entry of all new world hemorrhagegic Fever arenaviruses. J Virol. 86(7):4024-4028(2012)).

[0319] Although the epitopes of the three VHH-Fc variants tested showed a high degree of overlap, the individual contributions of each amino acid in hTfR1 differed slightly. Most surprisingly, D356 contributed the most to the binding of KB_A03 and KB_A09, while this amino acid was least associated with the binding of KB_A01.

[0320] Table 10. Based on Seqitope TM Data table position

[0321]

[0322] Example XIII: Generation of Protein A-Binding Antibody

[0323] Llama VHH is generally not associated with protein A-based resins like PrismA. TM Combination, but by changing the amino acids in the framework sequence, PrismA TMBy binding and thus altering protein A binding, VHHs can be generated. To explore the possibility of enabling protein A to bind to KB_A01, KB_A09, and KB_A11, as well as fusion proteins containing KB_A01, KB_A09, and KB_A11, these three VHHs were mutated based on a common protein A binding sequence, which is based on Henry et al., A.R.A.E., A.R.A.E., A.R.A.E., A.R.A.E., B.R.A.E., B.R.A.E., B.R.A.E., B.R.A.E., B.R.A.E., B.R.A.E., B.R.A.E., B.R.A.E., B.R.A.E., B.R.A.E., C ... KB_A01 was mutated from serine to arginine at position 19 and from asparagine to serine at position 84 (KB_A01: S19R, N84S, denoted as KB_A12 SEQ ID NO:73); KB_A09 was mutated from serine to arginine at position 19 (KB_A09: S19R, denoted as KB_A13 SEQ ID NO:74); and KB_A11 was mutated from serine to arginine at position 19 (KB_A11: S19R, denoted as KB_A14 SEQ ID NO:75). A positive control known to bind to protein A (SEQ ID NO:76) was also included (positive control 1). Green fluorescent protein (GFP) was expressed as an expression control and can be seen in the blot in Figure 12, but was not included in any other analyses. All variants were purified using a Ni-NTA-based method by fusing a short Gly2 adapter (GGHHHHHH, SEQ ID NO:77) with a His6 tag. Plasmids (pNIC28_Bsa4) with codon-optimized sequences were ordered from GeneArtThermo Fisher and used to transfect *E. coli* BL21(DE3)pRARE2 cells according to a standardized procedure at the Protein Science Facility, Karolinska Institute, Sweden. Briefly, pre-cultures were allowed to grow overnight and used to inoculate expression cultures. Expression cultures were cultured at 37°C in 2 × 1 ml Terrific Broth supplemented with 8 g / L glycerol and appropriate antibiotics. The next day, the temperature was lowered to 18°C, and cells were induced one hour later with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Protein expression was continued overnight at 18°C, followed by cell harvesting by centrifugation (4000 × g, 20 min). Cells were stored at -80°C.

[0324] Cells were suspended in lysis buffer (100 mM HEPES, 500 mM NaCl, 10% glycerol, 10 mM imidazole, 0.5 mM TTCEP, pH 8.0, 1 mg / ml lysozyme, 0.1% DDM, 1 mM MgSO4, and protease inhibitor (Rochec Omplete)). TM Lysis was performed using EDTA-free (0.5 Tb / mL) and Benzonase (0.125 U / mL) via a three-freeze-thaw cycle. Total cell lysates were clarified by centrifugation (4000 × g, 30 min). The supernatant was mixed with 25 μl of nickel-resin pre-equilibrated with equilibration buffer (20 mM HEPES, 500 mM NaCl, 10% glycerol, 10 mM imidazole, 0.5 mM TCEP, pH 7.5) in a 96-well plate equipped with a filter and incubated with gentle shaking at room temperature for 30 min. Unbound proteins were then collected by centrifugation (200 × g, 1 min) at 4 °C. The resin was washed three times with washing buffer (20 mM HEPES, 500 mM NaCl, 10% glycerol, 30 mM imidazole, 0.5 mM TCEP, pH 7.5) and resuspended in 40 μl elution buffer (20 mM HEPES, 500 mM NaCl, 10% glycerol, 500 mM imidazole, 0.5 mM TCEP, pH 7.5) and incubated at room temperature. The elution fraction was collected by centrifugation. Total cell lysates were analyzed by SDS-PAGE according to standard procedures. Figure 12A ), Clarified cell lysates ( Figure 12B ) and elution fractions ( Figure 12C (equal portions of the sample).

[0325] Wild-type (WT) elution fractions of KB_A01, KB_A09, and KB_A11, along with protein A mutant variants of KB_A01, KB_A09, and KB_A11 (hereinafter referred to as KB_A12, KB_A13, and KB_14, respectively), along with positive control 1, were pre-equilibrated with 25 μl of MabSelect PrismA bound to binding buffer (PBS; 10 mM phosphate, 140 mM NaCl, 2.7 mM KCl, pH 7.4). TMThe resin was mixed in a 96-well plate equipped with a filter and incubated with gentle shaking at room temperature for 45 minutes. Unbound protein was collected by centrifugation (200×g, 1 min) at 4°C. The resin was washed with 2×250 μl of wash buffer (PBS pH 7.4). To elute bound protein, the resin was resuspended in 40 μl of elution buffer (100 mM sodium citrate pH 3.0), neutralized in 1 M Tris-HCl pH 9.0, and incubated at room temperature. The elution fraction was collected by centrifugation. Aliquots of the flow-through and elution fraction were analyzed by SDS-PAGE. Figure 12B and 12C ).

[0326] The ability of WT KB_A01, KB_A09, and KB_A11, along with their corresponding protein A-enabled mutant variants KB_A12, KB_A13, and KB_A14, to bind target hTfR1 at single concentrations was evaluated using a Biacore T200 instrument via SPR analysis. Briefly, hTfR1 was covalently immobilized to the surface of a CM5 chip (Cytiva) using standard reagents via amine coupling. Human TfR1 was diluted to 25 μg / mL in 10 mM sodium acetate (pH 5.5) and immobilized for 600 seconds, producing a very high signal level of approximately 2300 reaction units (RU). Subsequently, all six VHHs were analyzed in run buffer (supplemented with 0.05%). Dilute to 100 nM in 20% PBS (PBS-T). Signals equal to or higher than the corresponding WT are considered “bindings similar to WT” (see Table 11).

[0327] The WT KB_A01, KB_A09, and KB_A11, as well as their corresponding protein A mutants KB_A12, KB_A13, and KB_A14, and the positive control 1 bound to PrismA were investigated in detail using SPR analysis with a Biacore T200 instrument. TM Resin capabilities. Each construct was diluted to 500 nM in running buffer (PBS-T, pH 7.4), then serially diluted four times at a 1:4 ratio (500, 125, 31.2, 7.81, and 1.95 nM) and tested on PrismA. TM The chip (Cytiva) was run on. The contact time for each concentration was 30 seconds at 30 μL / min, and the dissociation time was 30 seconds at 30 μL / min. Surface regeneration was achieved by washing with run buffer at 30 μL / min for 300 seconds.

[0328] Wild-type KB_A01, KB_A09, and KB_A11 purified based on Ni-NTA, and purified based on MabSelect PrismA. TMThe purification of the protein A-enabled mutant and positive control 1 on the resin yielded very pure products (KB_A01, KB_A12 and PrismA of positive control 1) in the elution fraction. TM For representative SDS-PAGE of the eluent, see [link to relevant documentation]. Figure 12B Based on PrismA TM Most of the WT KB_A01, KB_A09, and KB_A11 were found in the purified flow solution (see [link]). Figure 12C SDS-PAGE of flow fractions of KB_A01, KB_A12 and positive control 1 showed that mutations at positions 19 and 84 of KB_A01 and at position 19 of KB_A09 and KB_A11 produced fractions usable with PrismA. TM VHH is purified from resin.

[0329] Binding to hTfR1 was investigated using SPR at a single concentration of 100 nM. All three protein A-enabled mutant variants bound to hTfR with similar affinity to their corresponding WT VHH variants. In contrast, positive control 1 did not bind to hTfR1 (see Table 11).

[0330] It also utilizes pre-coated chips (Cytiva Series S Sensor Chip PrismA) TM The SPR of PrismA was validated. TM Resin bonding. WT KB_A01( Figure 12D KB_A09 and KB_A11 were similar to PrismA even at the highest concentration tested (500 nM). TM The resin bonding is very poor, while KB_A12 ( Figure 12E The binding signals of KB_A13 and KB_A14, as well as positive control 1, reached >1400 RU at the highest concentration (500 nM), confirming VHH-mediated binding with PrismA. TM Functional properties of resin bonding (Table 11).

[0331] Table 11. PrismA TM Supported VHH combined data verification function

[0332]

[0333] n / a = Not applicable (compared to itself)

[0334] Humanization of Example XIV-VHH

[0335] Seventeen VHH variants of KB_A01 (without His tag) were generated in CHO cells by ProteoGenix SAS, France (see Table 12, namely KB_A12, KB_A16-KB_A31). Each variant has one or more amino acid mutations compared to KB_A01. A positive control known to bind to protein A (SEQ ID NO: 84) (positive control 2) is also included. The cDNA encoding VHH was chemically synthesized, optimized for expression in CHO cells, and subcloned using ProteoGenix and Proteogenix proprietary mammalian cell expression vectors. (XtenCHO...) TM The transfection protocol transfects the vector into XtenCHO. TM In cells. Eight days after transfection, a total volume of 3.5 mL of culture medium was collected and used Mabselect PrismA. TM The resin (Cytiva) was purified. The clarified medium was filtered (0.22 μm) and then loaded onto pre-equilibrated (PBS pH 7.5) PrismA. TM On the resin. Bound VHH was eluted by pH changes, and the eluent was immediately neutralized by adding 1M Tris-HCl to pH 9.0. Protein concentration was determined by A280 spectrophotometry.

[0336] The ability of KB_A01 and 18 KB_A01 mutant variants (all carrying two protein A enabling mutations (S19R, N84S, see Example XII)) generated in CHO cells to bind to target hTfR1 was evaluated using a Biacore T200 instrument via SPR at a single concentration. E. coli-generated variants (Example XII) fused with the His6 tags of KB_A01, KB_A12, KB_A15, KB_A16, KB_A17, KB_A18, and KB_A19 were also included in the same experiment and run under the same conditions. Briefly, hTfR1 was covalently immobilized to the surface of a CM5 chip (Cytiva) using standard reagents via amine coupling. Human TfR1 was diluted to 15 μg / mL in 10 mM sodium acetate at pH 5.5, and the target immobilization level was 1000 reaction units (RU). Subsequently, all VHH were diluted in run buffer (supplemented with 0.05%). Dilute to 50 nM in 20% PBS. Signals similar to or higher than the corresponding WT KB_A01 (for E. coli-generated materials) or KB_A12 (for CHO-generated materials) are considered “bound” (see Table 12). KB_A12 is used as a reference for CHO-generated materials because WT KB_A01 is not generated as free VHH without the His6 tag, as it lacks PrismA.TM Integration capability. Similarly, KB_A15 was only tested for integration with His6 tags.

[0337] Binding of 18 mutant VHH variants of KB_A01 was analyzed using intermediate fixation with 1000 RU hTfR in a 50 nM screening mode. KB_A01 and KB_A15 were tested only as His marker proteins produced in E. coli, while KB_A12, KB_A15, KB_A17, KB_A18, and KB_A19 were tested as His marker proteins produced in E. coli and free VHH produced in CHO cells. KB_A20–KB_A31 were produced only in CHO cells and were analyzed by PrismA. TM Resin purification. As shown in Table 12, when analyzed at a single concentration of 50 nM, mutations at positions 1, 5, 11, 14, 19, 43, 44, 45, 46, 74, 78, 84, 86, and 109, individually and / or in combination with some of these mutations, did not interfere with binding to hTfR1. In contrast, above the 10 positions already mutated in KB_A18, mutations at positions 37, 49, 50, 96, and 97 completely disrupted binding to hTfR1. The mutation at position 47 resulted in a sharp loss of binding, while mutations at positions 58 and 87 resulted in a slight loss of affinity.

[0338] Table 12. Combined data of humanized VHH

[0339]

[0340] X = similar to the control, (X) = decreased binding affinity, (-) = severely decreased binding affinity, - = no obvious binding.

[0341] Table 13. Sequence Information

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353] The above embodiments are to be understood as illustrative examples of the present invention. Those skilled in the art will understand that various modifications, combinations, and changes can be made to the embodiments without departing from the scope of the invention. Specifically, where technically possible, different portions of solutions from different embodiments may be combined in other configurations. However, the scope of the invention is defined by the appended claims.

Claims

1. A heavy chain variable domain-only VHH antibody that specifically binds to transferrin receptor 1TfR1, said antibody comprising: The complementarity-determining region 1CDR1 has an amino acid sequence selected from the group consisting of GSIFGSKR as defined in SEQ ID NO:1 and GSIFGFNA as defined in SEQ ID NO:2; CDR2, having an amino acid sequence selected from the group consisting of ITYRGTT as defined in SEQ ID NO:3 and IAVAGST as defined in SEQ ID NO:4; and CDR3 has an amino acid sequence selected from the group consisting of WMFTTDNY as defined in SEQ ID NO:5 and WMYATANY as defined in SEQ ID NO:6, wherein If CDR1 has the amino acid sequence defined in SEQ ID NO:2, then CDR3 has the amino acid sequence defined in SEQ ID NO:6; The condition is that the VHH antibody does not contain CDR1 having the amino acid sequence defined in SEQ ID NO:2, CDR2 having the amino acid sequence defined in SEQ ID NO:4, and CDR3 having the amino acid sequence defined in SEQ ID NO:

6.

2. The VHH antibody according to claim 1, provided that the VHH antibody does not contain CDR1 having the amino acid sequence defined in SEQ ID NO:2, CDR2 having the amino acid sequence defined in SEQ ID NO:3, and CDR3 having the amino acid sequence defined in SEQ ID NO:

6.

3. The VHH antibody according to claim 1 or 2, wherein... The CDR1 has the amino acid sequence defined in SEQ ID NO:1; The CDR2 has an amino acid sequence selected from the group consisting of SEQ ID NO:3 and 4; and The CDR3 has the amino acid sequence defined in SEQ ID NO:

5.

4. The VHH antibody according to claim 3, wherein... The CDR1 has the amino acid sequence defined in SEQ ID NO:1; The CDR2 has the amino acid sequence defined in SEQ ID NO:4; and The CDR3 has the amino acid sequence defined in SEQ ID NO:

5.

5. The VHH antibody according to claim 3, wherein... The CDR1 has the amino acid sequence defined in SEQ ID NO:1; The CDR2 has the amino acid sequence defined in SEQ ID NO:3; and The CDR3 has the amino acid sequence defined in SEQ ID NO:

5.

6. The VHH antibody according to claim 1, wherein... The CDR1 has an amino acid sequence selected from the group consisting of SEQ ID NO:1 and 2; The CDR2 has the amino acid sequence defined in SEQ ID NO:3; and The CDR3 has the amino acid sequence defined in SEQ ID NO:

6.

7. The VHH antibody according to claim 6, wherein... The CDR1 has the amino acid sequence defined in SEQ ID NO:1; The CDR2 has the amino acid sequence defined in SEQ ID NO:3; and The CDR3 has the amino acid sequence defined in SEQ ID NO:

6.

8. The VHH antibody according to claim 1 or 2, wherein The CDR1 has the amino acid sequence defined in SEQ ID NO:1; The CDR2 has the amino acid sequence defined in SEQ ID NO:4; and The CDR3 has the amino acid sequence defined in SEQ ID NO:

6.

9. The VHH antibody according to any one of claims 1 to 8, wherein The VHH antibody has the following formula: Frame region 1, FR1,-CDR1-FR2-CDR2-FR3-CDR3-FR4; FR1 has the amino acid sequence QVQLQESGGG SVQAGGSLSLSCAAS as defined in SEQ ID NO:57; FR2 has the amino acid sequence MGWFRQAPG EQRDVVAT as defined in SEQ ID NO:58; FR3 has the amino acid sequence defined in SEQ ID NO:59: EYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYC; and FR4 has the amino acid sequence WGQGTQVTVSS as defined in SEQ ID NO:

22.

10. 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: 23 to 26.

11. A heavy chain variable domain-only VHH antibody that specifically binds to transferrin receptor 1TfR1, said antibody comprising: The complementarity-determining region 1CDR1 is composed of the amino acid sequence X1X2IX3GSKR as defined in SEQ ID NO:7, wherein X1 is G or E, X2 is S, D or I, and X3 is F or N. CDR2, which consists of the amino acid sequence ITX4X5GTT as defined in SEQ ID NO:8, wherein X4 is Y or V, and X5 is R, H, or G; and CDR3 consists of the amino acid sequence WMFTTX6NY as defined in SEQ ID NO:9, where X6 is D, T, or N.

12. The VHH antibody according to claim 11, wherein... The CDR1 consists of the amino acid sequence X1X2IX3GSKR defined in SEQ ID NO:7, where X1 is G or E, X2 is D or I, and X3 is F or N; The CDR2 consists of the amino acid sequence ITX4X5GTT defined in SEQ ID NO:8, where X4 is Y or V, and X5 is R, H, or G; and The CDR3 consists of the amino acid sequence WMFTTX6NY defined in SEQ ID NO:9, where X6 is D, T or N.

13. The VHH antibody according to claim 12, wherein... The CDR1 consists of the amino acid sequence GDIX3GSKR defined in SEQ ID NO:11, where X3 is F or N; The CDR2 consists of the amino acid sequence ITVX5GTT defined in SEQ ID NO:12, where X5 is R or G; and The CDR3 consists of the amino acid sequence WMFTTX6NY defined in SEQ ID NO:9, where X6 is T or N.

14. The VHH antibody according to claim 13, wherein... The CDR1 consists of the amino acid sequence GDINGSKR defined in SEQ ID NO:13; The CDR2 consists of the amino acid sequence ITRRGTT as defined in SEQ ID NO:14; and The CDR3 consists of the amino acid sequence WMFTTTNY as defined in SEQ ID NO:

10.

15. The VHH antibody according to claim 13, wherein... The CDR1 consists of the amino acid sequence GDIFGSKR defined in SEQ ID NO:15; The CDR2 consists of the amino acid sequence ITVGGTT defined in SEQ ID NO:16; and The CDR3 consists of the amino acid sequence WMFTTNNY as defined in SEQ ID NO:

54.

16. The VHH antibody according to claim 12, wherein... The CDR1 consists of the amino acid sequence EIINFGSKR defined in SEQ ID NO:17; The CDR2 consists of the amino acid sequence ITYHGTT defined in SEQ ID NO:18; and The CDR3 consists of the amino acid sequence WMFTTDNY as defined in SEQ ID NO:

5.

17. The VHH antibody according to any one of claims 11 to 16, wherein The VHH antibody has the following formula: Frame region 1, FR1,-CDR1-FR2-CDR2-FR3-CDR3-FR4; FR1 has the amino acid sequence QVQLQESGGG X7VQAGGSLX8LSCAAS as defined in SEQ ID NO:19, where X7 is S or L and X8 is S or R. FR2 has the amino acid sequence MGWFRQAPG X9X as defined in SEQ ID NO:

20. 10 RDX 11 VAT, where X9 is E, K, or Q, preferably K or Q, X 10 It is either Q or A, and X 11 V or L; FR3 has the amino acid sequence X as defined in SEQ ID NO:

21. 12 YX 13 DSVKG RFTISRDNAX 14 NTVYLQMNX 15 LKPEDTAX 16 YYC, where X 12 For E or K, X 13 For A or E, X 14 For K or N, X 15 It is N or S, and X 16 It is V or F; and FR4 has the amino acid sequence WGQGTQVTVSS as defined in SEQ ID NO:

22.

18. The VHH antibody according to claim 11, wherein the VHH antibody has an amino acid sequence selected from the group consisting of SEQ ID NO:23, 27 to 29, preferably selected from the group consisting of SEQ ID NO:27 to 29.

19. The VHH antibody according to any one of claims 1 to 18, wherein the VHH antibody specifically binds to human TfR1.

20. The VHH antibody of claim 19, wherein the VHH antibody specifically binds to the apical domain of the human TfR1.

21. The VHH antibody according to claim 19 or 20, wherein the VHH antibody has an affinity K selected from the range of 0.1 nM to 150 nM, preferably from 0.1 nM to 100 nM. D Combined with the aforementioned human TfR1.

22. The VHH antibody according to any one of claims 1 to 21, wherein the VHH antibody specifically binds to cynomolgus monkey TfR1.

23. The VHH antibody according to any one of claims 1 to 22, wherein the VHH antibody is a camel VHH antibody.

24. The VHH antibody according to any one of claims 1 to 22, wherein the VHH antibody is a humanized VHH antibody.

25. The VHH antibody of claim 24, wherein the VHH antibody comprises: According to the frame region 1FR1 of X1VQLX2ESGGGX3VQX4GGSL X5LSCAAS as defined in SEQ ID NO:78; According to FR2 of MGWFRQAPGX6X7X8X9VVATFR2 as defined in SEQ ID NO:79: According to EYADSVKGRFTISRDNX defined in SEQ ID NO:80 10 KNTX 11 YLQMNX 12 LX 13 FR3 of PEDTAVYYC; and According to WGQGTX as defined in SEQ ID NO:81 14 VTVSS FR4, of which X1 to X 14 At least one of them satisfies X1=E, X2=V, X3=L, X4=P, X5=R, X6=K, X7=G, X8=L, X9=E, X 10 =S,X 11 =I,X 12 =S,X 13 =R and X 14 =L; and X1 to X 14 Any remaining one of the following satisfies X1=Q, X2=Q, X3=S, X4=A, X5=S, X6=E, X7=Q, X8=R, X9=D, X 10 =A, X 11 =V,X 12 =N, X 13 =K and X 14 =Q.

26. The VHH antibody according to any one of claims 1 to 25, wherein the VHH antibody binds to protein A.

27. The VHH antibody according to claim 26, wherein the VHH antibody has a frame region 1FR1 according to QVQLQESGGGSVQAGGSLRLSCAAS according to SEQ ID NO:82, and a frame region 1FR3 according to EYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC according to SEQ ID NO:

83.

28. A fusion molecule comprising a VHH antibody according to any one of claims 1 to 27 linked to at least one molecule.

29. The fusion molecule of claim 28, wherein the fusion molecule comprises a VHH antibody of any one of claims 1 to 27, which is linked to the at least one molecule via a linker.

30. The fusion molecule according to claim 28 or 29, wherein the at least one molecule is selected from the group consisting of therapeutic agents and imaging agents.

31. The fusion molecule of claim 30, wherein the therapeutic agent is capable of treating central nervous system diseases or conditions.

32. The fusion molecule according to claim 30, wherein the imaging agent is selected from the group consisting of positron emission tomography tracers, single-photon emission computed tomography tracers, fluorescent probes, luminescent probes, probes containing metal complexes, and near-infrared fluorescent probes.

33. A pharmaceutical composition comprising: The fusion molecule according to any one of claims 28 to 31, wherein the at least one molecule is a therapeutic agent; and Pharmaceutically acceptable mediator.

34. A nucleic acid molecule encoding a VHH antibody according to any one of claims 1 to 27 or a fusion molecule according to any one of claims 28 to 31.

35. An expression vector comprising a nucleic acid molecule according to claim 34 operably linked to a promoter.

36. A host cell comprising the nucleic acid molecule of claim 34 or the expression vector of claim 35.

37. The fusion molecule according to any one of claims 28 to 31, used as a pharmaceutical agent, wherein the at least one molecule is a therapeutic agent.

38. The fusion molecule according to any one of claims 28 to 31, for treating diseases or conditions of the central nervous system (CNS), wherein the at least one molecule is a therapeutic agent capable of treating the CNS disease or condition.

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