Antibody passing through the blood-cerebrospinal fluid barrier

Abstract

The present invention relates to a binder that specifically binds to a folate transport complex. More specifically, an antibody or antibody fragment is disclosed that includes an immunoglobulin single variable domain (ISVD) antibody that binds to human folate receptor alpha (hFOLRα) present in choroid plexus epithelial cells. The present invention further relates to the antibodies and methods described herein for use in increasing the delivery of pharmaceutical compounds to the central nervous system through the process of receptor-mediated endocytosis and / or transcytosis.

Description

Technical Field

[0001] Field of the Invention The present invention relates to a binder that specifically binds to a folate transport complex. More specifically, an antibody or antibody fragment is disclosed that includes an immunoglobulin single variable domain (ISVD) antibody that binds to human folate receptor alpha (hFOLRα) present in choroid plexus epithelial cells. The present invention further relates to the antibodies and methods described herein for use in increasing the delivery of pharmaceutical compounds to the central nervous system through the process of receptor-mediated endocytosis and / or transcytosis.

Background Art

[0002] Background The development of drugs for the central nervous system (CNS) has proven to be extremely difficult. Historically, new CNS drugs have been plagued by a significantly lower success rate during development compared to drugs for indications outside the CNS. One of the main reasons is the existence of the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB), which are uniquely located in the brain microvascular endothelium. Such blood-brain interfaces significantly limit the bioavailability of pharmaceutical compounds in the brain. For example, due to the limited permeability of antibodies and small molecule compounds, even if they are effective, a large amount of these compounds needs to be administered to obtain a certain degree of effect. High-dose administration not only poses a risk of causing peripheral side effects to patients but also has an adverse impact on social costs. It can also strain the antibody production capacity, for example, in large-scale indications with millions of patients such as Alzheimer's disease (AD) and multiple sclerosis (MS), where the antibody production capacity can be an important limiting factor. Although numerous attempts have been made to find means and methods for efficiently shuttle transporting compounds across the BBB (for example, International Publication No. WO2015031673; International Publication No. WO2014033074; International Publication No. WO2015124540; International Publication No. WO2015191934), research on BCSFB transporters has not been extensively conducted. The BCSFB is located in the choroid plexus and is a highly vascularized structure that protrudes into the ventricles filled with cerebrospinal fluid (CSF). It consists of a single layer of choroid plexus epithelial (CPE) cells and the surrounding stroma and fenestrated capillaries. CPE cells are tightly connected by tight junctions and form a blood-CSF barrier that restricts the passage of molecules that can freely diffuse from the fenestrated capillaries into the stroma and into the brain parenchyma. The cells are polarized, with numerous microvilli on the apical side and numerous invaginations on the basal side to increase the surface area for ultrafiltrate of CSF and plasma, respectively.The most important function of the choroid plexus following the formation of the blood-CSF barrier is the transport of nutrients, ions, gases, proteins, and metabolites between the fenestrated choroidal vessels and the CNS.

[0003] The transcytosis pathway (e.g., receptor-mediated transcytosis) has attracted great interest in the field of CNS delivery because it has the potential to deliver large cargos, including pharmacological agents. One of the targets that may be found on the basolateral side of CPE cells is the folate receptor α (FRα) (Grapp et al 2013 Nat Comm 4:2123; Strazielle & Ghersi-Egea 2016 Curr Pharmaceut Design 22: 5463-5476). Therefore, it would be advantageous to hijack the folate transport system in the BCSFB in order to enhance the bioavailability of pharmacological compounds in the brain. SUMMARY OF THE INVENTION

[0004] Summary The present application discloses single domain antibodies, more particularly VHHs, that bind to the human folate receptor alpha (FRα), including the human FRα present in CPE cells. Accordingly, the antibodies described herein may be applied to deliver compounds, including therapeutic and / or diagnostic antibodies and small molecules, across the BCSFB following a single systemic administration to a mouse.

[0005] Thus, in a first aspect, the dissociation constant k off is less than 3x10 -2 / s, more particularly koff is 3x10 -2 ~1x10 -3Provided are folate receptor alpha (FRα) binders that can bind to human FRα at 1 / s. koff is measured by the biolayer interferometry method. In one aspect, the binder specifically binds to a human FRα epitope containing amino acid Q141 of SEQ ID NO: 1, and more specifically, to the following residues of SEQ ID NO: 1: R98, H99, E137, D138, Q141, E144, D145, R204, G205, Q211, W213, F214, D215, A217 and / or at least one or more of Q218, or binds to an epitope on FRα containing all of the residues. In the present invention, those epitope-binding ISVDs are characterized by containing a CDR3 sequence as depicted in SEQ ID NO: 5. In another aspect, the binding of the FRα binder to human FRα does not interfere with folic acid binding and / or folic acid transport by the human FRα. In another aspect, the binder can cross-react with primate and mouse FRα. Furthermore, the present invention has surprisingly revealed that the FR3 region, more specifically a portion of the so-called CDR4 loop, is important for the higher-order structural requirements for passing through the BCSFB, where the region is limited to FRs in which positions 72 and 73 are defined as amino acids D, E, P and N, G, respectively.

[0006] Thus, in certain aspects, in order to provide an agent for passing through the BCSFB, the ISVD contains a paratope consisting of the amino acid residues F29, S30, G31 and I33 of CDR1, the amino acid residues T52, S53, H54 and T56 of CDR2, the amino acid residues H95, F96, P97, G98, I101 and Y102 of CDR3, and / or the amino acid residues D72 and / or N73 of CDR4 according to Kabat numbering.

[0007] In certain embodiments, any of the above FRα binders are also provided to facilitate, enable, or improve the uptake of the biological or chemical entity to which it is conjugated into the cerebrospinal fluid (CSF) via the blood-CSF barrier (BCSFB). In another particular embodiment, the FRα binder also promotes the transport of the moiety to which it is conjugated into FRα-expressing cancer cells or improves the binding of the moiety to cancer cells expressing FRα. In another particular embodiment, the FRα binder comprises or consists of an immunoglobulin single variable domain or VHH.

[0008] In a second aspect, a blood-central nervous system (CNS) barrier shuttle comprising any of the above FRα binders is provided, and in a third aspect, any of the above FRα binders and blood-CNS barrier shuttles are provided for use as a medicament, more particularly for transporting one or more compounds across the CNS, more particularly the BCSFB. Also, any of the above FRα binders and blood-CNS barrier shuttles are provided for use in the treatment of neurological disorders. In certain embodiments, the neurological disorder is selected from the list consisting of Alzheimer's disease, stroke, dementia, muscular dystrophy, multiple sclerosis, amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, dystonia, Parkinson's disease, viral or microbial infection, inflammation, brain cancer, neuropathic pain, and traumatic brain injury.

[0009] In a fourth aspect, a composition for use in the treatment or diagnosis of a neurological disorder is provided, the composition comprising a human FRα binder conjugated to a neurological disorder drug or imaging compound, wherein the composition has a dissociation constant koff of less than 3x10 -2 / s, more particularly from 3x10 -2 to 1x10 -3It binds to human FRα with koff between / s. In certain embodiments, the neurodisease agent is a biological, small molecule, therapeutic agent, antisense oligonucleotide or test compound. In one embodiment, the binding to human FRα does not interfere with folate binding and / or transport by the human FRα. In another embodiment, the human FRα binder from the composition can cross-react with primate and mouse FRα. In yet another embodiment, the human FRα binder recognizes the same epitope on human FRα as an FRα binder consisting of the sequence as depicted in SEQ ID NO: 2. In another specific embodiment, the composition is a bispecific antibody comprising the human FRα binder and a second antigen-binding site that binds to a brain antigen. In a more specific embodiment, the brain antigen is selected from the group consisting of beta-secretase 1 (BACE1), amyloid beta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase 6. In yet another embodiment, the FRα binder from the composition comprises or consists of an immunoglobulin single variable domain or VHH.

[0010] Note The project leading to this application has received funding from the European Union's Horizon 2020 research and innovation program (grant agreement No. 721058).

Brief Description of the Drawings

[0011] Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0012] Detailed Description Definitions To make this specification easier to understand, certain terms are first defined. Further definitions are set forth throughout the detailed description. While the present invention is described with respect to specific embodiments and with reference to certain drawings, the invention is not limited thereto and is limited only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described herein are only schematic and non-limiting. In the drawings, the sizes of some elements may be exaggerated and not drawn to scale for illustrative purposes. Note that the terms "a" or "an" refer to one or more entities; for example, a "nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein. Further, as used herein, "and / or" is considered to be specifically disclosed for each of the two specified features or components, whether or not accompanied by the other. Thus, the term "and / or" as used in expressions such as "A and / or B" herein is intended to include "A and B", "A or B", "A (alone)", and "B (alone)". Similarly, the term "and / or" as used in expressions such as "A, B, and / or C" is intended to encompass each of the following aspects: namely, A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). When an indefinite or definite article (by way of example, "a" or "a", "the") is used to refer to a singular noun, this includes the plural of that noun unless something else is specifically stated. Further, the terms first, second, third, and other terms of the same kind in the specification and claims are used to distinguish like elements and are not necessarily for the purpose of describing a sequential or chronological order.The terms used in this way are interchangeable in appropriate circumstances, and it should be understood that the aspects of the invention described herein may operate in orders other than those described or illustrated herein.

[0013] In this specification, when an aspect or embodiment is described with the expression "comprising", it is understood that similar aspects or embodiments described with the expressions "consisting of" and / or "consisting essentially of" are also provided. When the term "comprising" is used in this specification and the claims, it does not exclude other elements or steps. Unless specifically defined herein, all terms used herein have the same meaning to those skilled in the art of the present invention.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide a general dictionary of many of the terms used in this disclosure to one of ordinary skill in the art. Those of skill should particularly refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., current Protocols in Molecular Biology (Supplement 100), John Wiley & Sons, New York (2012) for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than is understood by one of ordinary skill in the art.

[0015] Units, prefixes, and symbols are expressed in the form approved by the International System of Units (SI). Numerical ranges include the numbers defining the range. Unless otherwise specified, nucleotide sequences are indicated left to right in the 5' to 3' direction. Amino acid sequences are written left to right in the amino to carboxy direction. The headings provided herein do not limit the various aspects of the disclosure, which can be obtained by referring to the entire specification. Accordingly, the terms immediately defined below are more fully defined by reference to the entire specification.

[0016] As used herein, the term "about" is used to mean approximately, roughly, around, or in that region. When the term "about" is used with a numerical range, it modifies that range by extending the boundaries above and below the defined number. Generally, the term "about" can modify a numerical value by the variance above and below the recited value. For example, if the dissociation constant koff is about 1.50x10 -2 / s, it means that koff is in the range of 1.45x10 -2 ~1.55x10 -2 / s.

[0017] This application relates to antibodies that bind to folate receptors in mice, primates, and humans.

[0018] As used herein, the term "antibody" refers to an immunoglobulin (Ig) molecule or a molecule containing an immunoglobulin (Ig) domain that specifically binds to an antigen. An "antibody" can be an intact immunoglobulin derived from a natural or recombinant source and can be the immunoreactive portion of an intact immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. As used herein, the term "immunoglobulin (Ig) domain" refers to a globular region of an antibody chain or a polypeptide consisting essentially of such a globular region. Immunoglobulin domains are characterized by holding an immunoglobulin fold (named Ig fold herein) typical of antibody molecules, which fold consists of a two-layer sandwich of about 7-9 antiparallel β-strands arranged in two β-sheets and is optionally stabilized by conserved disulfide bonds. The term "immunoglobulin (Ig) domain" encompasses "immunoglobulin constant domains" and "immunoglobulin variable domains" (abbreviated as "IVD"), where the latter refers to an immunoglobulin domain consisting essentially of four "framework regions" (hereinafter referred to as "framework region 1" or "FR1"; "framework region 2" or "FR2"; "framework region 3" or "FR3"; and "framework region 4" or "FR4" in the art and herein, respectively); these framework regions are interrupted by three "complementary determining regions" or "CDRs" (hereinafter referred to as "complementary determining region 1" or "CDR1"; "complementary determining region 2" or "CDR2"; and "complementary determining region 3" or "CDR3" in the art and herein, respectively). Thus, the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulin variable domain(s) (IVD) that confers specificity for an antigen on an antibody by possessing an antigen-binding site.

[0019] The determination of CDR regions may be performed according to various methods, such as those based on contact analysis and designation based on binding site topography, as described by MacCallum et al. (J. Mol. Biol. (1996) 262, 732-745). Alternatively, the annotation of CDRs may be performed according to AbM (AbM is an antibody modeling package of Oxford Molecular Ltd., described at http: / / www.bioinf.org.uk / abs / index.html), Chothia (Chothia and Lesk, 1987; Mol Biol. 196: 901-17), Kabat (Kabat et al., 1991; 5th edition, NIH publication 91-3242), and IMGT (LeFranc, 2014; Frontiers in Immunology. 5(22): 1-22). The annotation further encompasses the depiction of CDR and framework regions (FRs) in immunoglobulin domain-containing proteins and is thus a method and system known to those skilled in the art that can apply these annotations to any immunoglobulin protein sequence without undue burden. These annotations are slightly different but each is intended to include the region of the loop involved in target binding.

[0020] The "immunoglobulin domain" of the present application also encompasses an "immunoglobulin single variable domain" (abbreviated as "ISVD") equivalent to the term "single variable domain", which defines a molecule in which the antigen-binding site is present on a single immunoglobulin domain and is formed by the single immunoglobulin domain. Thus, the immunoglobulin single variable domain is set apart from the "conventional" immunoglobulins or fragments thereof in which two immunoglobulin domains, particularly two variable domains, interact to form an antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form an antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both VH and VL contribute to the antigen-binding site, i.e., a total of six CDRs are involved in the formation of the antigen-binding site. Considering the above definition, the antigen-binding domains of conventional four-chain antibodies (such as IgG, IgM, IgA, IgD, or IgE molecules; known in the art), or Fv fragments such as Fab fragments, F(ab')2 fragments, disulfide-linked Fv or scFv fragments, or diabodies derived from such conventional four-chain antibodies (all known in the art) are not usually considered immunoglobulin single variable domains because the binding to each epitope of the antigen usually occurs not by one (single) immunoglobulin domain but by a pair of (associated) immunoglobulin domains such as the light and heavy chain variable domains, i.e., the VH-VL pair of immunoglobulin domains that jointly bind to the epitope of each antigen. In contrast, an immunoglobulin single variable domain can specifically bind to an epitope of an antigen without pairing with an additional immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain is formed by a single VH / VHH or VL domain. Therefore, the antigen-binding site of an immunoglobulin single variable domain is formed by three or fewer CDRs.Accordingly, a single variable domain can be a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof, as long as the single variable domain can form a single antigen-binding unit (i.e., a functional antigen-binding unit that essentially consists of a single variable domain and does not require interaction with another variable domain to form a functional antigen-binding unit). In one aspect of the invention, the immunoglobulin single variable domain is a heavy chain variable domain sequence (e.g., a VH-sequence); more specifically, the immunoglobulin single variable domain can be a heavy chain variable domain sequence derived from a conventional four-chain antibody or a heavy chain variable domain sequence derived from a heavy chain antibody. For example, the immunoglobulin single variable domain can be a (single) domain antibody (or an amino acid sequence suitable for use as a (single) domain antibody), a "dAb" or dAb (or an amino acid sequence suitable for use as a dAb), or a Nanobody (as defined herein, including but not limited to VHH); other single variable domains, or some suitable fragment thereof. In particular, the immunoglobulin single variable domain can be a Nanobody (as defined herein) or a suitable fragment thereof. Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx N.V. (Sanofi). For a general description of Nanobodies, reference is made to the prior art cited herein, such as the following further description (and, by way of example, those described in WO 2008 / 020079).

[0021] As used herein, the immunoglobulin domain also includes VHH, VHH domain, VHH antibody fragment, and the "VHH domain" also known as VHH antibody, which was originally described as the antigen-binding immunoglobulin (Ig) (variable) domain of "heavy-chain antibody" (i.e., "antibody lacking light chain"; Hamers-Casterman et al (1993) Nature 363: 446-448). The term "VHH domain" is chosen to distinguish these variable domains from the heavy-chain variable domain (referred to herein as "VH domain") present in conventional four-chain antibodies and the light-chain variable domain (referred to herein as "VL domain") present in conventional four-chain antibodies.For further explanation of VHH and Nanobody, reference is made to the review article by Muyldermans (Review in Molecular Biotechnology 74: 277-302, 2001), as well as the following patent applications which are cited as general background art: International Publication No. WO 94 / 04678, International Publication No. WO 95 / 04079 and International Publication No. WO 96 / 34103 of Vrije Universiteit Brussel; International Publication No. WO 94 / 25591, International Publication No. WO 99 / 37681, International Publication No. WO 00 / 40968, International Publication No. WO 00 / 43507, International Publication No. WO 00 / 65057, International Publication No. WO 01 / 40310, International Publication No. WO 01 / 44301, European Patent No. EP 1134231 and International Publication No. WO 02 / 48193 of Unilever; International Publication No. WO 97 / 49805, International Publication No. WO 01 / 21817, International Publication No. WO 03 / 035694, International Publication No. WO 03 / 054016 and International Publication No. WO 03 / 055527 of Vlaams Instituut voor Biotechnologie (VIB); International Publication No. WO 03 / 050531 of Algonomics N.V. and Ablynx N.V.; International Publication No. WO 01 / 90190 by the National Research Council of Canada; International Publication No. WO 03 / 025020 (= European Patent No. EP 1433793) by the Antibody Society; and International Publication No. WO 04 / 041867, International Publication No. WO 04 / 041862, International Publication No. WO 04 / 041865, International Publication No. WO 04 / 041863, International Publication No. WO 04 / 062551, International Publication No. WO 05 / 044858, International Publication No. WO 06 / 40153, International Publication No. WO 06 / 079372, International Publication No. WO 06 / 122786, International Publication No. WO 06 / 122787 and International Publication No. WO 06 / 122825 of Ablynx N.V., as well as further published patent applications by Ablynx N.V. As described in these documents, Nanobodies (in particular VHH sequences and partially humanized Nanobodies) can be characterized in particular by the presence of one or more "characteristic residues" in one or more framework sequences.The humanization and / or camelization of Nanobodies, as well as other modifications, parts or fragments, derivatives or "Nanobody fusions", multivalent constructs (including some non-limiting examples of linker sequences), and further descriptions of Nanobodies including different modifications for extending the half-life of Nanobodies and their preparations can be found, by way of example, in WO 08 / 101985 and WO 08 / 142164.

[0022] "Domain Antibodies", also known as "Dabs", "Domain Antibodies", "dAbs" (the terms "Domain Antibodies" and "dAbs" are used as trademarks by GlaxoSmithKline group companies), are described, by way of example, in European Patent No. 0368684, Ward et al. (Nature 341:544 - 546, 1989), Holt et al. (Tends in Biotechnology 21:484 - 490, 2003), WO 03 / 002609, and published patent applications such as WO 04 / 068820, WO 06 / 030220, WO 06 / 003388 of Domantis, etc., and are trademarks of GlaxoSmithKline. Domain Antibodies correspond essentially to the VH or VL domains of non-camelid mammals, particularly human four-chain antibodies. In order to bind an epitope as a single antigen-binding domain, i.e., without pairing with a VL or VH domain respectively, it is necessary to specifically select such antigen-binding properties, for example, by using a library of human single VH or VL domain sequences. Domain Antibodies, like VHHs, have a molecular weight of about 13 to about 16 kDa and, if derived from a fully human sequence, do not need to be humanized, for example, for use in human therapy. It should also be noted that a single variable domain can also be derived from certain sharks (see, for example, the so-called "IgNAR domain", for example, WO 05 / 18629).

[0023] Immunoglobulin single variable domains, such as Domain Antibodies and Nanobodies (including VHH domains and humanized VHH domains), exhibit macromolecules that have matured in vivo during their production, but affinity maturation can be further performed by introducing one or more modifications into the amino acid sequences of one or more CDRs, and these modifications improve the affinity of the resulting immunoglobulin single variable domain for each antigen compared to their respective parental molecules. The affinity matured immunoglobulin single variable domain molecules of the present invention may be prepared, for example, by methods known in the art as described by Marks et al. (Biotechnology 10:779-783, 1992), Barbas et al. (Proc. Nat. Acad. Sci, USA 91:3809-3813, 1994), Shier et al. (Gene 169:147-155, 1995), Yelton et al. (Immunol. 155:1994-2004, 1995), Jackson et al. (J. Immunol. 154:3310-9, 1995), Hawkins et al. (J. Mol. Biol. 226:889 896, 1992), Johnson and Hawkins (Affinity maturation of antibodies using phage display, Oxford University Press, 1996). Starting from an immunoglobulin single variable domain such as a Domain antibody or Nanobody, the process of designing / selecting and / or preparing a polypeptide is also referred to herein as "formatting" the immunoglobulin single variable domain; the immunoglobulin single variable domain that is part of the polypeptide is said to be "formatted" or in the "formatted state" of the polypeptide. Examples of ways in which an immunoglobulin single variable domain can be formatted, and examples of such formatting to avoid glycosylation as an example, will be apparent to those skilled in the art based on the disclosure herein.

[0024] Immunoglobulin single variable domains, such as Domain Antibodies and Nanobodies (including VHH domains), can be humanized, i.e., the degree of sequence identity with the closest human germline sequence can be increased. In particular, humanized immunoglobulin single variable domains, such as Nanobodies (including VHH domains), are as generally defined in the previous paragraph, but have humanized substitutions (as further defined herein) and / or corresponding thereto, and may be immunoglobulin single variable domains in which at least one amino acid residue (in particular, at least one framework residue) is present. Potentially useful humanized substitutions can be identified by comparing the sequence of the framework region of a naturally occurring VHH sequence with the corresponding framework sequences of one or more closely related human VH sequences, and then one or more of the potentially useful humanized substitutions (or combinations thereof) thus determined can be introduced into the VHH sequence (by any method known per se as further described herein), and the resulting humanized VHH sequence can be tested for affinity for the target, stability, ease and level of expression, and / or other desired properties. In this way, with a limited amount of trial and error, other or appropriate humanized substitutions (or appropriate combinations thereof) can be determined by those skilled in the art. Also, based on the foregoing, the framework region of an immunoglobulin single variable domain, such as a Nanobody (including a VHH domain), may be partially humanized or fully humanized. It should be noted that the immunoglobulin single variable domain and the antigen-binding chimeric protein of the present invention in a broad sense are not limited to a specific biological source or a specific preparation method.For example, and not for limiting purposes, immunoglobulin single variable domains, in particular the antigen-binding chimeric proteins of the present invention, can generally be obtained by: (1) isolating the VHH domain of a naturally occurring heavy chain antibody and further engineering the sequence to obtain an antigen-binding chimeric protein; (2) expressing a nucleotide sequence encoding a naturally occurring VHH domain in a format fused to the scaffold protein of the antigen-binding chimeric protein; (3) "humanizing" the naturally occurring VHH domain and / or scaffold protein, or such humanized VHH domain and / or scaffold protein, and / or expressing a nucleic acid encoding an antigen-binding chimeric protein; (4) "mutating" the naturally occurring VHH domain to reduce binding to existing antibodies, or engineering the fusion site of the scaffold protein to obtain an antigen-binding chimeric protein of the present invention with reduced binding to existing antibodies compared to native VHH; or (5) by a method using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se.

[0025] For the numbering of amino acid residues in an IVD, different numbering schemes can be applied. For example, it can be numbered according to the AHo numbering scheme for all heavy chain (VH) and light chain variable domains (VL) applied by Honegger, A. and Pluckthun, A. (J. Mol. Biol. 309, 2001) to the VHH domain of camelids. Another method for numbering the amino acid residues of the VH domain can be applied in a similar manner to the VHH domain and is known in the art. For example, by using the Kabat numbering system applied to the VHH domain of camelids in the paper by Riechmann, L. and Muyldermans, S., 231(1-2), J Immunol Methods. 1999, the FR and CDR sequences can be delimited. As is well known in the art for VH domains and VHH domains, the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions according to Kabat numbering may not be occupied by the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by Kabat numbering), and this should be noted. This generally means that the numbering by Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. The total number of amino acid residues in the VH domain and the VHH domain is usually in the range of 110 to 120, often between 112 and 115. However, it should be noted that for the purposes described herein, smaller and longer sequences may also be appropriate.

[0026] As used herein, an "epitope" refers to an antigenic determinant of a polypeptide that constitutes a binding site or binding pocket on a target molecule. An epitope can include 1, 2, or 3 amino acids in the unique spatial higher-order structure of the epitope. Generally, an epitope consists of at least 4, 5, 6, 7 such amino acids, and more usually at least 8, 9, 10 such amino acids. Methods for determining the spatial higher-order structure of amino acids are known in the art and include, for example, X-ray crystallography and multidimensional nuclear magnetic resonance. As used herein, a "higher-order structure epitope" refers to an epitope that includes amino acids of a spatial higher-order structure unique to the folded three-dimensional higher-order structure of a polypeptide. Generally, a higher-order structure epitope is composed of amino acids that are discontinuous in the linear sequence but come together in the folded structure of the protein. However, a higher-order structure epitope may also be composed of a linear sequence of amino acids that adopt a higher-order structure unique to the folded three-dimensional higher-order structure of the polypeptide (and do not exist in the denatured state). In a protein complex, a higher-order structure epitope is composed of amino acids that are discontinuous in the linear sequences of one or more polypeptides and come together when the variously folded polypeptides fold and associate in a unique quaternary structure. Similarly, a higher-order structure epitope may also consist of the linear sequences of amino acids of one or more polypeptides, and they come together to adopt a higher-order structure unique to the quaternary structure. The terms "higher-order structure" or "higher-order structure state" of a protein generally refer to the range of structures that a protein may adopt at a given moment. Those skilled in the art will recognize that the determinants of the higher-order structure or higher-order structure state include the amino acid sequence of the protein (including modified amino acids) and the primary structure of the protein reflected in the environment surrounding the protein. The higher-order structure or higher-order structure state of a protein is also related to structural features such as the secondary structure of the protein (e.g., α-helix, β-sheet, among others), the tertiary structure (e.g., the three-dimensional folding of the polypeptide chain), and the quaternary structure (e.g., the interaction of the polypeptide chain with other protein subunits).In particular, post-translational and other modifications to the polypeptide chain, such as ligand binding, phosphorylation, sulfation, glycosylation, or addition of hydrophobic groups, can affect the higher-order structure of the protein. Furthermore, environmental factors such as, in particular, the pH, salt concentration, ionic strength, osmotic pressure of the surrounding solution, and interactions with other proteins and cofactors can also affect the higher-order structure of the protein. The higher-order structure state of a protein may be determined by functional assays regarding activity and binding to other molecules, or by physical methods such as X-ray crystallography, NMR, spin labeling. For general considerations regarding the higher-order structure and higher-order structure state of proteins, see Cantor and Schimmel, Biophysical Chemistry, Part I: The Conformation of Biological Macromolecules, W.H. Freeman and Company, 1980, and Creighton, Proteins: Structures and Molecular Properties, W.H. Freeman and Company, 1993.

[0027] As used herein, the term "affinity" generally refers to the degree to which an antibody or other binding protein (further defined herein) binds to a target protein so as to shift the equilibrium of the target protein and the binding protein towards the existence of the complex formed by their binding. Thus, for example, when an antibody and an antigen are combined at relatively equal concentrations, a high-affinity antibody binds to the antigen and shifts the equilibrium in the direction of increasing the concentration of the resulting complex. The equilibrium dissociation constant K dis (also referred to as K D in this specification) is generally used to represent the affinity of a ligand for a target protein, or an antibody for an antigen. K dis is the calculated ratio of k off / k on n between an antibody and its antigen. The association constant (k on ) is used to evaluate the rate at which an antibody binds to a target. The dissociation constant (k off) is used to measure how quickly an antibody dissociates from a target and is expressed as the number of units dissociating from the target per second. Thus, the lower the k off , the higher the affinity for the target. k off , and thus K dis is also inversely proportional to the affinity. High-affinity interactions are characterized by a low K dis , a fast recognition rate (high k on ), and high stability of the formed complex (low k off ).

[0028] Within the scope of this application, the term "affinity" is used in the context of an antibody or antibody fragment that binds to an epitope of the folate receptor FRα. More specifically, it will be understood that the antibody or antibody fragment binds to its target via the paratope of its immunoglobulin (Ig) domain (typically with one or more CDRs) and is "functional".

[0029] As used herein, "amino acid" refers to the structural units (monomers) that make up proteins. These combine to form short polymer chains called peptides, or long chains called polypeptides or proteins. These chains are linear and unbranched, and each amino acid residue within the chain is attached to two adjacent amino acids. The 20 amino acids encoded by the universal genetic code are naturally incorporated into polypeptides and are called proteinogenic amino acids or natural amino acids. Natural amino acids or naturally occurring amino acids are glycine (Gly or G), alanine (Ala or A), valine (Val or V), leucine (Leu or L), isoleucine (Ile or I), methionine (Met or M), proline (Pro or P), phenylalanine (Phe or F), tryptophan (Trp or W), serine (Ser or S), threonine (Thr or T), asparagine (Asn or N), glutamine (Gln or Q), tyrosine (Tyr or Y), cysteine (Cys or C), lysine (Lys or K), arginine (Arg or R), histidine (His or H), aspartic acid (Asp or D), and glutamic acid (Glu or E).

[0030] As used herein, the terms "nucleic acid", "nucleic acid sequence" or "nucleic acid molecule" are used interchangeably and refer to polymers of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or analogs thereof. Nucleic acids may have any three-dimensional structure and may perform any function, whether known or unknown. Non-limiting examples of nucleic acids include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes and primers. Nucleic acid molecules may be linear or circular. Nucleic acids may include a promoter, intron, enhancer region, polyadenylation site, translation initiation site, 5' or 3' untranslated region, reporter gene, selectable marker, etc. Nucleic acids may include single-stranded or double-stranded DNA or RNA. Nucleic acids may include modified bases or modified backbones. Nucleic acids up to about 100 nucleotides in length are often also referred to as oligonucleotides. As used herein, "nucleotide" refers to the building blocks of oligonucleotides and polynucleotides and includes both naturally occurring and non-naturally occurring nucleotides for the purposes of the present invention. In nature, nucleotides such as DNA and RNA nucleotides contain a ribose sugar moiety, a nucleobase moiety, and one or more phosphate groups (not present in nucleosides). Nucleotides without a phosphate group are called "nucleosides" and are compounds that contain a nucleobase moiety and a sugar moiety. As used herein, "nucleobase" means a group of atoms that can be linked to a sugar moiety to form a nucleoside that can be incorporated into an oligonucleotide and that can bind to a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Naturally occurring RNA and DNA nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0031] As used herein, the terms "nucleotide sequence", "DNA sequence" or "nucleic acid molecule(s)" refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double-stranded and single-stranded DNA, (reverse) complementary DNA, and RNA. It also includes known types of modifications, such as methylation, and one or more "cap" substitutions of naturally occurring nucleotides with analogs. "Nucleic acid construct" means a nucleic acid sequence constructed to contain one or more functional units not found together in nature. Examples include circular, linear, double-stranded, episomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences derived from lambda phage), viral genomes containing non-native nucleic acid sequences, and the like. A "coding sequence" is a nucleotide sequence that, when placed under the control of appropriate regulatory sequences, is transcribed into mRNA and / or translated into a polypeptide. The boundaries of the coding sequence are determined by a translation start codon at the 5' end and a translation stop codon at the 3' end. The coding sequence can include, but is not limited to, mRNA, cDNA, recombinant nucleotide sequences, or genomic DNA, and may also contain introns under certain circumstances.

[0032] As used herein, an "expression cassette" includes any nucleic acid construct capable of inducing the expression of a gene / coding sequence of interest and is operably linked to a promoter of the expression cassette. An expression cassette generally preferably (from 5' to 3' in the transcription direction): a promoter region, a polynucleotide sequence operably linked to a transcription start region, a homolog, variant or fragment thereof, and a termination sequence including a stop signal and a polyadenylation signal for RNA polymerase, which is a DNA construct. It is understood that all of these regions must be functional in biological cells such as prokaryotic or eukaryotic cells to be transformed. Preferably, the transcription start region including the RNA polymerase binding site and the promoter region including the polyadenylation signal may be native to the biological cell to be transformed, or the region may be derived from another source that functions in the biological cell. Such a cassette can be constructed as a "vector". The term "vector" or alternatively "vector construct", "expression vector" or "gene transfer vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked and includes any vector known to those skilled in the art, including any suitable type, and examples include plasmid vectors, cosmid vectors, phage vectors such as lambda phage, adenovirus vectors, AAV vectors, viral vectors such as baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), P1 artificial chromosomes (PACs), etc., but are not limited thereto. Expression vectors include plasmids as well as viral vectors and generally contain the desired coding sequence and appropriate DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism (by way of example, bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system.Cloning vectors are generally used to manipulate and amplify a specific desired DNA fragment and may lack the functional sequences necessary for expression of the desired DNA fragment. Construction of expression vectors for use in cell transfection is also well known in the art and can thus be achieved via standard techniques (e.g., Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), and see the Ambion 1998 Catalog (Ambion, Austin, Tex.).

[0033] The terms "identical" or "identity" percent in the context of two or more nucleic acid or amino acid sequences refer to two or more sequences that are identical when compared and aligned for maximum correspondence (introducing gaps if necessary) without considering conservative amino acid substitutions as part of sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. A variety of algorithms and software known in the art can be used to obtain nucleotide or amino acid sequence alignments.

[0034] The terms "percent sequence identity" or "% sequence identity" or "percent identity" or "% identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences over a comparison window, taking into account any additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is a position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Since gaps are not nucleotides or amino acids, gaps shown in the target sequence are not counted. Similarly, gaps shown in the reference sequence are not counted since nucleotides or amino acids in the target sequence, not the reference sequence, are counted.

[0035] Such non-limiting examples of sequence alignment algorithms are described in Karlin et al., 1990, Proc. Natl. Acad. Sci., 87:2264-2268, modified in Karlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877, and incorporated into the NBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res., 25:3389-3402). In certain aspects, Gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or Megalign (DNASTAR) are more generally available software programs that can be used for sequence alignment. In certain aspects, the percent identity between two nucleotide sequences is determined using the GAP program of the GCG software package (e.g., using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 90, and length weights of 1, 2, 3, 4, 5, or 6). In certain alternative aspects, the percent identity between two amino acid sequences can be determined using the GAP program of the GCG software package incorporating the algorithm of the GAP program in the GCG software package incorporating the Needleman and Wunsch algorithm (J. Mol. Biol. (48):444-453 (1970)) (e.g., using either the BLOSUM 62 matrix or the PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5).Alternatively, in certain aspects, the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4:11-17 (1989)). For example, the percent identity can be determined using the ALIGN program (version 2.0), with PAM120 with residue table, gap length penalty of 12, and gap penalty of 4. One of ordinary skill in the art can determine appropriate parameters for maximum alignment using a particular alignment software. In certain aspects, the default parameters of the alignment software are used.

[0036] One of ordinary skill in the art will understand that the generation of sequence alignments for calculating percent sequence identity is not limited to binary sequence-sequence comparisons derived solely from primary sequence data. Sequence alignments can be derived from multiple sequence alignments. One program suitable for creating multiple sequence alignments is ClustalW2, available from www.clustal.org. Another suitable program is MUSCLE, available from www.drive5.com / muscle / . ClustalW 2 and MUSCLE are also available, for example, from the EBI (European Bioinformatics Institute).

[0037] In certain aspects, the percent identity "X" between a first nucleotide sequence and a second nucleotide sequence is calculated as 100×(Y / Z), where Y is the number of nucleotide residues scored as identical matches in an alignment of the first and second sequences (aligned by visual inspection or a particular sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percent identity between the first and second sequences will be higher than the percent identity between the second and first sequences. Different regions within one polynucleotide target sequence that align with a polynucleotide reference sequence can each have their own percent sequence identity. It should be noted that the values of percent sequence identity are rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, 80.14 are truncated to 80.1, and 80.15, 80.16, 80.17, 80.18, 80.19 are rounded up to 80.2. It should also be noted that the length values are always integers.

[0038] According to the present application, the degree of identity between a given reference nucleotide sequence and a nucleotide sequence that is homologous to the given nucleotide sequence is preferably at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of identity is preferably given for a nucleic acid region that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the full length of the reference nucleic acid sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is preferably given for at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or 200 nucleotides, preferably contiguous nucleotides. In certain embodiments, the degree / percentage of similarity or identity is given for the full length of the reference nucleic acid sequence.

[0039] As used herein, the term "amino acid identity" refers to the degree to which sequences are identical amino acid by amino acid over a comparison window. In this way, the "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) exist in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity. According to the present application, the degree of identity between a given reference amino acid sequence and an amino acid sequence that is a homolog of the given amino acid sequence is preferably at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of identity is preferably given for an amino acid sequence region that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the full length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of identity is preferably given for at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or 200 amino acids, preferably for contiguous amino acids. In certain embodiments, the degree / percentage of similarity or identity is given for the full length of the reference amino acid sequence.

[0040] A "homolog(s)" of a protein includes peptides, oligopeptides, polypeptides, proteins, and enzymes that have amino acid substitutions, deletions, and / or insertions compared to the unmodified protein in question and have biological and functional activities similar to those of the unmodified protein from which they are derived.

[0041] As used herein, the terms "defined by SEQ ID NO.", "present in SEQ ID NO. X", or "depicted in SEQ ID NO. X" refer to a biological sequence consisting of the amino acid or nucleotide sequence shown in SEQ ID NO. X. By way of example, a protein that is in / defined by SEQ ID NO. X includes the amino acid sequence shown in SEQ ID NO. X. A further example is an amino acid sequence that includes SEQ ID NO. X, which is an amino acid sequence that is longer than the amino acid sequence shown in SEQ ID NO. X but consists entirely of the amino acid sequence shown in SEQ ID NO. X (where the amino acid sequence shown in SEQ ID NO. X can be located at the N-terminus or C-terminus of the longer amino acid sequence or can be embedded within the longer amino acid sequence), or an amino acid sequence consisting of the amino acid sequence shown in SEQ ID NO. X.

[0042] The term "in vivo medical imaging" refers to the techniques and processes used to visualize the inside of a living organism (or a part and / or function thereof) for clinical purposes (e.g., disease diagnosis, prognosis prediction, treatment monitoring, etc.) or for medical research (e.g., anatomy and physiology research, etc.). Examples of medical imaging methods include invasive methods such as intravascular ultrasound (IVUS), as well as non-invasive methods such as magnetic resonance imaging (MRI), ultrasound (US), and nuclear medicine imaging methods. Examples of nuclear medicine imaging include positron emission tomography (PET) and single photon emission computed tomography (SPECT). In a preferred embodiment, nuclear medicine imaging methods are used for in vivo medical imaging. According to a specific embodiment, in vivo pinhole SPECT / micro-CT (computed tomography) imaging is used as an in vivo imaging approach.

[0043] As used herein, the term "radionuclide" relates to radioactive labels and is a chemical compound in which one or more atoms are replaced by radioisotopes. Radionuclides differ by their properties, including half-life, energy emission characteristics, type of decay, etc. This allows for the selection of radionuclides having a desired mixture of properties suitable for diagnostic and / or therapeutic use. For example, gamma emitters are generally used diagnostically, while alpha and beta emitters are generally used therapeutically. However, some radionuclides have both gamma emitters, alpha emitters, and / or beta emitters and may be suitable for both uses. Radionuclides used herein include, for example, actinium-225, astatine-209, astatine-210, astatine-211, bismuth-212, bismuth-213, chromium-76, cesium-137, carbon-11, chromium-51, cobalt-60, copper-64, copper-67, dysprosium-165, erbium-169, fermium-255, fluorine-18, gallium-67, gallium-68, gold-198, holmium-166, indium-111, iodine-123, iodine-124, iodine-125, iodine-131, iridium-192, iron-59, krypton-81m, lead-212, lutetium-177, molybdenum-99, nitrogen-13, oxygen-15, palladium-103, phosphorus-32, potassium-42, radium-223, rhenium-186, rhenium-188, samarium-153, technetium-99m, radium-223, rubidium-82, ruthenium-106, sodium-24, strontium-89, terbium-149, thallium-201, thorium-227, xenon-133, ytterbium-169, ytterbium-177, yttrium-86, yttrium-90, zirconium-89, but are not limited thereto. In certain embodiments, the radionuclide is selected from the group of radionuclides as described above.In certain embodiments, the radionuclide is selected from the group consisting of technetium-99m, gallium-68, fluorine-18, indium-111, zirconium-89, iodine-123, iodine-124, iodine-131, astatine-211, bismuth-213, lutetium-177, and yttrium-86.

[0044] For the purposes of this application, "patient" or "subject" relates to any mammal, including both vertebrates, particularly humans and other mammals, such as any of the rodents, rabbits, cows, sheep, horses, dogs, cats, llamas, pigs, or non-human primates (such as monkeys). In one embodiment, the patient is a human, rat, or non-human primate. Preferably, the patient is a human. In one embodiment, a patient is a subject having or suspected of having a disease or disorder, or an injury. In the context of this application, the disease is cancer, and more particularly, cancer characterized by FOLR1-expressing tumor cells.

[0045] The terms "treatment" or "treating" or "treat" can be used interchangeably and are defined as a therapeutic intervention that slows, interrupts, halts, controls, stops, reduces, or reverses the progression or severity of a sign, symptom, disorder, condition, injury, or disease, but not necessarily accompanied by complete elimination of all disease-related signs, symptoms, or disorders. The subject in need of treatment includes not only the subject already diagnosed with this disorder, but also the subject susceptible to or having a predisposition to this disorder, or the subject to be prevented from having the disorder. For example, in the treatment of a tumor (such as cancer), the therapeutic agent can directly reduce the pathology of the tumor cells or make the tumor cells more sensitive to treatment by other therapeutic agents or the subject's own immune system.

[0046] As used herein, the term "therapeutically effective amount" means an amount necessary to obtain the desired result or results when used in treatment.

[0047] As used herein, the terms "diagnosis", "prognosis", and / or "prediction" include diagnosing, prognosticating, and / or predicting a particular disease and / or disorder, thereby predicting the onset and / or presence of a particular disease and / or disorder, and / or predicting the progression and / or duration of a particular disease and / or disorder, and / or predicting the response of a patient suffering from a particular disease and / or disorder to treatment.

[0048] The term "statistically significantly" different is well known to those of ordinary skill in the art. Statistical significance plays a crucial role in statistical hypothesis testing. It is used to determine whether the null hypothesis should be rejected or maintained. The null hypothesis is the default assumption that nothing has occurred or changed. For the null hypothesis to be rejected, the observed results must be statistically significant. That is, the observed p-value must be less than a pre-specified significance level α. The p-value of the result is the probability of obtaining at least as extreme a result if the null hypothesis were true. In one embodiment, α is 0.05. In a more specific embodiment, α is 0.01. In an even more specific embodiment, α is 0.001.

[0049] Detailed description Folate receptor alpha (FRα) Folate is a type of compound that encompasses both natural folates such as vitamin B9 and folic acid (FA) as examples. These amino acids are essential for cells to produce nucleic acids and generate metabolic amino acids necessary for cell growth and division (Kamen 1997 Semin Oncol 24; Goh and Koren 2008 J Obstet Gynaecol 28). Folate is transported across the cell membrane in three ways. The main uptake pathway is via the folate carrier (RFC), which is distributed everywhere and supports the uptake of dietary folate (Matherly and Goldman 2003 Vitam Horm 66:403-456). The second pathway is via the proton-coupled folate transporter (PCFT), which utilizes the transmembrane proton gradient to mediate folate transport into the cell (Zhao et al 2011 Annu Rev Nutr 31:177-201). Finally, folate can be transported by folate receptors, and there are four types of glycopolypeptides in folate receptors (FRα, FRβ, FRγ, FRδ), with a molecular weight of 38-45 kDa (Ledermann et al 2015 Ann Oncol 26:2034-2043). Folate receptor alpha (FRα), which is the alpha isoform, is a protein encoded by the FOLR1 gene in humans. FRα is a cell surface receptor anchored to glycosylphosphatidylinositol and mediates the endocytosis of active folate (5-methyltetrahydrofolate or 5-MTF) in a clathrin-independent manner (Salazar and Ratnam 2007 Cancer Metastasis Rev 26:141-152; Kelemen 2006 Int J Cancer 119:243-250).Throughout this application, "folate receptor", "folate receptor alpha", "FolR", "FOLR", "FR", "FOLR1", "FR_α", or "FRα" are used interchangeably and refer to human folate receptor alpha as described above and depicted in SEQ ID NO: 1, unless otherwise specified. SEQ ID NO: 1: Human Folate Receptor Alpha (herein, hFOLRα or hFRα are used interchangeably) MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWAAWPFLLSLALMLLWLLS

[0050] This application provides antibodies and antibody fragments that bind to human folate receptor alpha, and more specifically target specific epitopes on FRα, such that the BCSFB intersects in search of a conjugate with specific affinity and higher-order structure in receptor binding. The development of antibodies against human FRα is part of a promising strategy for targeted treatment and immunotherapy. Indeed, in rapidly proliferating cells, since sufficient uptake of folate is required for one-carbon metabolic reactions, DNA biosynthesis, repair, and methylation, FRα is highly expressed in solid cancers such as ovarian cancer, breast cancer, and lung cancer (Cheung et al 2016 Oncotarget 7: 52553-52574).

[0051] FRα and cancer Approximately one-third of human cancers overexpress the folate receptor (Paulos, 2004). A variety of quantitative and semi-quantitative methods have been employed to measure FRα expression in tumor biopsies of patients who may benefit from FRα-targeted therapy (Parker, 2005). These methods include those using anti-FRα antibodies (e.g., IHC, radioimmunoassay, quantitative autoradiography, cytometric analysis, etc.), RT-PCR, FISH, radioligand binding assays, etc. (Parker, 2005). These approaches have demonstrated overexpression of FRα in ovarian cancer, renal cancer, lung cancer, brain tumors, endometrial cancer, colorectal cancer, pancreatic cancer, gastric cancer, prostate cancer, and breast cancer (Parker et al 2005 Anal Biochem 338). Overexpression of FRα in malignant cells confers a growth advantage to these cells in low folate medium. Indeed, increased expression of FRα in tumor tissue correlates with increased uptake of folate, an important nutrient for dividing cells (Farran, 2019). FRα also appears to be involved in cell migration and invasion, and in preclinical models, overexpression of FRα is associated with tumor progression (Scaranti, 2020). Furthermore, FRα may mediate cancer cell division, anchorage-independent growth, and adhesion properties (Scaranti, 2020).

[0052] Thus, since FRα is overexpressed in various solid epithelial tumors, it is an attractive and valuable anti-cancer drug target (Scaranti, 2020; Meric-Bernstam and Mills 2012 Nat Rev Clin Oncol 9:542-548). Furthermore, FRα has only a minimal physiological role in non-malignant tissues after embryonic development, and overexpression of FRα in tumors indicates poor patient prognosis (Hartmann, 2007). Additionally, FRα has a high affinity for non-physiological substrates (such as folic acid, for example) and is immunogenic (Farran, 2019). RFC and PCFT are not currently direct targets of anti-cancer drugs (Scaranti, 2020). Current research on FRα in cancer focuses on three aspects: (1) targeted anti-cancer drug therapy, (2) tumor imaging (which enables more accurate cancer surgery), and (3) predictive biomarkers (diagnostic markers) (Scaranti, 2020). There are many advantages to using FRα as a target for diagnosis and therapy (Popovici, 2020). One aspect is the location of FRα (on non-malignant epithelium, where the expression level of this protein is quite low or negligible (Parker, 2005)), which means that FRα is not accessible to the circulation (Popovici, 2020). Second, FRα binds to folic acid, and this small molecule can rapidly penetrate solid tumors. Third, internalized FRα takes up folic acid conjugates into the cell and is then rapidly recycled to the cell surface (Popovici, 2020).

[0053] FRα and CNS transport In normal tissues, the distribution of folate receptor alpha is low and restricted. Interestingly, among the few specialized epithelia that have FRα on their surface, choroid plexus epithelial (CPE) cells exhibit the highest level of expression. FRα is hypothesized to provide the major route for folate blood-CSF transport and can be detected on both the apical and basolateral membranes of CPE cells (Grapp et al 2013 Nat Comm). Furthermore, delivery of folate across the BCSFB has been shown to occur via exosome-mediated delivery: namely, folate is taken up by the basolateral membrane via endocytosis through FRα, transported to the luminal vesicles within multivesicular bodies, and ultimately released into the CSF in exosome vesicles (Grapp et al 2013 Nat Comm). Given that CPE cells form the BCSFB, FRα is a potential target for cargo delivery to the CSF via transcytosis from where it can diffuse homogeneously into the brain. Thus, the FRα-binding agents disclosed herein are particularly useful in diagnostic and / or therapeutic approaches where imaging compounds or pharmaceuticals are to be delivered to cancer tissues or into the brain, more specifically the CSF.

[0054] FRα binding agent In a first aspect, the present application discloses a binder, more particularly a binder comprising an antibody, even more particularly a binder comprising a single variable domain antibody, and most particularly a binder comprising a VHH, which recognizes and binds to mouse and / or human folate receptor alpha. These antibodies are FRα binders per se. In various embodiments, the FRα binder binds to FRα but does not functionally regulate FRα. In other embodiments, the FRα binder can also dissociate from FRα after binding to it. This is particularly useful in the process of folate receptor-mediated transcytosis, in which the folate receptor binds to cargo on the basolateral side of choroid plexus epithelial (CPE) cells, the cargo is transported through the cells, and the cargo is released on the apical side of the CPE cells. Thus, currently applied FRα binders are extremely useful in the delivery of drugs administered directly or indirectly into the brain into the peripheral blood. Currently applied FRα binders are also similarly useful in the delivery of therapeutic and / or imaging compounds to cancer cells. Accordingly, the present application also provides a composition comprising an FRα binder (described below). The composition can be a pharmaceutical composition and / or an imaging composition, and the present application contemplates their use in the treatment and / or study of various CNS diseases and / or FRα-expressing cancers.

[0055] In various embodiments, the FRα binder of the present application comprises a targeting moiety having an antigen recognition domain that recognizes an epitope present on FRα. In one embodiment, the antigen recognition domain recognizes one or more linear epitopes present on FRα. As used herein, a linear epitope refers to a continuous sequence of amino acids present on FRα. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on FRα. As used herein, a conformational epitope refers to one or more portions (which may be discontinuous) of amino acids that form a three-dimensional surface having characteristics and / or shape and / or tertiary structure that can be recognized by the antigen recognition domain.

[0056] In one aspect, the FRα binder of the present application includes a targeting moiety having an antigen recognition domain that recognizes one or more epitopes present on human FRα. In one aspect, human FRα includes the amino acid sequence of SEQ ID NO: 1. In a more specific aspect, human FRα consists of the amino acid sequence of SEQ ID NO: 1. In an even more specific aspect, the FRα binder of the present application does not compete with folic acid and thus does not bind to or interfere with the folic acid binding site of human FRα. In another aspect, the FRα binder of the present application competes with 2HFO42 or binds to the same epitope on human FRα or is alternatively represented by an FRα binder that includes or consists of the amino acid sequence as set forth in SEQ ID NO: 2. In a specific aspect, the FRα binder of the present application binds to a conformational epitope present on FRα, where the epitope includes the residue Q141 of SEQ ID NO: 1 or, more specifically, at least two or more residues selected from R98, H99, E137, D138, Q141, E144, D145, R204, G205, Q211, W213, F214, D215, and A217 of SEQ ID NO: 1. This means that the amino acid R at position 98, amino acid H at position 99, amino acid E at position 137, amino acid D at position 138, amino acid Q at position 141, amino acid E at position 144, amino acid D at position 145, amino acid R at position 204, amino acid G at position 205, amino acid Q at position 211, amino acid W at position 213, amino acid F at position 214, amino acid D at position 215, and amino acid A at position 217 of SEQ ID NO: 1 are part of the conformational epitope. In another specific aspect, the FRα binder of the present application binds to a conformational epitope present on FRα, where the epitope includes or consists of R98, H99, E137, D138, Q141, E144, D145, R204, G205, Q211, W213, F214, D215, and A217 of SEQ ID NO: 1.

[0057] In one aspect, the FRα binder of the present application encompasses a full-length multimeric protein comprising two heavy chains and two light chains. Each heavy chain includes one variable region (e.g., VH) and at least three constant regions (e.g., CH1, CH2, CH3), and each light chain includes one variable region (VL) and one constant region (CL). As described above in the Definitions section, the variable region determines the specificity of the antibody and includes three hypervariable regions also known as complementarity determining regions (CDRs) that contribute to the antibody binding specificity.

[0058] In some embodiments, the FRα binder comprises a targeting moiety that is an antibody fragment. The term "antibody fragment" refers to a part of any antibody or antibody-like structure that itself has high affinity for an antigenic determinant or epitope and contains one or more CDRs that account for such specificity. In some particular embodiments, the FRα binder of the present application is a single domain antibody, an immunoglobulin single variable domain, a heavy-chain-only antibody (VHH), a single-chain antibody (scFv), a shark heavy-chain-only antibody (VNAR), a microprotein (cysteine knot protein, knottin), a DARPin, a tetranectin, an affibody, an affimer, a transbody, an anticalin, an adnectin, an affilin, a targeting moiety that is a microbody, a peptide aptamer, an allosterase, a plastic antibody, a filomer, a stradbody, a maxibody, an evibody, a finomer, an armadillo repeat protein, a knotted domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a tribody, a pepbody, a vaxibody, a unibody, a duoibody, an Fv, a Fab, a Fab', an F(ab')2, a peptidomimetic molecule, or a synthetic molecule, as described in U.S. Patent No. 7,417,130, U.S. Patent Application Publication No. 2004 / 132094, U.S. Patent No. 5,831,012, U.S. Patent Application Publication No. 2004 / 023334, U.S. Patent No. 7,250,297, U.S. Patent No. 6,818,418, U.S. Patent Application Publication No. 2004 / 209243, U.S. Patent No. 7,838,629, U.S. Patent No. 7,186,524, U.S. Patent No. 6,004,746, U.S. Patent No. 5,475,096, U.S. Patent Application Publication No. 2004 / 146938, U.S. Patent Application Publication No. 2004 / 157209, U.S. Patent No. 6,994,982, U.S. Patent No. 6,794,144, U.S. Patent Application Publication No. 2010 / 239633, U.S. Patent No. 7,803,907, U.S. Patent Application Publication No. 2010 / 119446, and / or U.S. Patent No. 7,166,697, the contents of which are incorporated herein by reference in their entirety.See also Storz 2011 MAbs 3:310-317.

[0059] In certain embodiments, the FRα binder of the present application comprises a targeting moiety that is a single-domain antibody such as a VHH. The VHH may be derived from an organism that produces VHH antibodies such as camels or sharks, or may be a designed VHH. A VHH is a therapeutic protein derived from an antibody and contains the unique structural and functional characteristics of a naturally occurring heavy-chain antibody (see the definition section above). In some embodiments, the single-domain antibody described herein is an immunoglobulin single variable domain or ISVD. In the most specific embodiments, the FRα binder comprises a VHH as the targeting moiety.

[0060] In a specific embodiment, the FRα binder, more particularly, the binder comprising the ISVD or VHH of the present application, consists of a CDR3 having an amino acid sequence that differs from SEQ ID NO: 5 by at most two amino acids, or differs from SEQ ID NO: 5 by at most one amino acid, or consists of the amino acid sequence depicted in SEQ ID NO: 5, or comprises a CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 5. The CDR3 sequence represents an essential characteristic of the family of ISVDs, more particularly VHHs, that specifically bind to FRα at the same binding site.

[0061] VHHs and Nbs are often classified into different sequence families, or superfamilies, because they cluster clonally related sequences derived from the same progenitor cells during B cell maturation (Deschaght et al., 2017. Front Immunol. 10;8:420). This classification is often based on the CDR sequences of Nbs. As an example, each Nb family is defined as a cluster of (clonally) related sequences with a threshold of sequence identity in the CDR3 region. Thus, within one VHH family as defined herein, the CDR3 sequences are identical or very similar in amino acid composition, preferably having at least 80% identity, or at least 85% identity, or at least 90% identity in the CDR3 sequence, such that Nbs of the same family bind to the same binding site and have the same effect or functional impact.

[0062] Accordingly, the ISVD family is defined herein as a group of ISVD amino acid sequences having high similarity or identity in the CDR3 sequence. By default, ISVDs belonging to the same family bind to the same target epitope. Among the ISVD families, small deviations such as a single amino acid mutation occurring within one family may be of interest because they may improve these properties if the expression / stability / affinity / crystallization of a representative family is poor. One aspect relates to the ISVDs of the present application, including SEQ ID NO: 5, or homologs thereof having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% homology at the amino acid level, or humanized variants thereof.

[0063] In another specific embodiment, the FRα binder, more particularly, the agent comprising the ISVD or VHH of the present application, comprises a CDR3 having an amino acid sequence that differs from SEQ ID NO: 11, 14, 21, 26 or 30 by at most two amino acids, or differs from SEQ ID NO: 11, 14, 21, 26 or 30 by at most one amino acid, or comprises the amino acid sequence depicted in SEQ ID NO: 11, 14, 21, 26 or 30, or comprises a CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 11, 14, 21, 26 or 30. In another specific embodiment, the FRα binder, more particularly the ISVD or VHH of the present application, comprises a CDR3 sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% homology at the amino acid level of SEQ ID NO: 5, 11, 14, 21, 26 or 30, or a humanized variant thereof.

[0064] Table 1 provides an overview of the full-length and CDR sequences of the anti-FRα VHHs disclosed herein. VHH 2HFO42 and VHH 2HFO9 belong to family 3 and have a CDR1 with the consensus or conserved sequence SEQ ID NO: 113: X1SX2FX3GMX4MG, where X1 is G or E, X2 is G, T or P, X3 is S or I, and X4 is I or L; and have a CDR2 with the conserved sequence SEQ ID NO: 31: TX1TSHGTTNYADSVKG, where X1 is V or I, or have a CDR2 with the conserved sequence SEQ ID NO: 32: TX1TSX2GTTNYADSVKG, where X1 is V or I and X2 is H or G. In a further aspect related to the anti-FRα VHH, the FR sequences are defined as follows: namely, FR1 has a consensus or conserved sequence depicted as SEQ ID NO: 114: X1VQLX2ESGGGLVQX3GGSLRLSCAAS, where X1 is Q, E, D, X2 is Q or V, and X3 is A or P; FR2 has a conserved sequence depicted as SEQ ID NO: 115: WYRQX1PGKQRELVA, where X1 is V or A; FR3 has a conserved sequence depicted as SEQ ID NO: 116: RFTISRX1X2AKNTVX3LQMNSLX4PEDTAVYYC, where X1 is D, E, or P, X2 is N or G, X3 is L or Y, X4 is K or R; and FR4 has a conserved sequence depicted as SEQ ID NO: 117: WGX1GTX2VTVSS, where X1 is K or Q and X2 is Q or L.

[0065]

Table 1

[0066] 2HFO19, 3MFR73, 2MFR84, 2MFR63, 3HFO26, and 2MFRO7 of VHH belong to Family 1, have the conserved sequence SEQ ID NO: 33: GFPFSTX1YMS, where X1 is V or Y, for CDR1, have the conserved sequence SEQ ID NO: 34: GINX1X2GX3X4IDYADSVKG, where X1 is N or S, X2 is D or N, X3 is G or E, X4 is V or I, for CDR2, and have the conserved sequence SEQ ID NO: 35: ARGRX1FVATX2X3SSLR, where X1 is S or A, X2 is L or M, X3 is S or P, for CDR3, and comprise the sequences thereof.

[0067] In some embodiments, the FRα binder of the present application comprises a targeting moiety that is a VHH comprising a single amino acid chain having four "framework regions" and three "complementary determining regions" or CDRs. As used herein, "framework region" refers to the region within the variable domain located between CDRs. As used herein, "complementary determining region" or "CDR" refers to the variable loop region of a VHH containing an amino acid sequence that can specifically bind to an antigen target to form a paratope.

[0068] Thus, in a specific embodiment, the FRα binder comprises an ISVD that binds to the receptor via the residues of family 3 of ISVDs described herein, positions 29, 30, 31, and 33 of CDR1, positions 52, 53, 54, and 56 of CDR2, and positions 95, 96, 97, 98, 101, and 102 of CDR3, where Kabat numbering is used to define the amino acid positions of the ISVD as exemplified for SEQ ID NO: 2 of 2HFO4 in FIG. 22. In a further embodiment, as described in the examples, the "CDR4" or DE loop region located in FR3 affects the properties of the ISVD, specifically the ability to cross the BCSFB, and provides an ISVD further limited to a CDR4 sequence where position 72 is D, position 73 is N, or alternatively position 72 is E, position 73 is G, or alternatively position 72 is P, position 73 is G, according to the Kabat numbering referred to in SEQ ID NO: 2. More specifically, the CDR4 may be restricted to amino acid R at position 71, amino acid D, E, or P at position 72, amino acid N or G at position 73, amino acid A at position 74, amino acid K at position 75, amino acid N at position 76, and amino acid T at position 77 according to Kabat numbering (see FIG. 22 as an example).

[0069] In various embodiments, the FRα binder comprises a VHH having a variable domain that includes at least one CDR1, CDR2, and / or CDR3 sequence. In some embodiments, the CDR sequences of the ISVD of the FRα binder are known in the art and are defined by the CDRs of SEQ ID NO: 2 annotated according to Chothia, AbM, Maccallum, IMGT, or Kabat annotations as described and illustrated herein (FIG. 22).

[0070] In a further embodiment, the CDR1 sequence is selected from SEQ ID NO: 3, 9, 16, 24, or 28. In some embodiments, the CDR2 sequence is selected from SEQ ID NO: 4, 7, 10, 13, 17, 19, 25, 29, or 31. In some embodiments, the CDR3 sequence is selected from SEQ ID NO: 5, 11, 14, 21, 26, or 30.

[0071] In a more specific embodiment, the binder described herein relates to an anti-FRα VHH having a sequence that is a humanized variant or an affinity variant or an array-optimized variant as described and exemplified herein, and as provided in the sequence listing, the amino acid sequence is not limited to a tagged or fusion version, and is limited only to those CDR and FR sequences as provided in the format of ISVD which is FR1-CDR1-FR2-CDR2-FR3-'CDR4'-FR3-CDR3-FR4.

[0072] In certain embodiments, an FRα binder is provided, which has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to SEQ ID NO: 2 or 37, the agent comprises three complementarity determining regions (CDR1, CDR2, and CDR3), CDR1 is SEQ ID NO: 2 or 37, the agent comprises three complementarity determining regions (CDR1, CDR2, and CDR3), CDR1 comprises or consists of SEQ ID NO: 3, CDR2 comprises or consists of SEQ ID NO: 4, and CDR3 comprises or consists of SEQ ID NO: 5. In certain embodiments, the difference in the amino acid sequence between the homolog and SEQ ID NO: 2 is found in the framework region. The role of the framework region in specific binding to the target is rather limited, and similar effects of the ISVD can be obtained by variations in the framework sequence (see, for example, De Groeve et al 2010 J Nuclear Medicine 51:782; Saerens et al 2005 J Mol Biol 352:597-607). In certain embodiments, the difference in the amino acid sequence has been introduced, for example, for the purpose of humanization (see below). In even more specific embodiments, the difference in the amino acid sequence is limited to conservative amino acid substitutions (see below). In the most specific embodiments, an FRα binder is provided, wherein the FRα binder is represented by SEQ ID NO: 2 or 37.

[0073] In certain embodiments, an FRα binder is provided, which has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to SEQ ID NO: 6. The agent comprises three complementarity determining regions (CDR1, CDR2, and CDR3), where CDR1 comprises or consists of SEQ ID NO: 3, CDR2 comprises or consists of SEQ ID NO: 7, and CDR3 comprises or consists of SEQ ID NO: 5. In certain embodiments, the difference in the amino acid sequence between the homolog and SEQ ID NO: 6 is found in the framework region. In the most specific embodiment, an FRα binder is provided, which is represented by SEQ ID NO: 6.

[0074] In certain embodiments, an FRα binder is provided, which has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to SEQ ID NO: 36. The agent comprises three complementarity determining regions (CDR1, CDR2, and CDR3), where CDR1 comprises or consists of SEQ ID NO: 3, CDR2 comprises or consists of SEQ ID NO: 4, and CDR3 comprises or consists of SEQ ID NO: 5. In certain embodiments, the difference in the amino acid sequence between the homolog and SEQ ID NO: 36 is found in the framework region. In the most specific embodiment, an FRα binder is provided, which is represented by SEQ ID NO: 36.

[0075] In certain embodiments, an FRα binder is provided, which has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to SEQ ID NO: 12. The agent includes three complementarity-determining regions (CDR1, CDR2, and CDR3), where CDR1 includes or consists of SEQ ID NO: 9, CDR2 includes or consists of SEQ ID NO: 13, and CDR3 includes or consists of SEQ ID NO: 14. In certain embodiments, the differences in the amino acid sequences between the homolog and SEQ ID NO: 12 are found in the framework regions. In the most specific embodiment, an FRα binder is provided, where the FRα binder is represented by SEQ ID NO: 12.

[0076] In certain embodiments, an FRα binder is provided, which has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to SEQ ID NO: 15. The agent includes three complementarity-determining regions (CDR1, CDR2, and CDR3), where CDR1 includes or consists of SEQ ID NO: 16, CDR2 includes or consists of SEQ ID NO: 17, and CDR3 includes or consists of SEQ ID NO: 14. In certain embodiments, the differences in the amino acid sequences between the homolog and SEQ ID NO: 15 are found in the framework regions. In the most specific embodiment, an FRα binder is provided, where the FRα binder is represented by SEQ ID NO: 15.

[0077] In certain embodiments, an FRα binder is provided, which has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to SEQ ID NO: 18. The agent includes three complementarity determining regions (CDR1, CDR2, and CDR3), where CDR1 includes or consists of SEQ ID NO: 16, CDR2 includes or consists of SEQ ID NO: 19, and CDR3 includes or consists of SEQ ID NO: 14. In certain embodiments, the differences in the amino acid sequences between the homolog and SEQ ID NO: 18 are found in the framework regions. In the most specific embodiment, an FRα binder is provided, which is represented by SEQ ID NO: 18.

[0078] In certain embodiments, an FRα binder is provided, which has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to SEQ ID NO: 20. The agent includes three complementarity determining regions (CDR1, CDR2, and CDR3), where CDR1 includes or consists of SEQ ID NO: 16, CDR2 includes or consists of SEQ ID NO: 19, and CDR3 includes or consists of SEQ ID NO: 21. In certain embodiments, the differences in the amino acid sequences between the homolog and SEQ ID NO: 20 are found in the framework regions. In the most specific embodiment, an FRα binder is provided, which is represented by SEQ ID NO: 20.

[0079] In certain embodiments, an FRα binder is provided, the agent having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to SEQ ID NO: 22, the agent comprising three complementarity determining regions (CDR1, CDR2 and CDR3), CDR1 comprising or consisting of SEQ ID NO: 16, CDR2 comprising or consisting of SEQ ID NO: 19, and CDR3 comprising or consisting of SEQ ID NO: 21. In certain embodiments, the difference in the amino acid sequence between the homolog and SEQ ID NO: 22 is found in the framework region. In the most specific embodiment, an FRα binder is provided, the FRα binder being represented by SEQ ID NO: 22.

[0080] In certain embodiments, an FRα binder is provided, the agent having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to SEQ ID NO: 23, the agent comprising three complementarity determining regions (CDR1, CDR2 and CDR3), CDR1 comprising or consisting of SEQ ID NO: 24, CDR2 comprising or consisting of SEQ ID NO: 25, and CDR3 comprising or consisting of SEQ ID NO: 26. In certain embodiments, the difference in the amino acid sequence between the homolog and SEQ ID NO: 23 is found in the framework region. In the most specific embodiment, an FRα binder is provided, the FRα binder being represented by SEQ ID NO: 23.

[0081] In certain embodiments, an FRα binder is provided, which has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to SEQ ID NO: 27, the agent comprises three complementarity determining regions (CDR1, CDR2 and CDR3), CDR1 comprises or consists of SEQ ID NO: 28, CDR2 comprises or consists of SEQ ID NO: 29, and CDR3 comprises or consists of SEQ ID NO: 30. In certain embodiments, the difference in the amino acid sequence between the homolog and SEQ ID NO: 27 is found in the framework region. In the most specific embodiment, an FRα binder is provided, wherein the FRα binder is represented by SEQ ID NO: 27.

[0082] Humanization In one aspect, the FRα binder of the present application comprises a "humanized" immunoglobulin single variable domain or VHH, i.e., in order to increase the degree of sequence identity with the closest human germline sequence, one or more amino acid residues in the amino acid sequence of the VHH obtained by immunization are replaced with one or more amino acid residues present at corresponding positions in the VH domain derived from a conventional four-chain antibody of human origin. Potentially useful humanizing substitutions can be identified by comparing the sequence of the framework region of the naturally occurring VHH sequence with the corresponding framework sequences of one or more closely related human VH sequences (s), and then one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into the VHH sequence (by any method known per se as further described herein), and the resulting humanized VHH sequence can be tested for affinity for the target, stability, ease and level of expression, and / or other desired properties. In this way, with a limited amount of trial and error, other or appropriate humanizing substitutions (or appropriate combinations thereof) can be determined by those skilled in the art. Also, based on the foregoing, an immunoglobulin single variable domain (such as the VHH domain) (of the framework region) may be partially humanized or fully humanized.

[0083] Accordingly, in various embodiments, the FRα binder of the present application includes a targeting moiety that includes an amino acid sequence having one or more amino acid mutations with respect to SEQ ID NO: 2. In various embodiments, the FRα binder includes a targeting moiety that includes an amino acid sequence having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations with respect to SEQ ID NO: 2. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and cleavages. In some embodiments, the amino acid mutation is an amino acid substitution and may include conservative substitutions and / or non-conservative substitutions. In certain embodiments, the one or more amino acid mutations may be present in the CDR (e.g., CDR1, CDR2, or CDR3 region) of the targeting moiety. In other certain embodiments, the one or more amino acid mutations may be in the framework region (e.g., FR1, FR2, FR3, or FR4 region) of the targeting moiety. In the most specific embodiments, the one or more amino acid mutations are present only in the framework region of the FRα binder.

[0084] "Conservative substitution" may be made, for example, in accordance with the similarity of polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the amino acid residues involved. The 20 naturally occurring amino acids can be classified into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. As used herein, "conservative substitution" is defined as the exchange of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, by exchanging Asp for Glu, one negative charge remains in the modified polypeptide. Further, glycine and proline may be substituted for each other based on their ability to disrupt the α-helix.

[0085] As used herein, "non-conservative substitution" is defined as the replacement of an amino acid with another amino acid that is listed in a different one of the six standard amino acid groups (1)-(6) shown above.

[0086] In various embodiments, substitutions may include non-classical amino acids (by way of example, selenocysteine, pyrrolidine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, β-methylamino acids, C-α-methylamino acids, N-α-methylamino acids, and other designer amino acids, and amino acid analogs in general).

[0087] In the most specific embodiment, the mutation introduced into VHH_2HFO42 for the purpose of humanization is a deletion of residue M32 at position 40 of SEQ ID NO: 2 and / or a substitution of V with A.

[0088] In another most specific embodiment, the mutation introduced into VHH_2MFR67 for the purpose of humanization is a deletion of residue M32 at position 36, a substitution of E with D at position 72, and / or a substitution of D with N at position 73 of SEQ ID NO: 36.

[0089] In another most specific embodiment, the mutation introduced into VHH_2HFO19 for the purpose of humanization is a deletion of residue N52 and / or residue N53 of SEQ ID NO: 8.

[0090] The humanized version of 2HFO42 is provided as a VHH having an amino acid sequence as depicted in SEQ ID NO: 37 or alternatively as depicted in SEQ ID NOs: 38 - 65.

[0091] Humanization can be performed using humanization techniques known in the art. In some embodiments, possible humanizing substitutions or combinations of humanizing substitutions may be determined by methods known in the art, for example, by comparison of the VHH sequence with the sequence of a naturally occurring human VH domain, for illustrative purposes only and not by way of limitation. In some embodiments, the humanizing substitutions are selected such that the resulting humanized VHH still retains advantageous functional properties. Generally, as a result of humanization, the VHHs of the present application may become more "human-like" while still retaining favorable properties such as reduced immunogenicity compared to the corresponding naturally occurring VHH domains. In various embodiments, the humanized VHHs of the present application can be obtained by any suitable method known in the art and are thus not strictly limited to polypeptides obtained using a polypeptide comprising a naturally occurring VHH domain as a starting material. Indeed, amino acid sequence modifications may be achieved using techniques known in the art such as, for example, site-directed mutagenesis or PCR-based mutagenesis. Such techniques are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., 1989 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1989.

[0092] In various embodiments, for example, the mutations introduced to humanize an FRα binder do not substantially reduce the ability of the FRα binder of the invention to specifically bind to human FRα. In various embodiments, the mutations do not substantially reduce the ability of the FRα binder of the invention to specifically bind to FRα without neutralizing FRα.

[0093] Association kinetics of FRα binding agent In various embodiments, the binding affinity of the FRα binding agent of the present application for the full length and / or mature form and / or isoform and / or splice variant and / or fragment and / or monomer and / or dimer and / or tetramer and / or any other naturally occurring or synthetic analog, variant or mutant (including monomers and / or dimers and / or tetramers) of human FRα is determined by the equilibrium dissociation constant (K dis ) or alternatively by the dissociation constant k off . In various embodiments, the FRα binding agent binds to the full length and / or mature form and / or isoform and / or splice variant and / or fragment and / or other naturally occurring or synthetic analog, variant or mutant (including monomers and / or dimers and / or tetramers) of human FRα with a K D of less than 10 μM, more particularly less than 1 μM and / or 1 nM or greater. In other embodiments, the FRα binding agent of the present application comprises a targeting moiety that binds to the full length and / or mature form and / or isoform and / or splice variant and / or fragment and / or other naturally occurring or synthetic analog, variant and / or mutant (including monomers and / or dimers and / or tetramers) of human FRα, or binds to a variant (including monomeric and / or dimeric and / or tetrameric forms) with a K dis between 1 nM and 1 μM, or between 5 nM and 950 nM, or between 10 nM and 900 nM, or between 20 nM and 850 nM, or between 30 nM and 800 nM, or between 40 nM and 700 nM, or between 50 nM and 600 nM, or between 50 nM and 500 nM. In a more specific embodiment, the K dis for human FRα is between 55 nM and 350 nM. In the most specific embodiment, the K disis between 200 nM and 350 nM, more particularly between 250 and 300 nM. In other embodiments, the FRα binder has a K dis for the full length and / or mature form and / or isoform and / or splice variant and / or fragment and / or other naturally occurring or synthetic analogs, variants or mutants (including monomers and / or dimers and / or tetramers) of human FRα.

[0094] According to another aspect of the present application, the FRα binder of the present application has an affinity for mouse and human FRα in the range of about 1 nM to about 1 μM, or about 2 nM to about 700 nM, or about 2 nM to about 60 nM, or about 20 nM to about 300 nM, as measured, for example, by biolayer interferometry (BLI) and / or ELISA.

[0095] In various embodiments, the ISVD or VHH of the present application is not limited to a specific biological source or a specific preparation method. The ISDV or VHH sequence can generally be generated or obtained by preferably immunizing camelid species with mouse and / or human FRα molecules (i.e., to raise an immune response against FRα and / or increase heavy chain antibodies), obtaining a suitable biological sample (such as a blood sample or any sample of B cells) from a camelid, and starting from the sample to generate a VHH sequence directed against FRα using any suitable known technique. The VHH can also be obtained by expressing a nucleotide sequence encoding a naturally occurring VHH domain, "humanizing" a naturally occurring VHH domain, or expressing a nucleic acid encoding such a humanized VHH domain, using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known in the art, using nucleic acid synthesis techniques known in the art to prepare a nucleic acid encoding the VHH, then expressing the nucleic acid thus obtained, and / or by any combination of one or more of the foregoing.

[0096] Generation of folate receptor alpha binding agent The FRα binders of the present application, particularly FRα antibodies, and more particularly ISVD or VHH, are not limited to a specific biological source or a specific preparation method. Methods for producing the FRα binders of the present application are described herein. For example, the DNA sequence encoding the FRα binder of the present application can be easily prepared by techniques well known in the art such as cloning, hybridization screening, polymerase chain reaction (PCR). Standard techniques regarding cloning, DNA isolation, amplification and purification, enzyme reactions involving DNA ligase, DNA polymerase, restriction endonucleases, and various separation techniques are known to those skilled in the art and are commonly used. Many standard procedures are described in Sambrook et al. (1989), Maniatis et al. (1982), Wu (ed.) (1993), and Ausubel et al. (1992). Alternatively, the DNA sequence encoding the FRα binder of the present application can be chemically synthesized using methods known in the art. The synthetic DNA sequence can be ligated, for example, to other appropriate nucleotide sequences including expression control sequences to generate a gene expression construct encoding the desired FRα binder.

[0097] Accordingly, in various aspects, the present application provides an isolated nucleic acid comprising a nucleotide sequence encoding the FRα binder described in the present application. One aspect further discloses an expression cassette comprising the nucleic acid molecule. A more specific aspect discloses an expression cassette in which factors for cell-specific or tissue-specific expression are present. A further aspect relates to a vector comprising the expression cassette or the nucleic acid molecule. More specifically, the vector may be a viral vector, and even more specifically, a lentiviral vector or an AAV vector.

[0098] To produce currently applicable FRα binders, an expression vector containing a nucleic acid sequence encoding the FRα binder can be introduced into a host cell by gene transfer, transformation, or transduction techniques. Therefore, in various embodiments, the present application provides a host cell containing a nucleic acid encoding one of the FRα binders of the present application. For example, the nucleic acid encoding the FRα binder of the present application can be introduced into a host cell by retroviral transduction. Exemplary host cells are defined herein and include, by way of example, E. coli cells, Chinese hamster ovary (CHO) cells, yeast cells such as Pichia, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells. The transformed host cell can be grown under conditions in which the host cell can express the gene encoding the FRα binder of the present application.

[0099] Well-known techniques, for example, FRα binders can be collected and purified using affinity tags such as glutathione-S-transferase (GST) and histidine (His) tags, or chromatography, etc. Specific expression and purification conditions will vary depending on the expression system employed. For example, when the gene is expressed in E. coli, the gene is first cloned into an expression vector by placing the gene engineered downstream of an appropriate bacterial promoter, such as Trp or Tac and a prokaryotic signal sequence. In another example, when expressing the engineered gene in a eukaryotic host cell, such as CHO cells, it is first inserted into an expression vector containing, for example, an appropriate eukaryotic promoter, a secretion signal, an enhancer, and various introns. In one aspect, the FRα binder of the present application contains a His tag, a FLAG tag, and / or a Myc tag. In one aspect, the FRα binder of the present application contains a His tag and a proteolytic site that enables cleavage of the His tag.

[0100] Accordingly, the present application also provides a host cell comprising one of the FRα binders described herein. Also provided herein are host cells comprising any of the nucleic acid molecules or expression cassettes or vectors of the present application. The host cell can be either prokaryotic or eukaryotic. Representative host cells that may be used in the present invention include, but are not limited to, bacterial cells, yeast cells, plant cells, animal cells, etc. Bacterial host cells suitable for use in the present invention include cells of the genus Escherichia, Bacillus, Streptomyces, Erwinia, Klebsiella, Serratia, Pseudomonas, Salmonella, etc. Yeast host cells suitable for use in the present invention include species among Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia (e.g., Pichia pastoris), Hansenula (e.g., Hansenula polymorpha), Yarrowia, Schwaniomyces, Schizosaccharomyces, Zygosaccharomyces, etc. Saccharomyces cerevisiae, S. carlsbergensis, K. lactis are the most commonly used yeast hosts and are convenient fungal hosts. Animal host cells suitable for use in the present invention include insect cells and mammalian cells (especially derived from Chinese hamster (e.g., CHO, etc.)), human cell lines such as HeLa. Exemplary insect cell lines include, but are not limited to, Sf9 cells, baculovirus-insect cell lines (e.g., see Jarvis 2003 Virology 310:1-7). Non-limiting examples of plant cells include, inter alia, tobacco cells, Arabidopsis cells, tomato cells, corn cells, algal cells, etc. The host cell may be provided in suspension culture, flask culture, tissue culture, organ culture, etc. Alternatively, the host cell may be a genetically modified animal.

[0101] Animals or mammalian host cells suitable for harboring, expressing, and producing one of the FRα binders of the present application include Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCC CCL-61), DG44, etc. (Chasin et al 1986 Som Cell Mol Genet 12:555-556; Kolkekar et al 1997 Biochemistry 36:10901-10909), CHO-K1 Tet-On cell line (Clontech), CHO designated by ECACC 85050302 (CAMR, Salisbury, Wiltshire, UK), CHO clone 13 (GEIMG, Genova, IT), CHO clone B (GEIMG, Genova, IT), CHO-K1 / SF designated by ECACC 93061607 (CAMR, Salisbury, Wiltshire, UK), RR-CHOK1 named ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK), dihydrofolate reductase-negative CHO cells (CHO / -DHFR, Urlaub & Chasin 1980 PNAS 77:4216), and dp12.CHO cells (U.S. Patent No. 5,721,121); simian kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL-1651); human embryonic kidney cells (e.g., 293 cells, or 293T cells, or 293 cells subcloned for growth in suspension culture, Graham et al. 1977 J Gen Virol 36:59, or GnTI KO HEK293S cells, Reeves et al. 2002 PNAS 99:13419); baby hamster kidney cells (BHK, ATCC CCL-10); simian kidney cells (CV1, ATCC CCL-70); African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse Sertoli cells (TM4, Mather 1980 Biol Reprod 23:243-251); human cervical cancer cells (HELA, ATCC CCL-2); dog kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatocytes (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat hepatocytes (BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NYAcad.Sci, 383:44-68); MCR 5 cells; FS4 cells are included. According to a particular embodiment, the cells are mammalian cells selected from Hek293 cells or COS cells.

[0102] The above host cells can be transiently or stably transfected. Introduction of DNA such as such nucleic acid molecules, expression cassettes or expression vectors into prokaryotic and eukaryotic cells can be achieved using any technique known in the art including, but not limited to, standard bacterial transformation, calcium phosphate co-precipitation, electroporation, or liposome-mediated, DEAE-dextran-mediated, polycation-mediated, or virus-mediated transfection. For all standard techniques, see, for example, Molecular Cloning: A Laboratory Manual (Sambrook et al. 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R.I. Freshney. 1987. Liss, Inc. New York, N.Y.). The host cells may also be recombinant host cells, which involve cells that have been genetically engineered to contain the isolated DNA molecule, nucleic acid molecule, expression construct or vector of the present invention. The DNA can be introduced by any means known in the art suitable for a particular type of cell including, but not limited to, transformation, lipofection, electroporation, introduction via virus, etc.

[0103] Furthermore, in an alternative aspect, the use of an FRα binder, particularly an FRα antibody, more particularly a nucleic acid molecule, expression cassette or vector described herein encoding an FRα ISVD or VHH, is provided for the production of said FRα binder, antibody, ISVD or VHH. In certain aspects, the use is provided for intrabodym production. An intracellular antibody or “intrabodym” is an antibody or antibody fragment that is heterologously expressed within a designated intracellular compartment, and this process is made possible by in-frame incorporation of an intracellular trafficking signal. Intrabodyms can be expressed in any form or morphology, such as intact IgG molecules or Fab fragments, more particularly genetically engineered antibody fragments, such as single domain intrabodyms or VHHs. For a review, see Zhu, and Marasco, 2008 (Therapeutic Antibodies. Handbook of Experimental Pharmacology 181. Ed. Springer-Verlag Berlin Heidelberg).

[0104] Chimeras and fusions In various embodiments, any of the FRα binders of the present application is provided as part of a chimera or fusion with one or more other agents. In certain embodiments, the other agent is a cytotoxic agent, a therapeutic agent, an imaging agent, a radionuclide, an antisense oligonucleotide, an antibody or antibody fragment (also referred to herein as a multivalent or multispecific agent) including another VHH. In other specific embodiments, the other agent is a nanoparticle, a lipid nanoparticle or an exosome. Alternatively, in another expression, a composition, more particularly a pharmaceutical composition, is provided comprising any of the FRα binders of the present application coupled with one or more other agents. In one embodiment, the agent is a chemical entity. As used herein, the term "chemical entity" refers to simple or complex organic and inorganic molecules. Non-limiting examples of chemical entities used in the present application include peptides, peptidomimetics, proteins, antibodies (including antibody fragments such as ISVD and VHH), carbohydrates, nucleic acids or their derivatives, ligands, substrates, phosphates, agonists, antagonists, neurotransmitters, inhibitors, drugs. In one embodiment, the chemical entity is a biological substance, a small molecule, a therapeutic agent, an imaging agent, or a test compound.

[0105] As used herein, "biological" refers to a substance made from a living organism or its products. A biological can be composed of sugars, proteins, nucleic acids, or complex combinations of these substances, or can be a living entity such as a cell or tissue. Biologics can be isolated from various natural sources such as humans, animals, microorganisms, and can be manufactured by biotechnology methods and other state-of-the-art techniques. A non-limiting example of a biologic is an antibody.

[0106] As used herein, "small molecule" refers to an organic compound of low molecular weight (<900 daltons) that may control biological processes (similar to the fields of molecular biology and pharmacology). Most drugs are small molecules. Large structures such as nucleic acids, proteins, and many polysaccharides are not small molecules, but the monomers that make them up (ribonucleotides or deoxyribonucleotides, amino acids, monosaccharides, respectively) are considered small molecules. Small molecules can have various biological functions and uses, serving as cell signaling molecules, drugs in medicine, pesticides in agriculture, and playing many roles, such as inhibiting specific functions of proteins or inhibiting protein-protein interactions. These compounds can be natural products (such as secondary metabolites) or artificial products (such as peptide mimics).

[0107] As used herein, "therapeutic agent" refers to a substance that can delay, interrupt, stop, control, halt, reduce, or reverse the progression or severity of a sign, symptom, disorder, condition, injury, or disease, but does not necessarily involve the complete elimination of all signs, symptoms, conditions, or disorders associated with the disease. Non-limiting examples of therapeutic agents include pharmaceuticals, antibodies, antibody fragments, enzymes, antibiotics, anti-proliferatives, hormones, neurotransmitters, and small molecules.

[0108] An "imaging agent" is a compound that has one or more properties that enable direct or indirect detection of its presence and / or location. Examples of such imaging agents include proteins and small molecule compounds that incorporate a labeling site that enables detection, such as fluorescence or radioactivity.

[0109] The term "test compound" is used herein in the context of "drug candidate compound" or "candidate compound for lead optimization" in the therapeutic agents described in connection with the methods of the present invention. Thus, "test compounds" can be used for lead optimization, although they are not used as such in a commercial setting. These compounds include organic or inorganic compounds derived synthetically or from natural resources. Compounds include polynucleotides, lipids or hormone analogs characterized by low molecular weight. Other biological macromolecular organic test compounds include small peptides or peptidomimetic molecules (peptide mimetics) consisting of about 2 to about 40 amino acids, and large polypeptides containing about 40 to about 500 amino acids such as antibodies or antibody conjugates.

[0110] The above coupling between the FRα binder of the present application and the compound can be achieved by means of a chemical crosslinking agent or by generating a fusion protein. The covalent conjugation can be either direct conjugation or conjugation via a linker. In certain embodiments, the direct conjugation is by construction of a protein fusion (i.e., by genetically fusing two or more genes - one encoding the FRα binder of the present application and one or more other proteins - and expressing them as a single protein). In certain embodiments, the direct conjugation is by formation of a covalent bond between a reactive group on one or more portions of the FRα binder of the present application and a corresponding group or acceptor on a chemical entity (e.g., a neurological drug). In certain embodiments, the direct conjugation is by modifying (i.e., genetically engineering) one of the two molecules to be conjugated such that it contains a reactive group (non-limiting examples include a sulfhydryl group or a carboxyl group) that forms a covalent bond with one of the two molecules being conjugated under appropriate conditions. As one non-limiting example, a molecule (i.e., an amino acid) having a desired reactive group (i.e., a cysteine residue) may be introduced into a disulfide bond formed, for example, between an FRα antibody and a chemical entity (e.g., a neurological drug). Methods for covalent conjugation of nucleic acids and proteins are also known in the art (see, e.g., Zatsepin et al 2005 Russ Chem Rev 74:77-95 for photo-crosslinking). Non-covalent conjugation can be by any non-covalent addition means, including hydrophobic binding, ionic binding, electrostatic interactions, etc., as will be readily understood by those skilled in the art. The conjugation may be carried out using various linkers.For example, the FRα antibody and the nerve agent may be conjugated using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). The ligation of peptides containing 1 to 20 amino acids linked by peptide bonds may also be used. In certain such embodiments, the amino acids are selected from the 20 naturally occurring amino acids. In certain other such embodiments, one or more of the amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. The linker may be a "cleavable linker", for example, promoting the release of chemical entities during the delivery of the nerve agent to the brain or the delivery of the therapeutic agent to cancer cells. Acid-labile linkers, peptidase-sensitive linkers, photo-labile linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) are non-limiting examples that may be used.

[0111] According to certain embodiments, "coupling" can be achieved by generating a multivalent or multispecific antibody (e.g., a bispecific antibody). A multispecific antibody is an (monoclonal) antibody or antibody fragment that has binding specificities for at least two different sites. In one embodiment, the multispecific antibody comprises a first antigen-binding site that binds to mouse and / or human FOLR1 and a second antigen-binding site. In one embodiment, the second antigen-binding site is a brain antigen, and more particularly, a brain antigen selected from the list consisting of beta-secretase 1 (BACE1), amyloid beta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6. In another embodiment, the second antigen-binding site is a cancer antigen. A "cancer antigen" or "tumor antigen" refers to an antigenic substance produced by cancer or tumor cells, i.e., it induces an immune response in the host. Tumor antigens are tumor markers useful for identifying tumor cells in diagnostic tests and potential candidates for use in cancer treatment. Non-limiting examples of cancer antigens include MAGE-1, NY-ESO-1a, BAGE (see Renkvist et al 2001 Cancer Immunology).

[0112] Accordingly, in one aspect, the FRα binding agents described herein are multivalent or multispecific binding agents. The binding sites within the multivalent or multispecific agent may be proteinaceous and / or may be directly linked and / or may be fused by a linker or spacer. The compositions or binding agents(s) described herein may appear in “multivalent” or “multispecific” forms and thus are formed by binding two or more identical or different binding agents by chemical or recombinant DNA techniques. The multivalent forms may be formed by connecting building blocks directly or via a linker or by fusing the building blocks to an Fc domain coding sequence.

[0113] "Fc domain", "Fc region", or "Fc tail", when used interchangeably herein, refers to the single Fc chain and / or dimeric Fc domain of an Fc-containing protein. Specifically, in an antibody, the Fc domain thus undertakes antibody functions, and antibody Fc engineering represents the engineering functions of an antibody that are effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), and controls the serum half-life. Therefore, the Fc domain may exist in the form of mutants or variants containing amino acid substitutions, insertions, or deletions so as to enable different modifications of Fc, especially in terms of post-translational modification, dimerization behavior, effector function, serum half-life, etc. To indicate the mutations present in the Fc domain based on the sequence of naturally occurring IgG, conventional antibody numbering annotations are known in the art, such as IMGT numbering (LeFranc, 2014; Frontiers in Immunology. 5(22):1-22), Kabat numbering (Kabat, E.A. et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication n°91-3242, pp 662, 680, 689(1991)) or EU numbering (Edelman et al. (1969). The covalent structure of an entire gammaG immunoglobulin molecule. Proc Natl Acad Sci USA.; 63:78-85), etc.

[0114] Non-limiting examples of multivalent constructs include "bivalent" constructs, "trivalent" constructs, "tetravalent" constructs, and the like. The immunoglobulin single variable domains contained within a multivalent construct may be the same or different, and preferably bind to the same or overlapping binding sites. In another particular embodiment, the binding agents of the invention are in "multispecific" form and are formed by binding two or more building blocks or agents, at least one of which binds to FRα as shown herein and at least one of which binds to a further target or alternative molecule, so that when present in a multispecific fusion, present a binding agent or composition that can specifically bind both epitopes or targets, and thus include binding agents having different specificities. Non-limiting examples of multispecific constructs include "bispecific" constructs, "trispecific" constructs, "tetraspecific" constructs, and the like. To further illustrate this, any of the multivalent or multispecific (as defined herein) forms of the invention may be suitably directed against one or more different epitopes on the same FRα antigen, or may be directed against two or more different antigens, e.g., one building block against FRα and serum albumin, or one building block as a half-life extension against another target. As shown herein, the multivalent or multispecific ISVDs of the invention may also be obtained by the use of such multivalent or multispecific immunoglobulin single variable domains and have increased avidity and / or improved selectivity for the desired FRα interaction and / or for any other desired property or combination of desired properties, such as affinity or higher order structure requirements, or may be engineered and / or selected as such. In another embodiment, a polypeptide comprising any of the immunoglobulin single variable domains according to the invention in either monovalent, multivalent, or multispecific form is provided. Thus, polypeptides comprising monovalent, multivalent, or multispecific Nanobodies are included herein by way of non-limiting example.Multivalent or multispecific binding agents or components may be directly fused or conjugated by a suitable linker such that at least two binding sites can be simultaneously accessed or bound by the multivalent or multispecific agent.

[0115] Therapeutic and diagnostic uses of the FRα binding agents of the present application Blood-CSF Barrier Shuttle: FRα Binding Agent as a BCSFB Transporter Among all normal tissues expressing FRα, choroid plexus epithelial cells have the highest level of FRα. Furthermore, compared to other tissues such as the lung, retina, placenta, etc., the receptor is present on the basolateral surface of CPE that is in direct contact with folic acid in the peripheral circulation and any FRα binding agent. This is particularly advantageous considering the restricted brain bioavailability of systemically administered pharmaceutical compounds. Indeed, the transport and delivery of therapeutic agents to the brain are severely restricted by the blood-brain barriers such as the blood-brain barrier (BBB) and the blood-CSF barrier (BCSFB). Considering that the BCSFB is formed by FRα-expressing CPE, the FRα binding agent of the present application can shuttle a therapeutic agent, a diagnostic agent, or other compounds (to which the FRα binding agent is coupled) across the BCSFB and thus can be used to improve the brain delivery of the compound.

[0116] Accordingly, in one aspect of the present application, any of the FRα binders of the present application is provided for use in transporting a chemical entity across the blood-brain barrier, more specifically across the BCSFB, or for use in transporting a chemical entity to the brain. Along this line, the use of currently applicable FRα binders is provided for transporting a chemical entity across the blood-brain barrier, more particularly across the BCSFB, or to the brain. Also provided is the use of the FRα binders of the present application for promoting, enabling, increasing, or improving the uptake of a chemical substance into the CNS via the blood-brain barrier, more specifically via the BCSFB. The uptake is improved or increased when the chemical entity is present in the brain or CSF in a statistically significantly greater amount, or at least 10%, 15%, 20%, 25%, 50%, 75%, 100%, or at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold greater amount compared to a situation where the chemical entity is not coupled to one of the currently applicable FRα binders, in a situation where the chemical entity is coupled to one of the currently applicable FRα binders.

[0117] The FRα binders of the present application are also provided for use as a medicament, for use in in vivo medical imaging, for treating a neurological disease, neuropathic pain or cancer, particularly FRα-expressing cancer, or for use in preventing brain injury after a brain injury.

[0118] In the present application, it has been demonstrated that the FRα binders, more specifically FRα antibodies or fragments thereof, most specifically FRα-binding VHHs, need to meet certain criteria before they can be transported across the BCSFB. With respect to the family 3 VHHs disclosed herein, it has been revealed that the combination of epitope recognition and affinity for FRα is important for their BCSFB permeability. More specifically, FRα-binding VHHs that recognize the same FRα epitope as 2HFO42 disclosed herein have a dissociation constant (KD) determined by BLI of less than 5x10 -2 / s, more specifically less than 4x10 -2Less than 3.5x10 -2 Less than 3x10 -2 Less than 2.9x10 -2 Less than 2.8x10 -2 , 2.7x10 -2 , 2.6x10 -2 , 2.5x10 -2 , 2.4x10 -2 , 2.3x10 -2 , 2.2x10 -2 , 2.15x10 -2 , 2.1x10 -2 , 2x10 -2 , 1.9x10 -2 , 1.8x10 -2 , 1.7x10 -2 , 1.6x10 -2 , or less than 1.5x10 -2 / s and requires affinity for human FRα. In the most specific embodiments, the affinity for human FRα is between 8x10 -4 / s and 4x10 -2 / s, or between 9x10 -4 / s and 3x10 -2 / s, or between 1x10 -3 and 2.5x10 -2 / s, or between 2x10 -3 and 2x10 -2 / s. Antibodies comprising VHH bind to the epitope through the CDR3 region. Accordingly, this application contains at most two or fewer substitutions in the sequence depicted in SEQ ID NO: 5 and has an affinity of less than 5×10 -2 / s, more specifically less than 4×10 -2 , 3×10 -2 , 2×10 -2 or 1.5×10 -2 / s, or even more specifically between 8×10 -4 and 4×10 -2 / s or between 9×10 -4 / s and 3×10 -2 / s or between 1×10 -3 and 2×10 -2Provided is an FRα binder comprising a CDR3 sequence having affinity for human FRα at / s. In certain embodiments, when the FRα binder binds to the FRα, it does not interfere with folic acid binding and / or folic acid transport by human FRα. In another particular embodiment, the FRα binder can cross-react with primate and mouse FRα. In another particular embodiment, the FRα binder recognizes an epitope in human FRα containing Q at position 141 of SEQ ID NO: 1 and / or comprises a CDR2 sequence as depicted in SEQ ID NO: 31 and / or a CDR1 sequence as depicted in SEQ ID NO: 3. In the most particular embodiment, the FRα binder comprises, or consists of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the sequence to SEQ ID NO: 37. The FRα binder is also provided when coupled to a chemical entity to facilitate uptake of the chemical entity into cerebrospinal fluid (CSF) across the blood CSF barrier (BCSFB). The chemical entity can be a biological, small molecule, therapeutic agent, radionuclide, antisense oligonucleotide, imaging agent or test compound. In certain embodiments, the chemical entity is neurotensin or a neurotensin analog. In the most particular embodiment, the FRα binder comprises, or consists of, an antibody or antibody fragment, more specifically an immunoglobulin single variable domain or VHH.

[0119] The applicants who have elucidated the in vivo requirements necessary for BCSFB passage by transcytosis via FRα disclose herein a novel human blood-brain barrier shuttle. The shuttle efficiently delivers chemicals to the brain, particularly the meninges. Instead, the present application provides an FRα binder suitable for delivery of a chemical entity to the brain, which binder is one of the FRα binders disclosed in the present application. The transport of the chemical entity to the brain is significantly increased as compared to the transport of a chemical entity that is not part of the shuttle or a chemical entity that is not bound to the FRα binder of the present invention. In certain embodiments, the chemical entity is a drug for a neurological disorder.

[0120] The blood-brain barrier shuttle comprises an FRα binder that binds to the same epitope as 2HFO42 shown in SEQ ID NO: 2 (more specifically, having at most two amino acids different from SEQ ID NO: 5, or having at most one amino acid different from SEQ ID NO: 5, or comprising the CDR3 sequence depicted in SEQ ID NO: 5), and the shuttle has a dissociation constant k -2 less than 5×10 -2 , more specifically 4×10 -2 , 3.5×10 -2 , 3×10 -2 , 2.9×10 -2 , 2.8×10 -2 , 2.7×10 -2 , 2.6×10 -2 , 2.5×10 -2 , 2.4×10 -2 , 2.3×10 -2 , 2.2×10 -2 , 2.15×10 -2 , 2.1×10 -2 , 2×10 -2 , 1.9×10 -2 , 1.8×10 -2 , 1.7×10 -2 or 1.6×10 -2 / s for human FRα. In one aspect, the k off of the shuttle for human FRα is between 8x10 off / s and 4x10 -4 / s, or between 9x10 -2 / s and 3x10 -4 / s, or between 1x10 -2 and 2.5x10 -3 / s, or between 2x10 -2 and 2x10 -3 as determined by BLI. -2between 0 and 1000000000 / s. In addition to the FRα binder, the shuttle comprises a molecule or moiety that is transported to the CNS, more specifically through the BCSFB. In one aspect, the FRα binder of the blood-brain barrier shuttle comprises an ISVD having the CDR3, and the CDR4 loop (located in FR3) as defined herein comprises the amino acids D at position 72 and N at position 73 according to Kabat numbering, or the amino acids E at position 72 and G at position 73 according to Kabat numbering, or the amino acids P at position 72 and the amino acid G at position 73. In a further aspect, the FRα binder of the blood-CNS barrier shuttle further comprises an ISVD comprising a CDR2 sequence having at most 2 amino acids different from SEQ ID NO: 4 as depicted in SEQ ID NO: 32, or at most 1 amino acid different from SEQ ID NO: 4 as depicted in SEQ ID NO: 31, or at most 1 amino acid different from SEQ ID NO: 4 as depicted in SEQ ID NO: 4; and / or, by way of example, a CDR1 sequence having at most 4 amino acids different from SEQ ID NO: 3 as depicted in SEQ ID NO: 113, or a CDR1 sequence having 3 or 2 or 1 amino acids different from or as depicted in SEQ ID NO: 3. Specifically, the ISVD of the shuttle comprises, or consists of, an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity over the full length of the sequence to SEQ ID NO: 37 (excluding the last 9 amino acids (3xAla 6xHis sequence)). In certain embodiments, the amino acid differences are found outside the CDR regions, preferably within the FR regions but outside the CDR4 loop, preferably different from the amino acid positions shown herein that may affect the VHH affinity for FRα. The molecule or moiety that is part of the blood-brain barrier shuttle can be a neurotherapeutic agent, an imaging compound, a nanoparticle, an exosome.

[0121] The above-mentioned blood-brain barrier shuttle can alternatively be expressed as a blood-CNS barrier shuttle, a composition or a pharmaceutical composition, or more specifically, a blood-CSF barrier shuttle.

[0122] In another aspect, the blood-brain barrier shuttle, the blood-CNS barrier shuttle, the blood-CSF barrier shuttle, the composition or the pharmaceutical composition is provided for use as a medicine, more particularly for use in the treatment or diagnosis of neurological diseases. In one embodiment, the shuttle or composition comprises, in addition to any of the above-mentioned FRα binders, a neurological disease drug, a cancer drug, nanoparticles or an imaging compound. In certain embodiments, the neurological disease drug of the shuttle or composition is a biological agent, a small molecule agent, a therapeutic agent, a radionuclide, an antisense oligonucleotide or a test compound.

[0123] In another specific embodiment, the composition or shuttle is a bispecific antibody comprising the above-mentioned human FRα binder and a second antigen-binding site that binds to a brain antigen. Non-limiting examples of brain antigens include beta-secretase 1 (BACE1), amyloid beta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6. The bispecific antibody can also comprise a second or more antigen-binding sites that bind to a tumor antigen or a cancer antigen. The bispecific antibody is particularly advantageous for the treatment and / or diagnosis of brain cancer.

[0124] In certain embodiments, the molecule or moiety that is part of the shuttle or composition is neurotensin or a neurotensin agonist. In the present application, the neurotensin body temperature assay has been used as a sophisticated system for evaluating the activity of antibodies that cross the BCSFB. However, the VHH-neurotensin fusions described herein are also clinically relevant. First, research results indicate that a therapeutic effect can be expected for the induction of hypothermia by neurotensin (or a neurotensin agonist). Choi et al (2012 FASEB J 26:2799-2810) showed that administration of the NT agonist ABS-201 immediately after onset or up to 60 minutes after onset significantly reduced infarct formation and brain cell death in an animal model of focal ischemia and was effective in promoting long-term functional recovery in animals after stroke. Similar studies on the regulated hypothermia induced by NT agonists reduce brain oxidative stress during reperfusion from asphyxial cardiac arrest (Katz et al 2004 Brain Res 1017:85-91). Also, in a rat model of near-drowning, lowering the body temperature with neurotensin or an NT agonist provided improved neurological outcomes compared to short-term external cooling (Katz et al 2004 Crit Care Med 32:806-810). Thus, the shuttle or composition wherein the koff of human FRα as determined by BLI is 8×10 -4 ~4×10 -2 / s or 9×10 -4 / s~3×10 -2 / s or 1×10 -3~ 2.5×10 -2 / s or 2×10 -3 ~2×10 -2 / s is also provided for treating or preventing stroke, post-stroke brain cell death or post-brain injury brain damage. In certain embodiments, the shuttle or composition comprises neurotensin or a neurotensin agonist.

[0125] Nemeroff et al. (1979 PNAS 76:5368-5371) showed that in addition to its effect of inducing hypothermia, neurotensin is an important regulator of nociceptive transmission and is more potent than morphine as an anti-nociceptive agent. Neurotensin provides a strong analgesic effect when administered directly into the brain and reverses pain behaviors induced by the development of neuropathic pain and bone cancer pain in animal models (Demeule et al 2014 JIC 124:1199-1213). As part of a group of brain-penetrant neurotherapeutics (for example, by coupling with one of the currently applied FRα binders), neurotensin is effective in the clinical management of persistent and chronic pain. Thus, provided herein are shuttles or compositions comprising neurotensin or a neurotensin agonist, having a koff of 8×10 -4 ~4×10 -2 / s or 9×10 -4 / s~3×10 -2 / s or 1×10 -3 ~2.5×10 -2 / s or 2×10 -3~ 2×10 -2 / s for use in the treatment of neuropathic pain.

[0126] As used herein, "neurodegenerative disease" refers to a disease or disorder that affects the central nervous system or CNS, and / or a disease or disorder having an etiology in the CNS. "Central nervous system" or "CNS" refers to a complex of nerve tissues that control body functions, including the brain and spinal cord. Exemplary CNS diseases or disorders include neurodegenerative diseases (including, but not limited to, Lewy body disease, Parkinson's disease, tauopathies (including, but not limited to, Alzheimer's disease and supranuclear palsy), posterior column spinal cord syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, multiple system atrophy), striatonigral degeneration, prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, fatal familial insomnia), bulbar palsy, dystonia (including, but not limited to, DYT1 dystonia), motor neuron diseases (including, but not limited to, multiple sclerosis, Charcot-Marie-Tooth (CMT) disease, amyotrophic lateral sclerosis (ALS)), nervous system heterozygous degenerative diseases (including, but not limited to, Canavan disease, Huntington disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Hallervorden-Spatz syndrome, Lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia (including, but not limited to, Pick's disease, spinocerebellar ataxia), cancer (e.g., cancer of the CNS and / or brain, including brain metastases resulting from cancer in other parts of the body), neuropathy, amyloidosis, eye diseases or disorders, viral or microbial infections, inflammation, ischemia, seizures, behavioral disorders, lysosomal storage diseases, etc., but are not limited thereto).

[0127] "Neurodisorder drug" refers to a drug or therapeutic agent for treating one or more neurodegenerative disorders. Neurodisorder drugs contemplated in this application include, but are not limited to, antibodies, peptides, proteins, natural ligands of one or more CNS targets, modified versions of natural ligands of one or more CNS targets, aptamers, inhibitory nucleic acids or antisense oligonucleotides (i.e., small interfering RNA (siRNA), short hairpin RNA (shRNA) or gapmer), ribozymes, and small molecules, or active fragments of any of the foregoing that specifically recognize and / or act on (i.e., inhibit, activate, or detect) a CNS antigen or target molecule. "CNS antigen" or "intracerebral antigen" refers to an antigen expressed in the CNS including the brain, which can be targeted by antibodies and small molecules. Non-limiting examples of such CNS antigens or target molecules include amyloid precursor protein or a portion thereof, amyloid beta, beta-secretase BACE1, gamma-secretase, tau, alpha-synuclein, parkin, huntingtin, DR6, presenilin 1, presenilin 2, ApoE, glioma or other CNS cancer markers, and neurotrophins. Non-limiting examples of neurodisorder drugs and disorders for which they may be used in treatment include anti-BACE1 antibodies (e.g., International Publication No. WO 2009 / 121948, International Publication No. WO 2010 / 146058, International Publication No. WO 2012 / 064836) and anti-HER2 antibodies (e.g., trastuzumab) (e.g., International Publication No. WO 2003 / 087131).

[0128] In another aspect of the present application, a method of treating a subject is provided, the method comprising, when determined by BLI to include a neurodisorder drug, less than 5×10 -2 / s, or less than 4×10 -2 / s, 3.5×10 -2 , 3×10 -2 , 2.9×10 -2 , 2.8×10 -2 , 2.7×10 -2 , 2.6×10 -2 , 2.5×10 -2 , 2.4×10 -2 , 2.3×10 -2, 2.2×10 -2 , 2.15×10 -2 , 2.1×10 -2 , 2×10 -2 , 1.9×10 -2 , 1.8×10 -2 , 1.7×10 -2 , 1.6×10 -2 , or 1.5×10 -2 administering to the patient the above-described shuttle or (pharmaceutical) composition having a koff for human FRα of less than / s, wherein the subject has a neurological disorder. Also provided is a method for performing in vivo medical imaging of a body region or tissue of a subject, more particularly a brain region, the method comprising administering to the subject an effective amount of any of the blood-brain barrier shuttles disclosed herein comprising an imaging compound, and detecting the imaging compound in a body region of the subject. The method comprises collecting one or more images of the subject and displaying the one or more images of the subject. The images include a plurality of images over a period of time and may be taken over a period of time. The collection and display of the images can be performed by commercially available scanners and the associated computer hardware and software. For example, a PET scanner and a SPECT scanner may be used. The imaging compound can be any compound that enables efficient in vivo medical imaging. Non-limiting examples include radionuclides such as technetium (99mTC) or lutetium-177. Also provided is a method for transporting the compositions or shuttles described herein from the peripheral blood stream of a subject to the CSF, more particularly from the basolateral side to the apical side of CPE cells, the method comprising administering to the subject any one of the shuttles or (pharmaceutical) compositions described herein.

[0129] In one aspect of the above method, the composition or shuttle is administered to the patient using a route selected from the list consisting of oral administration, nasal administration, intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, intradermal administration, topical administration, and enteral administration. In one aspect, the composition is not administered intracranially or intraventricularly or extracranially, or by any alternative direct administration to the brain.

[0130] Anti-cancer approach: FRα-targeted anti-cancer therapeutic Surgery, chemotherapy, and radiotherapy have long been considered the best options for cancer treatment. However, these treatments are non-discriminatory tactics that overlap with harmful side effects and cannot protect against recurring cancer cells (Lecocq, 2019). The identification of molecular promoters of cancer cells such as HER2 has led to the development of molecular targeted treatments designed to bind to and inactivate the defective molecules of cancer cells (Lecocq, 2019). Modern cancer treatment and diagnosis focus on the targeted and thus specific delivery of high doses of chemotherapeutic agents or diagnostic agents to the tumor site while preserving normal tissues and thus overcoming the high systemic toxicity of the drugs. Targeted therapy in clinical practice requires highly affinity and tumor-specific agents and effective targeting vehicles to deliver therapeutic agents to the tumor site (Xing, 2018).

[0131] As described above, FRα is an attractive anti-cancer target for several reasons. These non-mucinous (serous and endometrioid) adenocarcinomas most stably express FRα (Elnakat, 2004), and thus anti-cancer therapeutics targeting FRα have been mainly developed for ovarian and endometrial cancers. FRα affinity ligands such as folic acid itself, or anti-tumor prodrugs conjugated to anti-FRα binding agents as described in the present application, can be taken up into FRα-expressing tumor cells based on the principle of the molecular “Trojan horse”. The endocytosis mechanism via FRα can be utilized as an entry point for delivering large amounts of anti-cancer therapeutics because FRα quantitatively circulates between the cell surface and intracellular compartments (Kalim et al 2017 Drug Des Devel Ther 11). Interestingly, in most growing healthy tissues except the kidney, FRα expression is limited to the epithelial luminal or apical surface that does not directly contact folic acid and circulating folate receptor-targeted agents (Elnakat and Ratnam 2004 Adv Drug Deliv Rev 56; Low and Kularatne 2009 Curr Opin Chem Biol). Furthermore, FRα-targeted agents do not accumulate in the epithelial cells of the proximal renal tubules. This is due to the circulating reabsorption process necessary to prevent the loss of folic acid in the urine (Sega and Low 2008 Cancer Metastasis Rev 27). Therefore, systemically administered FRα-targeted agents should be non-nephrotoxic and have minimal systemic toxicity (Salazar and Ratnam 2007 Cancer and Metastasis Reviews 26).

[0132] The first folate-conjugated cytotoxic agent to be evaluated in tumor therapy was a maytansinoid conjugate (Reddy et al 2007 Cancer Res 67). Since then, a series of chemotherapeutic agents have been conjugated to folate or anti-FRα mAb for FRα tumor targeting (Cheung et al 2016 Oncotarget 7). Non-limiting examples include vintafolide, a folate conjugate of desacetylvinblastine monohydrazide (DAVLBH), a derivative of the microtubule destabilizing agent vinblastine (Vlahov et al 2006 Bioorg Med Chem Lett 16), IMGN853, an anti-FRα mAb conjugated to the microtubule stabilizing agent maytansinoid (Ab et al 2015 Mol Cancer Ther 14), mirvetuximab soravtansine, MOR-ab-202, and others. Mirvetuximab soravtansine is a conjugate of the maytansinoid DM4 to a humanized anti-FRα mAb via a cleavable linker (Scaranti, 2020#739). Preclinical studies have demonstrated antitumor activity, and tolerability has been proven in a phase 1 clinical trial (Scaranti, 2020). MOR-ab-202 is a new generation of Ab conjugated with farletuzumab and eribulin, a microtubule targeting agent. MOR-ab-202 has improved specificity in vivo and shows a durable and potent antitumor effect in xenograft models (Farran, 2019#261).

[0133] Second, the role of non-conjugated FRα-specific monoclonal antibodies (mAbs), such as the fully humanized IgG1 antibody farletuzumab, in passive immunotherapy has been studied. Passive anti-FRα immunotherapy is based on the administration of mAb therapy that can selectively target FRα-positive cancers (Farran, 2019). The antitumor activity is due to antibody-dependent cell cytotoxicity (ADCC) (Ebel et al 2007 Cancer Immun 7). FRα can be passively targeted by chimeric, mouse, and human antibodies alone or as conjugates to deliver T cells, radionuclides, and cytokines to cancer tissues (Farran, 2019). Farletuzumab has been evaluated in a Phase I clinical trial and had a slow clearance rate due to an estimated terminal half-life of 121 to 260 hours (Scaranti, 2020). Farletuzumab has also been evaluated in a Phase II clinical trial of ovarian cancer women for combination therapy with carboplatin and taxane agents and maintenance therapy with farletuzumab alone (Scaranti, 2020). MOv18 is also an IgG1 antibody and was generated by vaccinating mice with ovarian cancer cells (Scaranti, 2020). When a chimeric labeled with a radioisotope was administered intravenously or intraperitoneally to ovarian cancer patients to evaluate the feasibility of radioimmunoscintigraphy, this method was proven to be safe in several early-phase trials (Scaranti, 2020). Next, the IgE form of MOv18 was developed to cause a rapid allergic hypersensitivity reaction by mast cells and was more effective than the IgG1 isotype in preclinical trials (Scaranti, 2020).

[0134] In another aspect, any of the FRα binders of the present application is provided for use in in vivo medical imaging or for treating FRα-expressing cancers selected from the list consisting of cancer, particularly FRα-expressing cancer, more particularly ovarian cancer, breast cancer, pleural cancer, lung cancer, cervical cancer, endometrial cancer, colon cancer, kidney cancer, bladder cancer, and brain tumors. In the present application, it is taught that for transport to the CNS via the BCSFB, the FRα binder of the present application should have a dissociation constant koff within a specific range. However, this specific dissociation constant is not an essential feature for binding to FRα or for binding on the surface of FRα-expressing cancer cells. Thus, any FRα binder disclosed herein is provided for use in cancer diagnostic and treatment approaches, for example, by coupling with an anti-cancer agent or an imaging compound.

[0135] In one aspect, the FRα binder provides a K of 50 nM to 500 nM for human FRα dis and the binder coupled to a chemical entity improves the uptake of the chemical entity into FRα-expressing cancer cells or the binding of the chemical entity to the surface of FRα-expressing cancer cells. In certain aspects, the FRα binder is one of the FRα binders of the present application. In the most particular aspect, the FRα binder is one of the VHHS of the present invention.

[0136] VHHs have been widely studied in the context of targeted cancer therapy and immunotherapy. In the fight against cancer, VHHs have been employed in various types of strategies: namely, (1) attenuating oncogenic signals, (2) delivering a lethal punch to cancer cells, (3) designing cancer vaccines, (4) engaging cytolytic cells, and (5) preventing immunosuppressive events (Lecocq, 2019).

[0137] VHHs that target cancer cells despite lacking antagonist properties have been coupled to other technology platforms to deliver a targeted lethal punch to cancer cells (Lecocq, 2019). VHHs have been coupled to cell death-inducing ligands (such as TRAIL), truncated forms of Pseudomonas exotoxin A, various drugs and drug-loaded nanoparticles, photosensitizers (i.e., when exposed to light of a specific wavelength in an oxygenated environment, photosensitizers form ROS), therapeutic radionuclides (i.e., radioactive labels such as lutetium-177, iodine-131, astatine-211, actinium-225, and bismuth-213 that can emit their energy near cancer cells, thereby causing irreparable DNA damage.), and enzymes for prodrug activation (such as β-lactamase that converts the prodrug 7-(4-carboxybutanamide)cephalosporin mustard in phenylenediamine mustard), various drugs, and drug-loaded nanoparticles (Lecocq, 2019). Similar to photosensitizers, branched gold nanoparticles kill cancer cells when excited with near-infrared light but generate heat instead of reactive oxygen species (Lecocq, 2019). VHHs can also reduce potential adverse effects because they can bring these toxic sites closer to cancer cells while minimizing toxic effects on healthy tissues (Lecocq, 2019).

[0138] Some bifunctional molecules have been designed (such as anti-EGFR VHHs coupled to TRAIL) (Lecocq, 2019). Drugs frequently used in the treatment of various types of cancer include cisplatin and its analogs, carboplatin, oxaliplatin, doxorubicin, RTK inhibitors, deeffector molecules, etc. Since these drugs lack selectivity, VHHs have been used as targets for cancer cells (Lecocq, 2019).

[0139] In various embodiments, provided is a pharmaceutical composition comprising any of the FRα binders of the present application coupled to a chemotherapeutic agent for use as a medicament, more particularly for treating cancer, and even more particularly for treating FRα-expressing cancer.

[0140] Non-limiting examples of such chemotherapeutic agents include alkylating agents such as thiotepa, cytoxan, cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, piposulfan; aziridines such as benzodopa, carboquone, meturedopa, uredopa; ethyleneimines and methylamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolmelamine; acetogenins (e.g., bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; calicheamicin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycin (e.g., cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobenbitin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin gamma 1 and calicheamicin omega 1; dynemicin including dynemicin A); bisphosphonates such as clodronate; antibiotics such as esperamicin;Also, neocarzinostatin chromophore and related chromoproteins (enediyne antibiotics chromophore), actinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, cardinophilin, chromomycin, daunorubicin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, adriamycin, doxorubicin (including morpholino-doxorubicin, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, keramycin, rhodomycin, streptozotocin, tubercidin, ubenimex, dinostatin, zorubicin and other mitomycins; antimetabolites such as methotrexate, 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, drostanolone propionate, epithiostanol, mepithiostane, testolactone; antiadrenalins such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; dexamethasone; diacontin; elformithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;Podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; lizoxine; sizofiran; spirigermanium; tenuazonic acid; triazicon; 2,2',2''-trichloroethylamine; trichothecenes (e.g., T-2 toxin, verruculin A, loline A and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., taxoids, e.g., taxol, paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), abraxane, albumin-engineered nanoparticle formulation of paclitaxel without cremophor (American Pharmaceutical Partners, Schaumberg, Ill. and taxotere, docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemzar, gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; navelbine, vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (camptosar, CPT-11) (including treatment regimens of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin (including oxaliplatin treatment regimen (FOLFOX)); lapatinib (tykerb);Inhibitors of PKC-α, Raf, H-Ras, EGFR (for example, erlotinib (Tarceva) which inhibits cell proliferation, etc.) and VEGF-A, and pharmaceutically acceptable salts, acids or derivatives of any of the above. Furthermore, the treatment method can further include the use of photodynamic treatment.;

[0141] In some embodiments, the FRα binder or pharmaceutical composition described herein acts synergistically when co-administered with another therapeutic agent. In such embodiments, the FRα binder and the additional therapeutic agent may be administered at a lower dose than the dose employed when the agent is used in the context of monotherapy.

[0142] FRα Targeting for Tumor Imaging When diagnosing cancer, it is desirable to know as much as possible about the tumor, such as the presence of the target tumor antigen and the immune status, in order to plan and monitor the most effective treatment (Lecocq, 2019). FRα targeting for non-invasive imaging of FRα-positive primary and metastatic tumors enables reliable patient selection for individualized anti-cancer treatment with FRα-targeted therapeutic agents and enables systemic monitoring of the FRα expression status of the tumor throughout the treatment (Cheung et al 2016 Oncotarget 7). Imaging techniques based on FRα-specific agents also help surgeons perform better resections in patients with tumors that express FRα (Scaranti, 2020).

[0143] One approach is the use of FRα-targeted contrast MRI (Scaranti, 2020). Here, a target folate-conjugated tracer (such as a dendrimer-polycinate, for example) accumulates in FRα-expressing tumors and improves contrast enhancement (Scaranti, 2020). Other examples available in the art include (1) folic acid coupled with iron oxide having carboxylate in breast cancer, (2) superparamagnetic iron oxide nanoparticles incorporated into heparin-folate micelles, (3) radioactive tracers such as folic acid derivatives of radioisotopes (e.g., 99mTc, which is cheaper and easier to produce than 111In) used in whole-body SPECT analysis (Scaranti, 2020). In clinical trials, the tolerance of 99mTc-ertafolitide (a peptide derivative of folic acid) is good (Scaranti, 2020). Folic acid radio-conjugates have been proposed as a promising therapeutic strategy for patients with FRα-positive cancers (more details). Also, non-radioactive labeling approaches involving fluorescent probes linked to folic acid that enable intraoperative visualization of tumors have also been demonstrated to be successful (Scaranti, 2020).

[0144] Another approach is the use of labeled FRα binders. Antibody-based diagnostics have provided promising levels of tumor-to-background resolution, such as 89Zr-DFO-M9346A (Scaranti, 2020), for example. Radioimmunoscintigraphy (RIS) using radiolabeled monoclonal antibodies targeting FRα has already been used in clinical trials and has shown success in ovarian cancer patients (Crippa et al 1991 Eur J Cancer 27; van Zanten-Przybysz et al 2001 Int J Cancer 92).

[0145] VHH-based imaging has been widely studied for detecting cancer cells in preclinical trials (e.g., antigens CEA, EGFR, HER2, PSMA, CD20, CD38, etc.) (Lecocq, 2019). For clinical purposes, the most advanced VHH-based imaging agent is the 68Ga-coupled anti-HER2 Nanobody 2Rs15d for PET imaging of BC patients (Lecocq, 2019). In the first clinical trial in 2016, it was revealed that HER2 in primary tumors or local or distant metastases could be detected and imaged in a short time of 60 minutes after injection, with high specificity and without side effects such as nephrotoxicity and tracer-induced antibodies (Lecocq, 2019). Furthermore, background uptake was very low, except for the signals observed in the kidneys, intestines, and liver (Lecocq, 2019). Recently, a Phase II clinical trial to evaluate the potential of 68Ga-NOTA-2Rs15d for detecting brain metastases (NCT03924466) has been initiated (Lecocq, 2019). By performing VHH-based imaging of cancer markers, especially since targeted therapies have been developed for many of these cancer markers, some of which are based on the use of VHHs (e.g., anti-HER2 VHH for targeted therapy), it can serve as a guide for treatment selection (Lecocq, 2019). Furthermore, VHH-based probes have been developed for imaging the expression of immune checkpoints (Lecocq, 2019).

[0146] For non-invasive imaging, VHH needs to be labeled with an imaging probe that can consist of (1) a radioisotope, (2) a fluorescent dye, (3) a microbubble, (4) a chemical substance such as gadolinium, which enables imaging by techniques such as single-photon emission computed tomography (SPECT), positron emission tomography (PET), optical imaging (OI), ultrasound (US), and MRI (Lecocq, 2019). Most of the imaging studies using VHH use SPECT and PET because these radioisotope-based techniques provide high sensitivity, high resolution, and quantitative information (Lecocq, 2019). In preclinical studies, VHH often contains a C-terminal hexahistidine tag inserted for purification purposes and can be complexed with 99mTc(CO3), a γ-emitting radionuclide that can be easily detected using SPECT (Lecocq, 2019). In the more clinically important PET, VHH is labeled with positron-emitting radionuclides (18F (half-life 68 minutes), 64Cu, 68Ga (half-life 110 minutes), 89Zr) (Lecocq, 2019). The half-life coincides with the biological half-life of VHH when injected intravenously (Lecocq, 2019). Site-specific labeling is desired to obtain a uniform and consistent tracer (for example, ligation via transpeptidase sortase A, catalyzing peptide bond formation between the LPXTG peptide motif expressed at the C-terminus of VHH and the N-terminal oligo-glycine motif on the label) (Lecocq, 2019). As an alternative to radioisotope labeling of VHH, there is a method that uses a fluorescent dye and can be combined with optical imaging (OI).In in vivo imaging, near-infrared emitting fluorescent dyes (such as IRDye-680RD or -800CW, Cy5, Alexa Fluor 680, etc.) are selected as labels because they provide signal detection at depths from several hundred micrometers to 1 cm, as well as strong contrast and resolution (Lecocq, 2019). The advantage of OI is that, in contrast to imaging via radioisotopes, it does not require a dedicated facility, making it flexible, convenient, and cost-effective (Lecocq, 2019). OI is often used for the study of surface lesions during surgery or endoscopic procedures because, compared to imaging using radioisotopes, the tissue penetration ability of OI dyes is limited (Lecocq, 2019). US can be used as an alternative to radioisotope-labeled VHH while maintaining the ability to obtain high-resolution images (Lecocq, 2019). In US, it is necessary to conjugate VHH to US contrast agents, microbubbles, and nanobubbles to reveal the molecular characteristics of the vascular wall (after intravenous administration) (Lecocq, 2019). In MRI imaging, VHH-coated superparamagnetic nanoparticles enable antigen detection in xenograft tumors (Lecocq, 2019).

[0147] FRα-binding VHH for use in seranostics An emerging modality in precision oncology is the development of seranostics that enable patient selection, treatment, and monitoring (Lecocq, 2019). In this approach, labeled compounds and imaging technologies are used for patient diagnosis and the selection of optimal treatment regimens, and related compounds are used to target cancer cells and tumor stroma for treatment (Lecocq, 2019). Against this background, VHH and VHH-directed therapeutic agents have attracted attention (Lecocq, 2019). This interest is due to their high antigen specificity, small size, ease of labeling and engineering design, which enable the design of specific imaging and treatment methods targeting antigens on tumor cells, immune cells, and proteins within the TME (Lecocq, 2019).

[0148] In oncology, there is increasing interest in targeted radionuclide therapy (TRNT), which selectively delivers radioactivity to kill malignant cells while minimizing damage to healthy cells (Ersahin et al., 2011). This therapeutic strategy has gained attention because of the widespread availability of therapeutic radionuclides (Tomblyn et al., 2012). Radioimmunotherapy (RIT) is a TRNT strategy that uses radiolabeled monoclonal antibodies (mAbs) that interact with tumor-associated proteins expressed on the surface of cancer cells and are thus readily accessible to these circulating agents. RIT for the treatment of B-cell non-Hodgkin's lymphoma (NHL) consists of the radiolabeled anti-CD20 mAbs 90 Y-ibritumomab tiuxetan (Zevalin) and 131 I-tositumomab (Bexxar). Zevalin is currently approved by the FDA as a treatment for relapsed refractory NHL and as a late-line addition to unlabeled anti-CD20 mAb rituximab. Because lymphomas are highly radiosensitive, relatively low absorbed doses are required to achieve an objective response. Recent clinical trials have shown that combination therapy with rituximab and zevalin is beneficial compared to rituximab monotherapy (Tomblyn et al., 2012), but Zevalin is only approved for patients with advanced disease (patients in whom the disease has relapsed or patients who are refractory to chemotherapy or immunotherapy with rituximab).

[0149] VHH has excellent properties in in vivo cell targeting compared to classical mAbs and their derived fragments (De Vos et al., 2013). Regarding cancer molecular imaging, VHHs have been directed against various membrane-bound cancer cell biomarkers such as CEA, EGFR, HER2, and PSMA (D'Huyvetter et al., 2014). Due to their excellent targeting specificity and the fact that they remain functional after radiolabeling with radionuclides, VHHs have become a valuable tool for nuclear imaging and TRNT (D'Huyvetter et al., 2014).

[0150] Diagnostic tests such as IHC are current practice, but cannot depict whole tumor expression levels, which is even worse in the case of metastatic lesions (Lecocq, 2019). Indeed, this could explain why the outcome response cannot be accurately predicted in all patients. Whole-body non-invasive imaging methods such as PET, SPECT, MRI, OI using VHH-based tracers meet these drawbacks and can be performed repeatedly without the need to take invasive biopsies (Lecocq, 2019). Interestingly, many of the described VHHS retain the potential to be used as both molecular imaging probes and therapeutic agents. The term "theranostic" was originally proposed to describe the development of diagnostic tests in parallel with the application of therapies targeting specific molecular features (Lecocq, 2019). Currently, the term theranostics is used in a much more stringent sense and rather refers to agents that are identical or closely related and have the potential to be used for both diagnostic and therapeutic purposes (Lecocq, 2019). VHHS targeting cancer-targeting membrane proteins (e.g., HER2) have been evaluated in both imaging and therapeutic applications (Lecocq, 2019). The clearest example of VHH theranostics is when both the diagnostic tracer and the therapeutic compound are radiolabeled in the TRT approach (Lecocq, 2019). The radiolabels can be different (e.g., gallium-68 or Fluor-18 for PET imaging, actinium-225 for α-TRT), but sometimes the radiolabels are the same, such as iodine-131-labeled VHH, which is first used at a low dose in SPECT imaging for diagnosis and dosimetry and then at a high dose for TRT (Lecocq, 2019). Importantly, diagnostic and therapeutic VHH radiopharmaceuticals have similar pharmacokinetic and biodistribution profiles (Lecocq, 2019).

[0151] In one aspect, there is provided any of the FRα binders of the present application coupled to a radionuclide. In one aspect, the FRα binder is coupled or fused to the radionuclide directly or via a coupling agent and / or a linker and / or a tag. In a specific aspect, the FRα binder is fused to the radionuclide via a His tag. The methods used to radiolabel the FRα binder are conventional methods and are known to those skilled in the art. Any available methods and chemical properties may be used for the association or conjugation of the radionuclide and the FRα binder. As an example, tricarbonyl chemistry may be used for radiolabeling (Xavier et al. 2012). In certain aspects, the FRα binder is coupled to a radionuclide that damages cells or is otherwise cytotoxic, and the FRα binder targets the radionuclide to FRα-expressing cells, preferably cancer cells. The FRα binder labeled with a radioisotope is used, for example, but not limited to, targeting a radionuclide that damages cancer tissue in order to preferentially damage or kill cancer cells.

[0152] According to certain aspects, any of the FRα binders described herein is useful for targeted radionuclide therapy. As used herein, "targeted radionuclide therapy" refers to the targeted delivery of a radionuclide to a disease site, followed by damage to target cells and adjacent cells (the bystander effect). In targeted radiotherapy, also referred to as systemic targeted radionuclide therapy (STaRT), a biological effect is obtained by the energy absorbed from the radiation emitted by the radionuclide. Non-limiting exemplary radionuclides are iodine-131, astatine-211, bismuth-213, lutetium-177, or yttrium-86. Exemplary radionuclides that can be used to damage cells such as cancer cells are high-energy emitters. For example, a high-energy radionuclide is selected and targeted to cancer cells. High-energy radionuclides preferably act over a short distance such that the cytotoxic effect is localized to the target cells. In this way, radiotherapy is delivered more locally to reduce damage to non-cancerous cells.

[0153] The present invention also relates to the use of the FRα binders described herein for disease diagnosis and / or prognosis and / or treatment prediction in a subject. As a non-limiting example, a subject having or at risk of having cancer can be determined based on the expression level, pattern, or profile of FRα in a test sample derived from the subject compared to a predetermined standard or standard level in a corresponding non-cancerous sample. In other words, the FRα polypeptide can be used as a marker indicating the presence or absence of cancer or the risk of developing cancer, and also as a marker for evaluating the prognosis of cancer and predicting the optimal treatment method.

[0154] In a further related aspect, the present disclosure contemplates pharmaceutical compositions comprising any of the FRα binders as described herein in relation to a pharmaceutically acceptable carrier. Thus, the FRα binder alone or conjugated with a chemical agent (see above) may be formulated in a physiological or pharmaceutically acceptable carrier suitable for in vivo administration. In certain embodiments, such compositions are suitable for oral, intravenous or intraperitoneal administration. In other embodiments, such compositions are suitable for direct local administration to the tumor site. In certain embodiments, such compositions are suitable for subcutaneous administration.

[0155] Method of treatment In another aspect of the present application, a method of treating a subject is provided, the method comprising administering to the patient a composition comprising one of the FRα binders of the present application conjugated with an anti-cancer agent, wherein the subject is suffering from cancer.

[0156] Also provided is a method of binding an FRα binder to cancer tissue, more particularly FRα-expressing cancer tissue, the method comprising administering to the cancer tissue a composition comprising one of the currently applied FRα binders. Also provided is a method of inducing a compound in cancer cells or tissue, more particularly FRα-expressing cancer cells or tissue, the method comprising administering to the cancer cells or tissue a composition comprising a compound conjugated with any of the currently applied FRα binders. In one embodiment, the cancer cells or tissue are present in a mammal, more particularly a human. In another embodiment, the cancer cells or tissue are in vitro cancer cells or tissue. In yet another embodiment, the compound is any of the cytotoxic or chemotherapeutic compounds or imaging compounds described herein.

[0157] In one embodiment, a method of administering or delivering or inducing an anti-cancer agent or imaging compound to FRα-expressing cancer cells is provided, which comprises administering to a subject a composition comprising any of the FRα antibodies disclosed herein conjugated to an anti-cancer agent or imaging compound.

[0158] In one aspect of the above method, the composition is administered to the patient using a route selected from the list consisting of oral administration, nasal administration, intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, intradermal administration, topical administration, and enteral administration. In one aspect, the composition is not administered intracranially or intraventricularly or extracranially, or by any alternative direct administration to the brain.

[0159] In vivo medical imaging method In another aspect, the present disclosure provides an in vivo medical imaging method. The method includes administering to a subject, such as a human or non-human subject, an effective amount of a labeled FRα binder described herein. The effective amount is an amount sufficient to label a desired cell or tissue, meaning that the labeled structure is detectable over the analysis period. The method includes collecting one or more images of the subject and displaying one or more images of the subject. The images include a plurality of images over a period of time and may be taken over a period of time. The collection and display of the images are performed by commercially available scanners and the associated computer hardware and software. For example, a PET scanner and a SPECT scanner may be used. The imaging compound can be any compound that enables efficient in vivo medical imaging. Non-limiting examples include radionuclides, such as technetium (99mTc) or lutetium-177. Further, to further improve the usefulness of the generated images, CT, X-ray, or MRI may be used simultaneously or sequentially to provide additional information, such as a description of the structural features of the subject. For example, a dual PET / CT scanner can be used to collect relevant data and display an image that overlays the data obtained from the two modalities. As an example, when selecting a radionuclide for in vivo imaging, a radionuclide that emits gamma rays or positrons, or a radionuclide that decays by electron capture, may be preferred. Then, for example, positron emission tomography (PET) or single photon emission computed tomography (SPECT) can be used to easily detect the radiation. Generally, the half-life of the radionuclide is long enough to be produced and used in the test, but it is desirable that it is not so long that radioactivity remains in the patient for a significant period of time after the test is performed. Further, by adjusting the amount of radioactivity used for labeling, the total radiation dose used to obtain the desired effect can be minimized.

[0160] Although specific embodiments, specific configurations, and materials and / or molecules are discussed herein with respect to the cells and methods of the invention, it should be understood that various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention. The following examples are provided to better illustrate specific embodiments and should not be considered as limiting the present application. The present application is limited only by the claims.

[0161] Example Example 1. Immunization of alpacas with folate receptor alpha (FRα) and construction of a VHH library. Two llamas were subcutaneously injected with a mixture of 50 μg of human FRα (Acro Biosystems, catalog number FO1-H5229), 50 μg of human FRα (Sino Biological Inc., catalog number 11241-H08H), 50 μg of mouse FRα (R&D Systems, catalog number 6936-FR), and 50 μg of mouse FRα (Sino Biological Inc., catalog number 50573-M08H) per injection on days 0, 7, 14, 21, 28, and 35. All proteins used for immunization contained a His6 tag and were emulsified with Gerbu adjuvant-P before injection. On day 40, blood was collected from the llamas into anticoagulant tubes for subsequent lymphocyte preparation.

[0162] Separate VHH libraries were constructed from the lymphocytes of each llama, and the presence of antigen-specific VHHs was selected and screened. For this purpose, first-strand cDNA synthesis was performed using oligo(dT) primers with total RNA from peripheral blood lymphocytes as a template. Using this cDNA, the sequence encoding VHH was amplified by PCR, digested with PstI and NotI, cloned into the PstI and NotI sites of the phagemid vector pMECS, and a VHH library was created by transformation into E. coli. To enrich these libraries for FRα (or FOLR1, used interchangeably herein)-specific VHHs, a combinatorial panining approach against recombinant human and mouse FOLR1 was used, followed by random selection and screening of clones by ELISA for the presence of antigen-specific VHHs in their periplasmic extracts (PE). To measure cross-reactivity against human and mouse FOLR1, each clone was tested against both antigens.

[0163] Example 2. Isolation of human and mouse FRα-specific VHHs. After sequencing, 98 conjugates were identified and classified into 11 different families based on the sequences of complementarity-determining region (CDR) 3. Most of the VHHs belonged to one large family (Family 1), and the other families were composed of 1 to 4 VHHs.

[0164] Of the 98 VHHs, 91 showed cross-reactivity with both human and mouse FOLR1, and this included all members of families 1, 3, 7, 9, and 10. Families 5, 6, 8, and 11 each contained only one human FOLR1-specific VHH. Family 2 contained one cross-reactive VHH and three human FOLR1-specific VHHs, and family 4 was composed of mouse FOLR1-specific VHHs and human FOLR1-specific VHHs. Based on these data, in combination with the absence of internal restriction sites in the VHH sequences, a large number of VHHs with a wide range of dissociation rates from family 1 were included, and six VHHs were selected for further characterization from four different cross-reactive families (Table 2). These VHHs were subcloned into an expression vector with cMyc and His8 tags at the C-terminus, produced in E. coli BL21(DE3)pLysS cells, and purified by immobilized metal affinity chromatography (IMAC).

[0165]

Table 2

[0166] After panning of the core 76 / 77 against human folate receptor alpha (FRα) and mouse folate receptor alpha (FRα), 95 colonies were randomly selected from the second and third rounds, and the presence of FRα-specific VHHs in their periplasmic extracts (PE) was analyzed by ELISA for both human and mouse FRα. As a result, 98 hits were identified. For these 98 hits, the dissociation rate (k off ) in the PE was determined by biolayer interferometry (BLI) with both mouse and human FRα directly immobilized on the AR2G biosensor. The dissociation rate was calculated using a 1:1 binding model. In the PE-ELISA, the ratio of the signal from wells coated with human or mouse FRα to the signal from uncoated blocked wells (negative control) is shown. Abbreviations: SE: standard error.

[0167] Example 3. The FRα-binding VHH binds to recombinant and native FRα and does not compete with folic acid (FA) binding. As a first step, the binding affinity and cross-reactivity of the purified VHH for human and mouse FRα were evaluated by ELISA using detection of the cMyc tag (Figure 1A-B). All VHHs bound to human and mouse FRα, and their EC 50 was in the range of nM to pM and comparable to the natural affinity of folic acid (oxidized folic acid; <1 nM) and 5-methyltetrahydrofolic acid (reduced folic acid; 1-10 nM) for human FRα (Wang et al 1992 Biochemical pharmacology 44:1898-1901; Gates et al 1996 Clinical cancer research 2:1135-1141; Kamen and Caston 1986 Biochemical pharmacology 35:2323-2329). Most of the VHHs tested showed stronger binding to mouse FRα compared to human FRα. 2HFO19 (family 1) bound best to mouse and human FRα among all VHHs tested, with EC 50 of 0.1 nM and 0.4 nM, respectively (Figure 1A-B). 2HFO42 (family 3) and 3MFR60 (family 10) showed binding affinities comparable to 2MFR7 (family 1) for both human and mouse FRα. Next, since there is a high level of sequence conservation between FRα and FRβ, binding to human and mouse folate receptor beta (FOLR2, encoded by FOLR2 and used interchangeably herein as FRβ) was investigated to evaluate the specificity of the VHHs. In contrast to FOLR1, FOLR2 is mainly expressed in myeloid cells such as neutrophils and macrophages (O'Shannessy et al 2015 Journal of ovarian research 8:29; Ross et al 1999 Cancer 85:348-57). Since none of the VHHs bound to human FRβ (data not shown), it was shown that the VHHs are highly specific for FRα and have a low potential for off-target effects.

[0168] As a next step, the kinetic binding constants were determined using BLI (Table 3). Within the panel, 2HFO19 and 3MFR60 showed the highest affinity for mouse FRα, and the equilibrium dissociation constants (K dis or K D ) were 2.97 nM and 2.59 nM, respectively. The lowest K D was observed for 2HFO42 (23.8 nM), which is still a good binder. Similar to the ELISA results (Figure 1A - B), higher binding affinities were measured for mouse FRα than for human FRα, and in most cases, the K D values increased by one order of magnitude. 3HFO 26 and 3MFR60 showed an almost 100 - fold difference in K D between mouse FRα and human FRα, from 7.86 nM to 662 nM and from 2.59 nM to 169 nM, respectively. For 3HFO28, binding to human FRα could not be confirmed by BLI, so this VHH was excluded from further characterization.

[0169]

Table 3

[0170] Association and dissociation rates (k on and k off ) and equilibrium dissociation constant (K D ) of anti - folate receptor alpha (FRα) VHHs measured by biolayer interferometry (BLI) using mouse and human (m / h) FRα directly immobilized on a 10 μg / ml AR2G biosensor. Kinetic parameters were calculated using a 1:1 binding model.

[0171] Furthermore, since the binding to the solid-phase immobilized recombinant substance does not necessarily reflect the native higher-order structure of the antigen, the binding to FRα expressed on the cell membrane was evaluated by flow cytometry. Mouse or human FOLR1 was transiently transfected into HEK293T cells that do not endogenously express FRα, and dose-dependent VHH binding based on the His tag was detected (Figure 1C-D). All VHHs were able to bind to cell-expressed mouse FRα, and their EC 50 values were in the low nM range, while for native human FRα, the EC 50 values ranged from 4.5 nM to 26.4 nM.

[0172] Since the inhibition of the endogenous transport of folic acid via FRα can have a significant impact on brain homeostasis, the competition of representative family members with FA for mouse FRα binding was evaluated by ELISA (Figure 1E). After pre-incubation with excess FA to fully saturate the receptor, the tested VHHs retained full binding ability and did not compete with FA in mouse FRα binding.

[0173] Example 4. FRα-binding VHHs bind to partially overlapping epitopes. VHHs with high sequence identity in the CDR3 region, i.e., the VHH family, are derived from the same B cell lineage and have diverged from each other by somatic hypermutation or affinity maturation. Typically, VHHs of the same family recognize the same epitope on the target, but other properties may be different (De Genst et al 2006 PNAS 103:4586-4591; De Gents et al 2005 J Biol Chem 280:14114-14121). To investigate whether VHHs from different families bind to different epitopes, epitope binning was performed using the tandem binding method by BLI. One VHH from each family was bound to mouse FRα on the biosensor, followed by binding of the second VHH (Table 4).

[0174]

Table 4

[0175] Competition between different VHHs of the complementarity-determining region (CDR) 3 family was evaluated by a tandem binding assay using biolayer interferometry (BLI). A streptavidin biosensor was loaded with biotinylated mouse FRα (Acro Biosystems, Cat.nr.FO1-M82E9), followed by binding of an anti-FRα VHH (VHH of the first association). The biosensor was then transferred to a solution containing a second VHH mixed 1:1 with the first VHH (VHH of the second association). Using the ForteBio Data Analysis Software, the absolute value of the additional binding signal when the second association was overlaid on the first association was calculated. The data were normalized for each VHH in the first association using the following formula: (signal - self) / (max - self), where self is the value of the VHH in the first association combined with the same VHH in the second association. The numerical values are presented as percentages.

[0176] 2MFR32 (SEQ ID NO: 96) showed further binding to all other VHHs, indicating that it recognizes another epitope on mFRα. 2HFO19, 2HFO42, and 3MFR60 did not bind to mFRα simultaneously and showed competition in all different combinations, suggesting that they bind to similar epitopes. Since different conformational states have been reported for FRβ (Wibowo et al 2013 PNAS 110:15180-15188), these epitopes can be either identical, partially overlapping, or mutually exclusive higher-order structures. Thus, the VHHs can be pooled into two different epitope bins: 2HFO19, 2HFO42, and 3MFR60, which recognize (partially) overlapping epitopes, and 2MFR32, which binds to a different epitope.

[0177] Example 5. Human FRα Q141 is essential for the binding of anti-FRα VHH. To identify the exact region where the VHH binds to FRα, epitope mapping experiments were performed. Since the VHH can bind to human FRα but not to FRβ, seven human FOLR1 / 2 chimeric constructs in which the region of human FOLR1 was replaced with the corresponding human FOLR2 (FRβ) region: Chimera 1 (SEQ ID NO: 67), Chimera 2 (SEQ ID NO: 68), Chimera 3 (SEQ ID NO: 69), Chimera 4 (SEQ ID NO: 70), Chimera 5 (SEQ ID NO: 71), Chimera 6 (SEQ ID NO: 72) and Chimera 7 (SEQ ID NO: 73) were generated (Figure 2A): Three additional Chimera 1 constructs: Chimera 1a (replacement of fragment 31 - 40; SEQ ID NO: 74), Chimera 1b (replacement of fragment 41 - 50; SEQ ID NO: 75) and Chimera 1c (replacement of fragment 51 - 64; SEQ ID NO: 76) were generated. These constructs were transiently transfected into HEK293T cells and the dose-dependent binding of the VHH to wild-type FRα and FRβ (WT) or the chimeric constructs was evaluated by flow cytometry via detection of the tag (Figure 2B). When the expression of the constructs was confirmed with an anti-FRα antibody (mAb), most of the chimeras showed similar expression levels. Chimera 6 (SEQ ID NO: 72) could not be detected with the mAb, but the tested VHH showed binding, indicating that this region is the epitope of the antibody (data not shown). Different anti-FRα VHHs were able to bind to the FRα-FRβ chimeras expressed in cells to the same extent as WT FRα, with the exception of Chimera 4 (SEQ ID NO: 70), where binding completely disappeared with all VHHs. This means that this region (amino acids 140 - 150) plays an important role in the binding of the VHHs to their targets.

[0178] To further narrow down which specific residues may play a role, single residues of human FRα in the four non-conserved chimeric regions between FRα and FRβ were mutated to their human FRβ counterparts. All VHHs retained binding to E140Q (SEQ ID NO: 77), R147H (SEQ ID NO: 79), and Y150H (SEQ ID NO: 80) of human FRα (data not shown), but introduction of the Q141R mutation (SEQ ID NO: 78) completely abolished binding (Figure 2C). To verify the role of this residue in VHH binding, the R135 residue of human FRβ was substituted with glutamine (SEQ ID NO: 81), and VHH binding was analyzed. Surprisingly, introduction of this single residue into human FRβ restored the binding ability of the VHH. In addition to the (partial) overlap of the epitopes, all VHHs require residue Q141 for binding.

[0179] Example 6. Anti-FRα VHH 2HFO42 can cross the blood-CSF barrier in vivo. To examine the in vivo blood-CSF barrier permeability of human-mouse FRα cross-reactive VHHs, an in vivo screening was set up as described by Wouters et al (2022 Fluids and barriers of the CNS;19(1):79). Briefly, the FRα-binding VHH was genetically fused to the neuropeptide neurotensin (NT; Figure 3A), a bioactive tridecapeptide. After central administration, NT elicits a measurable hypothermic effect in mice when acting on the hypothalamic neurotensin receptor (NTSR) (Bissette et al 1976 Nature 262:607-609; Nemeroff et al 1979 PNAS 76:5368-5371; Prange et al 1979 Pharmacology,biochemistry,and behavior 11:473-477). This hypothermic effect is mediated by NT binding to both neuronal NTSR1 and astrocytic NTSR2 (Tabarean 2020 Neuropharmacology 171:108069). However, upon peripheral administration, NT itself cannot cross the blood-brain barrier (Pardridge 1998 Journal of neurochemistry 70:1781-1792). Thus, the presence of a hypothermic effect after intravenous injection of the VHH-NT fusion protein indicates the potential of the VHH to cross the barrier and subsequent target engagement of NT. Since FOLR1 is expressed only in choroid plexus epithelial (CPE) cells that form the blood-CSF interface and not in the BBB (Weitman et al 1992 Cancer research 52:3396-3401; Weitman et al 1992 Cancer research 52:6708-6711; O'Shannessy et al 2011 Oncotarget 2:1227-1243; Grapp et al 2013 Nature Comm 4:2123), the decrease in body temperature suggests that the VHH reaches the brain by crossing the blood-CSF barrier rather than the BBB.

[0180] To analyze the penetration ability in vivo, different human / mouse cross-reactive anti-FRα VHH-NT and control VHH-NT (anti-eGFP VHH) fusion proteins were intravenously injected into TLR4- / - mice at a dose of 250 nmol / kg (~4.2 mg / kg), and body temperature was continuously measured using implanted temperature probes until 4 hours after injection (Figure 3B). Among the VHHs tested, only 2HFO42-NT showed a clear decrease in body temperature compared to the negative control VHH, indicating that NT is involved in the receptor in the brain and suggesting that 2HFO42 may pass through the blood-brain barrier. The integrity of the VHH-NT fusion protein was verified by mass spectrometry (data not shown), and it was confirmed that the lack of a decrease in body temperature was due to the lack of action potential and not due to the loss of NT. Next, the dose-dependence of the hypothermic response was examined. 2HFO42-NT and anti-eGFP-NT were intravenously injected at doses of 50, 150, 250, and 500 nmol / kg, and body temperature was measured (Figure 3C-D). In the case of 2HFO42, the degree of body temperature decrease increased with increasing dose, but the anti-eGFP control VHH could not cause a decrease in body temperature at all the doses tested, confirming that there is no passive uptake in the central nervous system.

[0181] These results indicate that among the human-mouse cross-reactive anti-FRα VHHs tested, only 2HFO42 can pass through the blood-CSF barrier. This is because 2HFO19 (family 1) and 2HFO42 (family 3) have similar k off values (3.93x10 -3 / s vs. 4.67x10 -3 / s), respectively, and 2MFR7 (family 1) and 2HFO42 (family 3) have similar K D values (1.67x10 -8 vs. 2.38x10 -8) This indicates that the ability to pass through the BCSFB is not solely related to the affinity of the VHH for mFRα. Interestingly, since other members of the 2HFO42 family 3 also did not induce hypothermia in mice, it was shown that the epitope alone is not the only determining factor for BCSFB passage.

[0182] Example 7. Requirements for the dissociation rate of binding for family 3 VHHs to pass through the blood-CSF barrier. Among the VHH family 3, 2HFO42 is the only member confirmed to pass through the BCSFB in mice, meaning that the passing ability also depends on features other than the CDR3 sequence and epitope recognition. Therefore, the kinetic parameters of binding were hypothesized to affect receptor-mediated transcytosis and passing ability, similar to those known for BBB-passing compounds.

[0183] From the binding results obtained by ELISA, BLI, and FACS, VHHs can be ranked according to their optimal affinity for mFRα. The affinity of 2HFO9 (fam 3) for mFRα is too low (K D 213 nM; koff 8.2x10 -3 / s), and that of 2MFR67 is too high (K D 23.3 nM; koff 2.22x10 -3 / s) to enable functional passage compared to that of 2HF042 (K D 4.3 nM; koff 9.87x10 -4 / s). Since the sequences of the three family members are conserved in CDR3, the residues of 2HFO9 and 2MFR67 different from those of 2HFO42 were systematically mutated to the corresponding residues of 2HFO42 in all possible combinations to investigate whether a more similar affinity of 2HFO42 determines the passing ability in the NT model.

[0184] 2HFO9 differs from 2HFO42 at three residues at positions 40, 51, and 54 (by Kabat numbering) (Figure 4A). Different single, double, and triple mutants were generated and produced as NT fusion proteins in E. coli (as described in Wouters et al. Fluids Barriers CNS. 2022;19(1):79). The integrity of the fusion proteins was confirmed by SDS-PAGE and mass spectrometry, and the binding to human and mouse FRα was determined by ELISA (data not shown) and BLI (Figure 5A). Looking at the dissociation rates of the 2HFO9 variants on mouse FRα, the introduction of G54H improved the dissociation rate for both the single mutant and the double mutant form (k off 2.04 10 -3 to 2.36x10 -3 / s), falling within the same range as the dissociation rate of 2HFO42. In contrast, the introduction of A40V and / or I51V had no effect on the dissociation rate.

[0185] Next, different mutant NT fusions were intravenously injected into TLR4- / - mice at a dose of 250 nmol / kg to confirm their ability to cross the BCSFB and measure changes in body temperature. In contrast to 2HFO9, the 2HFO9 (I51V-G54H) variant with a k off value similar to that of 2HFO42 showed a similar body temperature decrease as 2HFO42-NT (Figure 4B). The production of 2HFO9 (G54H)-NT was not successful in this experiment, but its passage ability was later confirmed in production using HEK293-F cells. 2HFO9 (I51V) and 2HFO9 (A40V, I51V) did not show a clear decrease in body temperature, indicating that these VHHs were unable to cross into the brain. These results indicate that the presence of H54 is beneficial for crossing and increases the affinity for FRα.

[0186] 2MFR67 differs from 2HFO42 at four positions, at the 40th and at positions 72 - 74. The E72D mutation decreased the affinity of 2MFR67 for mouse FRα by about 2.5 - to 4-fold in the single, double, and triple mutant forms (Figure 5B), and as a result, the dissociation rate was shown to be in the same range as that of 2HFO42. Introduction of the E72D mutation into 2MFR67 resulted in passage through the BCSFB in the single, double, and triple mutant forms, as determined by the marked decrease in body temperature upon intravenous injection (Figure 4C). In contrast, the 2MFR67 variants D73N and S74A were unable to pass through the mouse BCSFB, as judged by the lack of a decrease in body temperature when fused to the NT. These results indicate that the presence of A74 is disadvantageous for passage and that A74 slightly increases the affinity for FRα.

[0187] As a conclusion, the results using point mutants suggest that the dissociation rate is an important determinant in family 3 VHHs and indicate a narrow window in the KD / k ratio of the optimal dissociation rate to support BCSFB passage of family 3 VHH variants in the mouse system. The k values of mouse and human FRα can be found in Table 5 and in the BCSFB passage ability of the NT fusions in mice. off ratio for the optimal dissociation rate to support BCSFB passage of family 3 VHH variants in the mouse system. The k values of mouse and human FRα can be found in Table 5 and in the BCSFB passage ability of the NT fusions in mice. off can be found in Table 5 and in the BCSFB passage ability of the NT fusions in mice.

[0188]

Table 5

[0189] Example 8. 2HFO42 and 2MFR67 are concentrated in the CSF of non-human primates upon peripheral administration. As a first step towards clinical application, it was evaluated whether 2HFO42, which passes through the BCSFB in mice, could pass through the blood-CSF barrier in non-human primates. First, binding to cynomolgus monkey FRα was confirmed by ELISA (Table 6). Next, to isolate CSF, a catheter was placed near the ventricle of cynomolgus monkeys. To analyze uptake into CSF over time, a mixture of VHHs unrelated to 2HFO42 was intravenously injected at a dose of 8 mg / kg for each VHH, and plasma and CSF were sampled continuously (Figure 6A-B). Bioanalysis of the VHHs was performed by ELISA for each antigen and detection of the tag. All VHHs showed similar pharmacokinetic profiles in plasma, with a half-life of approximately 20 minutes, consistent with the prediction for VHHs that do not prolong the half-life. Surprisingly, high 2HFO42 VHH levels were measured in CSF compared to control VHH levels and were detectable in CSF 24 hours after injection. These data indicate that 2HFO42 can retain the ability to cross the blood-CSF barrier between different species.

[0190] 2MFR67, a family member of 2HFO42, cannot cross the blood-cerebrospinal fluid (CSF) barrier in mice and has a much higher affinity for mFRα than 2HFO42. However, its affinity and dissociation rate for human and rhesus FRα (Table 6) are within the range of 2HFO42 on mouse FRα (Table 5). To evaluate whether this dissociation rate enables 2MFR67 to cross the blood-CSF barrier in non-human primates, VHH was tested together with control VHH following the same procedure as described above for 2HFO42 (Figure 6C-E). Both 2MFR67 and control VHH showed similar kinetic profiles in plasma, and similar amounts of VHH were detected upon intravenous administration (Figure 6C). The plasma levels of both 2MFR67 and control VHH dropped below the lower limit of quantification at 24 hours post-injection, indicating that the VHH was removed from circulation at that time. In CSF, control VHH was also detected (Figure 6D), but significantly higher levels of 2MFR67 were detected compared to control VHH. The CSF-to-plasma ratio of the VHH amount was also higher for 2MFR67 compared to control VHH, indicating that the higher levels of 2MFR67 in CSF were not due to higher levels in plasma (Figure 6E). These data indicate that 2MFR67 does not show blood-CSF barrier crossing in mice but can reach the CSF upon peripheral administration in rhesus monkeys, and this VHH has a dissociation rate optimal for blood-CSF barrier crossing in non-human primates and, by extension, in humans, and that the dissociation rates optimal for BCSFB crossing in non-human primates and mice overlap.

[0191]

Table 6

[0192] Association and dissociation rates (k on and k off ) and equilibrium dissociation constant (K D) was captured on a mouse Fc biosensor using an anti-FLAG tag antibody, and then measured by biolayer interferometry (BLI) for cynomolgus monkeys, mice, and humans (rh / m / h). Kinetic parameters were calculated using a 1:1 binding model.

[0193] Example 9. Sequence optimization of 2HFO42 for human FRα. Considering that the optimal affinity range for BCSFB passage differs between mice and humans, an in vitro transcytosis assay using human CPE cells was set up (see Example 11). First, the inventors started constructing a number of sequences based on current VHH data and tested the effect of the variants on the affinity for human FRα. These optimized sequences were particularly humanized and sites that were prone to post-translational modifications were removed. During the design, although the binding to human FRα was intended to be further optimized, the binding to human FRβ was avoided as the specificity was maintained. The sequences can be found in Table 7.

[0194]

Table 7-1

Table 7-2

Table 7-3

Table 7-4

Table 7-5

[0195] The binding of the humanized variants of 2HF042 was evaluated basically as described above by biolayer interferometry (BLI) through kinetic binding analysis to mouse and human FRα proteins biotinylated via the Avi tag and captured by a streptavidin biosensor. The VHHs were analyzed at a concentration of 100 nM, and the dissociation rates were determined using fitting of a 1:1 Langmuirian interaction. The dissociation constants were compared to 2HF042 (Q1E, Q5V, Q108L) (P01500004) and 2MFR67 (Q1E, Q5V, Q108L) (P01500005), respectively. The results are depicted in Table 8.

[0196] Second, the variants were analyzed for their ability to compete with the binding of the family 3 VHH 2MFR67 to human FRα in a competitive AlphaLISA, which is a homogeneous assay without a washing step. Here, human FRα biotinylated with an Avi tag (FO1-H82E2, AcroBiosystems) was captured on streptavidin-coated Alpha Donor beads (PerkinElmer, catalog number 6760002), and 2MFR67 was captured on anti-Flag antibody AlphaLISA acceptor beads (PerkinElmer, catalog number AL112C). The binding of 2MFR67 and FRα causes energy transfer from one bead to the other, ultimately generating a fluorescence signal. For each well of a white low-binding 384-well microtiter plate (F-bottom, Greiner catalog number 781904), 5 μl of serial diluted VHH was mixed with 5 μl of 2.5 nM monovalent 2MFR67-Flag3-His6 and 5 μl of 15 nM biotinylated human FRα protein. After incubation for 1 hour at room temperature, streptavidin-coated Alpha Donor beads and anti-Flag AlphaLISA acceptor beads were added to a final volume of 25 μl to a final concentration of 20 μg / mL each and incubated for 1 hour at room temperature in the dark. The interaction between the beads was evaluated after irradiation at 680 nm and reading at 615 nm on an EnSight device. Curve fitting was performed using 4PL non-linear regression analysis in Graphpad Prism 9.0.

[0197] The dose-dependent competition of different FRα-binding VHHs was evaluated to determine the respective IC50 values (Table 8). Figure 7 shows the dose-dependent inhibition of different VHHs that compete with the binding of MFR67 (family 3) and hFRα in AlphaLISA. As a result, 2HF019 (Q1E, Q5V, Q108L) (P01500003, fam 1) was found to compete with 2MFR67, confirming that the epitopes of these VHHs overlap. Furthermore, the dose-dependent binding to mouse and cynomolgus FRα was evaluated by ELISA (Figure 8A-B). The thermal stability of the variants was analyzed by examining the melting temperature in PBS at neutral pH and in acetate buffer at pH 5.5 (Table 8).

[0198]

Table 8

[0199] Example 10. Affinity optimization of the 2HFO42 variant for human FRα. Furthermore, affinity optimization for human FOLR1 was performed by a library approach by substituting the amino acids at positions forming CDR1, CDR2, and the extra loop of framework 3 (the so-called "CDR4" loop, an alias of the humanized variant of 2HFO42 (P01500001)). In fact, the FR3 region of the heavy-chain antibody corresponds to the fourth loop region, the DE loop, which is located on the same side of the Ig fold in its three-dimensional structure, and thus is considered a potentially involved "CDR4" region that may be involved in or affect antibody / antigen interaction. The FR3 region forming the DE loop contains positions 71 - 78 according to Kabat numbering (Kelow et al., 2020; MABS 12 / 1,e1840005), and as previously observed, substitution of at least residues 72 - 74 affected affinity and / or passage. Therefore, for affinity optimization of 2HFO42, CDR3 was not changed to avoid the issue of binding specificity, but these sequence regions of 2HFO42 for the functionality of this VHH in the complex with FRα were analyzed by changing CDR1, CDR2, and the so-called CDR4 region.

[0200] In the first round, the amino acids of different CDR regions were substituted with all 20 amino acids, i.e., in separate single-site saturation libraries. In the AffMatCDR1 library, 9 residues (G26 - I33, G35) were substituted, in AffMatCDR2, 9 residues (T50 - N58) were substituted, and in AffMatCDR4, 6 positions (D72 - T77) were substituted. After screening, the mutations of interest were combined into a new combinatorial library and screened again to see if the binding to human FOLR1 was improved.

[0201] For the construction of single-site saturation libraries, mutations were introduced by site-directed mutagenesis PCR. In the case of single-site saturation libraries, the codon of interest was mutated with three different primers containing degenerate codons NDT, VHG, and TGG at a molar ratio of 12:9:1 (22-ctrick method). This primer combination encodes all 20 amino acids without stop codons, and there is only one redundant set for valine (GTT and GTG) and leucine (CTT and CTG). Using h1 2HFO42 (P01500001) as a template, individual PCR reactions were set up for each position. The PCR products were purified using a gel extraction kit, and equimolar amounts of the purified PCR products were pooled for each CDR region. In each library pool, parental plasmid DNA was removed by performing DpnI digestion at 37 °C for 60 minutes, followed by purification using a PCR purification kit (Qiagen). The newly synthesized DNA was ligated using T4 ligase and transformed into electrocompetent E. coli TOP10 to generate a library size that covers at least 5-fold of the theoretical diversity. For each AffMat library, the nucleotide sequences of 90 colonies were determined as a quality control to confirm the diversity of the library.

[0202] Single colonies were picked and then cultured in 96-well plates. To well cover different variants, libraries with a diversity exceeding 4-fold were selected. For each of the AffMatCDR1 and AffMatCDR2 libraries, nine 96-well plates were prepared with 90 clones per plate to enable the screening of single-site variants. Five 96-well plates were used for the AFFMATCDR4 library. As in-plate controls, the parental strain P01500001 and the family member 2MFR67 P01500005 were included on the same plate. From the glycerol stock, variants were generated overnight at 37 °C and 250 rpm in a 96-Deep well plate containing 1 ml of 2xTY / canamycin, and then periplasmic extracts were prepared from the bacterial cell pellets.

[0203] Variants were screened in a competitive AlphaLISA (Figure 9). Subsequently, clones with improved affinity for human FOLR1 compared to the in-plate control P01500001 were selected, subjected to sequence analysis, and the presence or absence of mutations was determined. The nucleotide sequences of the hit clones were determined, and then the dissociation rates of the single clones in BLI for human and mouse FRα were analyzed. Some of the variants with a dissociation rate improvement of 1.5-fold or more for human FOLR1 were also analyzed for binding to human FOLR2 in BLI to confirm whether the introduced mutations bind to human FOLR2. None of the point mutants showed binding to human FOLR2.

[0204] The selected mutations identified to improve the dissociation rate for human FRα were combined in one combinatorial library. In the combinatorial library, site-directed mutagenesis was repeated to introduce mutations at eight different positions. In CDR4, the mutations were introduced in pairs in six combinations to prevent the introduction of unwanted post-translational modification sites. As a first step, the combinations of D72-N73, D72-P73, E72-G73, P72-G73, and P72-N73 were introduced into P01500001 in six separate PCR reactions using specific primers. Subsequently, CDR1 and CDR2 mutations were added to each of these six reactions, resulting in a theoretical library size of 576. Single colonies were picked to create 12 96-well plates of the AffMatCombo library for screening, as was done for the single-site CDR library. To evaluate the quality of the library, the nucleotide sequence was determined for one plate.

[0205] Screening of the AffMatCombo library was performed using competitive AlphaLISA with the periplasmic extract diluted 1:80. Figure 9A shows a representative example of the screening results in the competitive AlphaLISA of the combinatorial library, compared with the CDR1, CDR2, and CDR4 libraries.

[0206] The nucleotide sequences of the clones with improved affinity for human FOLR1 compared to the in-plate controls P0150001- and P0150005 were determined and the combinatorial mutations were decoded. Using biotinylated FOLR1 protein captured on streptavidin biosensors, 100 selected clones were subjected to dissociation rate analysis for binding to human FOLR1 and mouse FOLR1 in BLI.

[0207] Selected variants with optimized affinity for human FRα, having different combinations of up to four substitutions in the CDR1, CDR2, and CDR4 regions, were generated as purified flag3-His6-tagged VHHs for further characterization (Figure 10, Table 9). Nanobodies were produced at 0.5 L scale in TG-1 E. coli and purified from the periplasmic fraction using standard affinity chromatography on a 2 mL Ni Sepharose FastFlow column and then desalted. Purity was determined by SDS-PAGE.

[0208] The dissociation rates of the purified variants were determined in BLI for human, mouse, and cynomolgus FRα. Furthermore, the binding of the variants with optimized affinity to human FRα expressed on HeLa cells was measured by flow cytometry (Figure 10B).

[0209] As a result, so-called CDR4 region substitutions, particularly the D72E-N73G and D72P-N73G substitutions, were indicated to most strongly improve the dissociation rate for human FR compared to the basic variant (P01500006).

[0210]

Table 9-1

Table 9-2

[0211] Example 11. In vitro transcytosis assay using human HIPCPP cells. To evaluate the ability of FRα-binding VHHs to cross the BCSFB in humans, the in vitro transcytosis assay using immortalized HIBCPP cells was performed with modifications to Dinner et al. (2016 J Vis Exp e54061). As controls for transcytosis, anti-Tfr1 Nb188 (De Wilde et al. 2020) and an irrelevant control VHH (IRR4) were used. All samples were analyzed in replicates.

[0212] For VHH uptake and transport measurements, 1×10 5 HIBCPP cells were seeded onto transwell culture inserts (growth area 0.3 cm 2 , pore size 0.4 mm, pore density 4×10 6 pores / cm 2 , polyester membrane). Cells were trypsinized with 0.25% trypsin at 37 °C for 20 minutes, then washed and seeded onto the filter (0.1 ml, seeding density 1x10 5(Cells / Insert). In the case of standard cell culture, 0.5 ml of medium is added to a 24-well plate, and the insert is placed in each well. For reverse cell culture, the cells are plated onto the aforementioned insert placed in a 12-well plate that has been inverted and filled with medium. The cells are incubated at 37 °C in 5% CO2 for 24 hours. The next day, for the cells cultured in the standard setup, 0.1 ml of fresh medium is added to each insert to provide nutrients. In the case of the reverse setup, the culture insert is inverted again and transferred to a 24-well plate (containing 1 ml of cell culture medium), and 0.2 ml of fresh medium is added to each insert. HIBCPP is cultured in DMEM / F12 + GlutaMax (Gibco, 31331) supplemented with 10% FBS and 5 μg / mL insulin for 3 days, and then switched to folate-free RPMI medium (Gibco, 27016) supplemented with 1% FBS and 5 μg / mL insulin. When the cells reach confluence and the TEER value reaches 500 Ω.cm 2 When it reaches, 100 nM VHH in the culture medium is added to the basolateral compartment and cultured at 37 °C in 5% CO2 for 4 hours to evaluate transcytosis. Samples are collected from the basolateral and apical sides and stored at -20 °C until further analysis.

[0213] To quantify VHH in the cell culture medium from the apical side, a sandwich ELISA method was used. An 80 ng of AffiniPure Goat anti-alpaca IgG, VHH domain, polyclonal antibody (Jackson ImmunoResearch, catalog number 128-005-232) was coated onto a 96-well Nunc-Immuno™ (MaxiSorp) plate at 4 °C overnight. The samples were incubated at room temperature for 1 hour. Detection was performed at room temperature for 30 minutes using 100 ng of anti-His antibody [HRP], mAb, mouse (GenScript, A00612). Interpolation into the standard curve was performed using 4PL analysis of GraphPad Prism 9.0.

[0214] In the HIBCPP transcytosis assay, a panel of affinity-optimized variants of 2HFO42 was analyzed. In addition to anti-hTfr Nb188 (P01500022) used as a positive control, P01500042, P01500045, P01500047, and P01500006 were found to be transported to the apical compartment (Figure 11).

[0215] Example 12. Co-crystal structure of FRα and FRα-binding VHH. To determine the exact epitope of the FRα binders disclosed herein, co-crystallization studies were performed on human FRα. Recombinant human FRα (29-234) protein was produced in a special low-glycosylated HEK293 cell line. The protein was completely deglycosylated using EndoH. Nanobodies were produced at 2L scale in TG-1 E. coli and purified from the periplasmic fraction using standard affinity chromatography with a 2 mL Ni Sepharose FastFlow column. After analysis by SDS-PAGE, the pool was injected onto a Superdex 75 size exclusion column equilibrated with 50 mM MES pH 6.0, 150 mM NaCl and concentrated to 10 mg / mL.

[0216] The co-crystal structure analysis of hFRα with 2HFO42 (Q1E, Q5V, Q108L) (P01500004), 2HFO19 (Q1E, Q5V, Q108L) (P01500003), and 2MFR67 (Q1E, Q5V, Q108L) (P01500005) was initiated. Nanobodies were added to the FOLR1 protein at a 1.2-fold molar excess. Crystallization was performed using the sitting-drop vapor diffusion method with a Mosquito crystallization robot to set up crystallization drops containing 0.1 ul of the protein sample + 0.1 ul of the bottom solution. Crystallization screening was carried out using a screening kit commercially available from Molecular Dimensions. X-ray data were collected at 100 K at the synchrotron facilities of Soleil and Diamond. The X-ray data were processed using the xdsme processing pipeline (Legrand, 2017) or the autoPROC+Staraniso processing pipeline (Vonrhein et al, 2011, Vonrhein et al, 2018).

[0217] The structures of the 2HFO19-FOLR1 complex and the 2HFO42-FOLR1 complex were analyzed using the Phaser program (McCoy et al., 2007) of the Phenix suite (Adams et al., 2010), using PDB entries 4lrh and 7s0e as models of FOLR1 and 2HF)19, respectively, with molecular replacement. Subsequently, the structure was manually built using Coot (Emsley and Cowtan, 2004) and refined using phenix.refine (Afonine et al., 2012) of the Phenix suite. Subsequently, the structure of the 2MFR67-FOLR1 complex was analyzed by the molecular replacement method using the 2HFO42-FOLR1 structure, manually built with Coot, and refined using phenix.refine.

[0218] 2HFO19(P01500003)-FOLR1 crystallized at a resolution of 2.8 Å at neutral pH (6.5). From these experiments, it was revealed that 2HFO19 binds to a conformational epitope on hFRα that includes the amino acid residues P94, A95, R98, H99, E137, D138, E140, Q141, W143, E144, D145, R147, T148, R204, W213 and F214 (Figure 13). The paratope of 2HFO19 includes the amino acid residues of the CDR1, CDR2 and CDR3 regions, more specifically F27, P28, T31, V32 and Y33 (from CDR1), N52, N53, G56 and V57 (from CDR2), and R98, R99, R100, S101, F102, V103, L106, S108 and S109 (from CDR3).

[0219] 2HFO42(P01500004)-FOLR1 crystallized at a resolution of 2.23 Å at neutral pH (6.5). The AU contains 2 copies of FOLR1 and 2 copies of Nb42, forming two Nb42-FOLR1 complexes. 2HFO42 passing through the BCSFB binds to a conformational epitope on hFRα that includes the amino acid residues R98, H99, E137, D138, Q141, E144, D145, R204, G205, Q211, W213, F214, D215, A217 and Q218 (Figure 12). The paratope of 2HFO42 includes the amino residues of F29, S30, G31, I33 (from CDR1), T52, S53, H54, T56 (from CDR2), and H95, F96, P97, G98, I101, Y102 (from CDR3, according to Kabat numbering).

[0220] In two copies of 2HFO42, the higher-order structure of the CDR4 region located within framework 3 is different, indicating the flexibility of this loop. CDR4 is either in a "closed" higher-order structure facing the CDR2 region or in an "open" higher-order structure where CDR4 is separated, and K75 forms an H-bond with S30. The higher-order structure of CDR4 does not affect the higher-order structure of CDR1 or CDR2. In either higher-order structure, H-bonds are formed between the backbones of CDR1 and CDR2 and the side chains of R71 and N76 of CDR4. In the closed higher-order structure, an additional H-bond is formed between the backbone of A74 of CDR4 and the backbone of S53 of CDR2. The higher-order structure of CDR4 does not affect the interface with FOLR1, but it results in different surfaces, and the "open" higher-order structure creates a positively charged cavity between CDR1-CDR2 and CDR4 (Figure 17).

[0221] To investigate the relevance of CDR4 amino acids and orientation to functionality, the co-crystal structure of the extracellular domain of human FOLR1 with 2MFR67 was determined at a resolution of 3.09 Å at neutral pH 6.5. The sequence of 2MFR67 differs from 2HFO42 at positions 40 and 72, 73, and 74 of the CDR4 loop within framework 3. The interface with human FOLR1 is similar for both 2HFO42 and 2MFR67 family 3 members: H-bonds are formed between S30, T52, H54, T56, H98, P97, G98, I101, Y102 (by Kabat numbering) of 2MFR67 and R98, Q141, E144, D145, R147, R204, D215, Q218 of FOLR1. From this structure, it was shown that in 2MFR67, the CDR4 region exists only in an open higher-order structure away from the CDR region and does not interact with FOLR1.

[0222] 2MFR67 and 2HFO42 bind to FOLR1 in the same manner, but differently from the binding mode of the 2HFO19 Nanobody family 1. They all bind to the "core epitope" surrounding human FOLR1 Q141, but the paratopes are different, and 2HFO42 and 2MFR67 pick up additional interactions with FOLR1 residues 204 and 213 - 218 via the CDR3 region (Figure 18).

[0223] Example 13. Binding to breast cancer-derived cell lines expressing FRα. It has been proven that epithelial-derived cancers such as ovarian cancer, breast cancer, pleural cancer, lung cancer, cervical cancer, endometrial cancer, colorectal cancer, kidney cancer, bladder cancer, and brain tumors overexpress FOLR1. Considering that the VHHs described herein bind to new FRα epitopes, it was evaluated whether FRα-binding VHHs exhibit improved binding properties to FOLR1-expressing cancer cells, more specifically, cell lines derived from FOLR1-positive breast cancer. This was evaluated by dot blot of 0.5 μg of human MCF7 and MDA-MB-231 cell lysates. Human FOLR1-expressing MCF7 and MDA-MB-231 breast tumor-derived cells were homogenized at 20 Hz for 3 minutes in 0.5% CHAPS / PBS using a Tissue Lyser II and then centrifuged at 13,000 rpm at 4 °C for 5 minutes. Only cell lysate (500 ng) or lysis buffer ((-) control) was spotted onto a nitrocellulose membrane, dried for 15 minutes, and then blocked with 3% BSA in TBS 0.1% Tween (TBS-T) for 1 hour at room temperature. The membrane was then incubated overnight at 4 °C with 75 μg / ml of VHH or 0.75 μg / ml of monoclonal antibody in 1.5% BSA in TBST. After washing 3 times with TBST, the membrane was incubated with an anti-His 1:5000 antibody for 1 hour at room temperature. The membrane was washed 3 times with TBST and then incubated with a secondary antibody conjugated to Alexa Fluor 680 for 1 hour at room temperature. After washing 3 times with TBST, the blot was scanned and quantified with ImageJ software. A human FRα-binding monoclonal antibody (mAb) was used as a positive control. The loss of binding of the monoclonal antibody to FRα chimera 6 (described in Example 5) demonstrates that the mAb binds to a different epitope. Surprisingly, as can be seen in Figure 15, FRα-binding VHHs of family 1 and family 3 that recognize the novel FRα epitope showed improved binding to MCF7 and MDA-MD-231 cells compared to the positive control.

[0224] Example 14: Isothermal titration calorimetry. From this structure, it is indicated that the high affinity of 2MFR67 for human FOLR1 is not due to the direct interaction of the CDR4 residue with the receptor, but rather that this residue affects the higher-order structure of the CDR4 region. To rule out the possibility of an additional binding site via the CDR4 region, isothermal titration calorimetry (ITC) measurements of the stoichiometry of the interaction between the humanized variants h1 2HFO42 (P01500001) and h1 2MFR67 (P01500005) and human FOLR1 were performed using a glycoprotein-modified human FOLR protein (AcroBiosystems Cat.Nr FO1-H52H1). All proteins were dialyzed overnight in PBS buffer and concentrated using an Amicon Ultra 10 kDa cut-off centrifugal filtration device. Human FOLR1 was used at a concentration of 6.5 μM in the cell. The titration involved 26 injections of 1.5 μL of the VHH protein into the cell, performed at 90-second intervals. The first injection (0.4 μL) of the ligand was made and discarded during data analysis. All data were performed at room temperature. The data were fitted to a single binding site model using the PEAQ ITC analysis software provided by the manufacturer. The results are shown in Figure 19.

[0225] According to the ITC measurements, the stoichiometry of the 2HFO42-FOLR1 interaction is 1:1, indicating that no second interaction occurs in solution. Similarly, a 1:1 stoichiometry was determined for the 2MFR67-FOLR1 interaction. Comparing the thermodynamic parameters, the binding of 2MFR67 to FOLR1 is accompanied by an entropic penalty, while the entropic contribution of the humanized 2HFO42-FOLR1 binding is favorable. This is presumably the result of the high degree of freedom of the CDR4 loop in 2MFR67, which results in an entropic penalty upon binding. On the other hand, this degree of freedom allows the paratope to fit better, resulting in a higher entropic contribution. In conclusion, from these data, it was confirmed that the CDR4 region within 2MFR67 is not involved in direct receptor interaction and that a 1:1 stoichiometry exists.

[0226] Example 15: Biophysical properties of the affinity-optimized 2HFO42 variant. Protein stability in the transcytosis process with acidification during transport is important for functionality. To evaluate the effect of the introduced substitutions on the stability of the affinity-optimized variant, temperature-induced unfolding (T m ) and aggregation (T agg ) were measured. The fluorescence of endogenous tryptophan was monitored during temperature-induced protein unfolding using an UNcle instrument (Unchained Labs; Pleasanton, CA, USA). Briefly, 10 μL of 0.5 mg / mL variant in PBS (pH 7.4) and 10 mM acetate buffer (pH 5.5) was added to a sample cuvette, and a linear temperature gradient from 25 °C to 95 °C was initiated at a rate of 0.1 °C / min while performing a 180-second pre-incubation. To determine the melting temperature (T m ) and the aggregation onset temperature (T agg ), respectively, the barycentric mean (BCM) signal and the static light scattering (SLS; 266 nm and 473 nm) signal were plotted against temperature.

[0227] The results are shown in Table 10. In conclusion, for most variants, the introduced substitutions do not significantly affect thermal stability, except for the V51R mutation, which has a negative effect. The combination of N73P and G28T-S30I has the highest Tm value regardless of the tag type.

[0228]

Table 10

[0229] Example 16. In vivo cross of the affinity-optimized variant in a recombinant human FOLR1 mouse model. To enable the testing of the ability to cross the BCSFB for each in a genetically engineered mouse containing a human FOLR1 knock-in mouse, variants with optimized affinity for 2HFO42 were generated as neurotensin fusions. Based on the results of in vitro transcytosis assays using HIBCPP cells, variants with a wide range of dissociation rates for human FOLR1 were selected based on binding affinity and stability evaluations. In particular, AffMat variants with substitutions only in CDR1, as well as variants containing mutations in both CDR1 and CDR4 regions, were included. The variants were cloned into a pcDNA3.4 expression vector containing a C-terminal Flag3-His6-GS-NT(8-13) tag and expressed in HEK-F cells at a 300 mL scale to obtain endotoxin-free protein P01500070-79. During recloning, an E1D mutation was additionally introduced to avoid the formation of pyroglutamic acid. Purification was performed by the method described above in this specification. All purified variants P01500070-79 were analyzed by SDS-PAGE and MS analysis, and the purity and correct mass were confirmed. As a quality control, P01500070-79 was analyzed for binding to human and mouse FOLR1 and thermal stability in dissociation rate analysis by BLI (Table 11).

[0230] The generation of gene - recombinant human FOLR1 mice was performed using the CRISPR / Cas9 method. Since our VHH is cross - reactive with mouse and human FOLR1, in order to obtain gene - recombinant mice, it was essential to first knockout mouse FRα and replace it with human FRα. Homozygous hFOLR1 tg / tg mice were used in the neurotensin hypothermia model, and probes were implanted as described in Example 6. Figure 20 shows the results of body temperature changes after intravenous injection of different variants at a dose of 250 nmol / kg. Anti - mTFR VHH - NT served as the system control and showed hypothermia. P01500079, which is humanized 2HFO42, did not induce hypothermia in hFRa tg / tg mice, indicating that its affinity for human FRα was not optimal. In contrast, the affinity - optimized variant P01500076 containing four substituents [G26E, G28T, S30I, I33L] in CDR1 induced hypothermia, and its dissociation rate for hFRα (1.12E - 03 1 / s) was shown to be optimal for BCSFB passage in this humanized system. In the mouse system, no cross - reaction with P01500076 was observed because the affinity for mFRa was too high.

[0231]

Table 11

[0232] Example 17. Influence of size on the cross - reaction of 2HFO42. To analyze whether it has the potential to be used as a shuttle into the CSF, the influence of size on the BCSFB passage ability of 2HFO42 was analyzed. For this purpose, 2HFO42 (P01500004) was engineered into a bivalent format by gene fusion. Here, 2HFO42 was fused to a flexible [GGGGS]3 linker, followed by the introduction of either an irrelevant VHH, anti - eGFP VHH, or a second 2HFO42 VHH, and introduced into an expression vector containing a C - terminal Flag3 - His6 - GS - NT 8-13 peptide.

[0233] The NT fusion protein was produced in E. coli and purified from the periplasmic fraction using standard affinity chromatography with a 2 mL Ni Sepharose FastFlow column. The purity and integrity of the purified protein were verified by SDS-Page and MS analysis, respectively. For quality control, binding to mouse FOLR1 protein was measured using BLI.

[0234] To evaluate functional passage into the brain, the divalent 2HFO42 NT fusion was analyzed for induction of hypothermia in the neurotensin mouse model described in Example 6. Each construct was injected intravenously at a dose of 250 nmol / kg (n = 4). As a reference, monovalent 2HFO42-NT was used. The results are shown in Figure 21. These results indicate that divalent 2HFO42 fused with a non-FOLR1 VHH (anti-eGFP VHH) as a cargo can pass through the BCSFB, but the divalent format of 2HFO42 VHH does not show a functional response in the NT model due to such high binding affinity of the divalent 2HFO42 Nb to receptors with inefficient release.

[0235] Example 18. Removal of post-translational methionine oxidation sites in 2HFO42. Within the CDR1 region of 2HFO42, the conserved methionine at position 32 is prone to oxidation in forced oxidation experiments, and substitution is desirable for manufacturability. For this purpose, a single-site substitution library was created by substituting M32 of the humanized variant (P0150001) of 2HFO42 with other amino acids excluding cysteine. For all variants, dissociation rate analysis against mouse FOLR1 and human FOLR1 was performed using BLI as described above. Subsequently, variants with the most beneficial substitutions M32P, M32I, and M32L (Table 13) were purified from large-scale E. coli production for further characterization (P01500012, P01500013, and P01500014, respectively). Furthermore, the M32I / P mutation was introduced into two VHHs, 2MFR67 (resulting in P01500016 and P01500017), which have higher affinity for different CDR4 residues and FOLRα, and the affinity-optimized variant P01500019 with [G26E, G28P, D72E, N73G] substitutions (resulting in P01500015 and P01500016, respectively).

[0236] The purified Nanobodies were evaluated for binding to human and mouse FOLRα by BLI, binding to cynomolgus monkey FOLRα by ELISA, and stability by Tm analysis using Uncle. The binding results showed that, in all backgrounds, substitution of M32 with proline or isoleucine was associated with loss of binding to FolRα, a decrease in binding level, and an increase in dissociation rate. The penalty for binding appears to be greater in 2MFR67 and the affinity-improved variant P01500019, indicating that the local context of the CDR4 region is important. The panel stability assessment indicated that the M32P mutation gave a strong decrease in Tm compared to the wild-type variant, while the M32I mutation was benign and in the case of P01500019 further increased the Tm value.

[0237] To overcome the negative impact of the M32 mutation on stability and FOLRα binding affinity, the inventors introduced additional compensatory mutations predicted by the algorithm FoldX based on the structure of the 2HF42-hFOLR1 complex to compensate for either the M32P or M32I substitution (Schymkowitz et al. 2005; Nucleic Acids Res.; 33, Issue suppl_2, W382-W388). The variants listed in Table 12 were generated as Flag3-His6-tagged proteins for binding assays and Tm determination, and the results are also indicated.

[0238] [Table 12]

[0239] [Table 13-1] [Table 13-2]

Claims

1. Dissociation constant k determined by the biolayer interferometry method off is less than 3×10 -2 / s, and specifically binds to human folate receptor alpha (FRα), wherein the binder specifically binds to a human FRα epitope containing amino acid Q141 of SEQ ID NO: 1, a folate receptor alpha (FRα) binder.

2. The FRα binder according to claim 1, wherein the binder specifically binds to human FRα at an epitope of at least two or more amino acids selected from R98, H99, E137, D138, Q141, E144, D145, R204, G205, Q211, W213, F214, D215, A217 and / or Q218 of SEQ ID NO:

1.

3. The FRα binder according to any one of claims 1 or 2, comprising an immunoglobulin single variable domain (ISVD) that specifically binds to human FRα.

4. The FRα binder according to claim 3, wherein the ISVD binds to human FRα via a paratope comprising the amino acid residues F29, S30, G31 and I33 of CDR1, and the amino acid residues T52, S53, H54 and T56 of CDR2, and the amino acid residues H95, F96, P97, G98, I101 and Y102 of CDR3 according to the Kabat numbering of VHH 2HFO42 shown in SEQ ID NO:

2.

5. The FRα binder according to any one of claims 3 or 4, wherein the ISVD comprises the amino acids D72 and N73, or E72 and G73, or P72 and G73 in FR3 according to the Kabat numbering.

6. The FRα binder according to any one of claims 3 to 5, wherein the ISVD comprises the amino acids R71, A74, K75, N76, and T77 in FR3, and the amino acids D72 and N73, or E72 and G73, or P72 and G73 in FR3 according to the Kabat numbering.

7. The FRα binder according to any one of claims 3 to 6, wherein the ISVD comprises the CDR3 sequence shown in SEQ ID NO: 5 or consists of an amino acid sequence that differs from SEQ ID NO: 5 by at most two amino acids.

8. An FRα binder according to any one of claims 3 to 7, wherein k off is 3x10 -2 to 1x10 -3 / s, the FRα binder.

9. The FRα binder according to any one of claims 3 to 8, wherein the binding to human FRα does not interfere with folic acid binding and / or folic acid transport by the human FRα.

10. The FRα binder according to any one of claims 3 to 9, wherein the binder can cross-react with FRα of primates and mice.

11. An FRα binder according to any one of claims 3 to 10, wherein the binder comprises an ISVD comprising a CDR3 sequence according to SEQ ID NO: 5, SEQ ID NO: 31 and / or SEQ ID NO: 32, and / or a CDR2 sequence as depicted in SEQ ID NO: 4, and a CDR1 sequence as depicted in SEQ ID NO: 113 or SEQ ID NO:

3.

12. An FRα binder according to any one of claims 3 to 10, wherein the ISVD comprises CDR1, CDR2, and CDR3 sequences as present in SEQ ID NO: 2, wherein the CDRs are annotated according to Chothia, AbM, MacCallum, IMGT, or Kabat.

13. An FRα binder according to claim 11, wherein the ISVD comprises an FR1 sequence according to SEQ ID NO: 114, an FR2 sequence according to SEQ ID NO: 115, an FR3 sequence according to SEQ ID NO: 116, and an FR4 sequence according to SEQ ID NO: 117, or a sequence having at least 90% identity over the full length of the sequence, wherein the CDR1, 2, 3, and 4 regions are identical.

14. An FRα binder according to any one of claims 1 to 13, wherein the ISVD comprises the amino acid sequence SEQ ID NO: 2 or SEQ ID NOs: 118 to 121, or a homolog having at least 90% identity over the full length of the sequence, or consists thereof, wherein the CDR1, 2, 3, and 4 regions are identical to any one of SEQ ID NO: 2 or SEQ ID NOs: 118 to 121, or a humanized variant thereof.

15. An FRα binder according to any one of claims 1 to 14, wherein when the FRα binder is coupled to a chemical entity, it promotes the uptake of the chemical entity into cerebrospinal fluid (CSF) via the blood-CSF barrier (BCSFB), or into FRα-expressing cancer cells, or enables the binding of the chemical entity to FRα-expressing cancer cells.

16. An FRα binder according to claim 15, wherein the chemical entity is a biological, small molecule, therapeutic agent, radionuclide, antisense oligonucleotide, imaging agent, or test compound.

17. An FRα binder according to any one of claims 15 or 16, wherein the chemical entity is neurotensin or a neurotensin analog.

18. An FRα binder according to any one of claims 1 to 14, wherein the binder comprises or consists of an antibody or an antibody fragment, and / or the binder is a bispecific binder.

19. A blood-brain barrier (CNS) shuttle consisting of an amino acid sequence as depicted in SEQ ID NO: 2, comprising an FRα binder comprising an ISVD that binds to the same epitope on human FRα as VHH 2HFO42, wherein the shuttle has a dissociation constant k for human FRα of less than 3x10 -2 / s as determined by biolayer interferometry off and / or, according to Kabat numbering, comprises amino acids D72 and N73, or E72 and G73, or P72 and G73 of FR3.

20. A blood-CNS barrier shuttle according to claim 19, wherein the FRα binder comprises a CDR3 sequence as depicted in SEQ ID NO: 5 or consists of an amino acid sequence that differs from SEQ ID NO: 5 by at most two amino acids.

21. The blood-CNS barrier shuttle according to any one of claims 19 or 20, wherein k off is 3x10 -2 to 1x10 -3 / s, the blood-CNS barrier shuttle.

22. A blood-CNS barrier shuttle according to any one of claims 19 to 21, further comprising a molecule that is transported to the CNS, more particularly through the BCSFB.

23. A blood-CNS barrier shuttle according to any one of claims 19 to 22, wherein the FRα binder recognizes an epitope of human FRα that contains Q at position 141 of SEQ ID NO:

1.

24. A blood-CNS barrier shuttle according to any one of claims 19 to 23, wherein the FRα binder comprises a CDR2 sequence as depicted in SEQ ID NO: 31 and / or SEQ ID NO: 32, and / or a CDR1 sequence as depicted in SEQ ID NO: 4 or SEQ ID NO:

113.

25. A blood-CNS barrier shuttle according to any one of claims 19 to 24, comprising or consisting of an amino acid sequence of SEQ ID NO: 2 or 118 - 121 or a homolog having at least 90% identity over the full length of said sequence, wherein the CDR1, 2, 3, and 4 regions are identical to any one of SEQ ID NO: 2, 118 - 121, or their humanized variants.

26. A blood-CNS barrier shuttle according to any one of claims 19 to 25, wherein the molecule is a neurodisease drug, a cancer therapeutic, or an imaging compound.

27. A blood-CNS barrier shuttle according to any one of claims 19 to 26, wherein the blood-CNS barrier shuttle is a BCSFB shuttle.

28. The FRα binder according to any one of claims 1 to 18, or the blood-CNS barrier shuttle according to any one of claims 19 to 27, for use as a medicament.

29. The FRα binder according to any one of claims 1 to 18, or the blood-CNS barrier shuttle according to any one of claims 19 to 27, for use in transporting one or more compounds to the CNS, more particularly through the BCSFB.

30. The FRα binder according to any one of claims 1 to 18, or the blood-CNS barrier shuttle according to any one of claims 19 to 27, for use in the treatment of neurological diseases.

31. The FRα binder according to any one of claims 1 to 18, or the blood-CNS barrier shuttle according to any one of claims 19 to 27, for use according to claim 30, wherein the neurological disease is selected from the list consisting of Alzheimer's disease, stroke, dementia, muscular dystrophy, multiple sclerosis, amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, dystonia, Parkinson's disease, viral or microbial infection, inflammation, brain cancer, neuropathic pain and traumatic brain injury.

32. A composition for use in the treatment or diagnosis of a neurological disorder, the agent comprising a human FRα binder in which a drug is coupled to a neurological disorder drug or an imaging compound, wherein the FRα binder binds to the same epitope on human FRα as 2HFO42 consisting of the amino acid sequence as depicted in SEQ ID NO: 2, and wherein the composition has a dissociation constant k determined by biolayer interferometry off of less than 3x10 -2 / s for binding to human FRα, the composition.

33. The composition according to claim 32 for use according to claim 32, wherein the composition is a multispecific antibody comprising the human FRα binder and a second antigen-binding site that binds to a brain antigen and / or a cancer antigen.

34. The composition according to claim 33 for use according to claim 32, wherein the brain antigen is selected from the group consisting of beta-secretase 1 (BACE1), amyloid beta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR) and caspase 6.

35. A composition according to claim 33 or 34 for use in the treatment or diagnosis of a neurological disorder, wherein the agent comprises a human FRα binder coupled to a neurological disorder drug or an imaging compound, wherein the neurological disorder drug is a biological, small molecule, therapeutic agent, radionuclide, antisense oligonucleotide or test compound.

36. A nucleic acid molecule encoding an FRα binder, a blood-CNS barrier shuttle or a multispecific binder according to any one of claims 1 to 35.

37. A vector comprising the nucleic acid molecule according to claim 35.

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

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