Heavy chain variable domain that binds a free light chain

Abstract

The present invention relates to antibodies binding to, or pairing with, free light chains, such as free kappa light chains and / or (kappa) amyloidogenic light chains, and formulations comprising said antibodies. The present invention further relates to the use of said anti-light chain antibodies and formulations in therapy and / or diagnosis.

Description

[0001] Invention Field

[0002] This invention relates to antibodies that bind to or pair with free light chains, such as free κ light chains and / or (κ) amyloidogenic light chains, and formulations comprising said antibodies. The invention further relates to the use of said anti-light chain antibodies and formulations in therapeutic and / or diagnostic applications. Background of the Invention

[0004] Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, affecting approximately one in 100,000 people annually (Gertz, 2018). AL amyloidosis is classified as plasma cell (PC) dyscrasia, in which mutated PC clones aberrantly proliferate, leading to the synthesis of high concentrations of free light chains (FLCs) (Fotiou et al., 2020). Under standard conditions, light chains (LCs) and heavy chains (HCs) are produced in nearly equal proportions within plasma cells and subsequently incorporated into antibodies. However, in AL amyloidosis, the abnormally large excess of FLCs produced, along with the presence of destabilizing mutations, triggers the aggregation of FLCs into amyloid deposits (also called fibrils), which are deposited systemically in almost every organ, such as the heart, kidneys, skin, liver, and nerves. Such amyloid fibril deposits in organs lead to dysfunction in those organs. For example, more than three-quarters of patients with AL amyloidosis have such deposits in the heart, leading to the development of rapidly progressive cardiomyopathy with a rather poor prognosis (Merlini et al., 2013). In another instance, AL amyloidosis, which accounts for approximately 85% of cases, is the most common form of amyloidogenesis in the kidneys (Gurung and Tingting, 2022).

[0005] There is currently no cure for AL amyloidosis. Unfortunately, patients diagnosed with this disease typically have a very poor prognosis, with a typical survival of 6 months to 3 years (Weiss et al., 2016). Current treatments focus on symptom management and / or aiming to further limit the production of FLC. The latter type of treatment therefore focuses on the depletion of plasma cell clones, which is often poorly tolerated, especially in patients with severe cardiac deposits. Although there are no commercially available treatments designed to clear amyloid deposits, this area is currently under investigation, including via passive immunotherapy. For example, anselamimab is currently in a phase III clinical trial for primary systemic amyloidosis, targeting λ- or κ-derived amyloid fibrils to clear them (Edwards et al., 2021). The antibody birtamimab is also in a phase III clinical trial for Mayo stage IV AL amyloidosis, and it appears to show some benefit in patients with AL amyloidosis who have advanced cardiac involvement (Gertz et al., 2022). However, it is noteworthy that birtamimab does not target the fibrils themselves, but rather serum amyloid protein (SAP) co-localized with amyloid deposits. Broggini et al. (2023) recently identified a llama-derived nanobody that binds to and stabilizes H3 (a λ-induced amyloidosis LC).

[0006] Therefore, there remains a need for antibodies (e.g., VH chains or nanobodies) that specifically target free light chains (e.g., free κ light chains and / or (κ) amyloidogenic light chains), stabilize these free light chains, and thereby prevent and / or inhibit the formation of (amyloid) fibrils. Such antibodies would be extremely useful in diagnosis and / or treatment, including preventative and therapeutic interventions in inflammatory diseases or conditions, particularly those involving inflammatory diseases or conditions with altered FLC profiles. Summary of the Invention

[0007] This invention addresses the aforementioned identified need by providing novel antibodies that bind to or pair with free light chains, such as free κ light chains and / or (κ) amyloidogenic light chains. These antibodies can be useful in diagnosis or treatment, particularly in the treatment of conditions involving fibrils based on free (κ) (amyloidogenic) light chains. These antibodies exhibit remarkably high specificity for free light chains (especially free κ light chains) and prevent and / or inhibit the formation of light chain dimers and the resulting light chain fibrils.

[0008] In a first aspect, the present invention provides antibodies that specifically bind to or pair with free light chains, wherein the antibody comprises a heavy chain variable region comprising: i) a CDR-H1 consisting of SEQ ID NO: 1, 4, 7, 10, or 13, a CDR-H2 consisting of SEQ ID NO: 2, 5, 8, 11, or 14, and a CDR-H3 consisting of SEQ ID NO: 3, 6, 9, 12, or 15; or ii) CDR-H1, CDR-H2, and CDR-H3 sequences having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to any of the sequences listed in i. Preferably, the antibody is selected from the group consisting of: i) antibody moieties containing a VH domain but not a VL domain, or ii) nanobodies.

[0009] In a second aspect, the present invention provides antibodies that specifically bind to or pair with free light chains, wherein the antibodies comprise: i. CDR-H1 composed of SEQ ID NO: 1; CDR-H2 composed of SEQ ID NO: 2; and CDR-H3 composed of SEQ ID NO: 3; ii. CDR-H1 composed of SEQ ID NO: 4; CDR-H2 composed of SEQ ID NO: 5; and CDR-H3 composed of SEQ ID NO: 6; iii. CDR-H1 composed of SEQ ID NO: 7; CDR-H2 composed of SEQ ID NO: 8; and CDR-H3 composed of SEQ ID NO: 9; iv. CDR-H1 composed of SEQ ID NO: 10; CDR-H2 composed of SEQ ID NO: 11; and CDR-H3 composed of SEQ ID NO: 12; v. CDR-H1 composed of SEQ ID NO: 13; CDR-H2 composed of SEQ ID NO: 14; and CDR-H3 composed of SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 10; SEQ ID NO: 11; and CDR-H3 composed of SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; and SEQ ID NO: 19; ... 15 of the CDR-H3; or any of the sequences listed in vi. and i. to v. having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to the CDR-H1, CDR-H2 and CDR-H3 sequences.

[0010] In a third aspect, the present invention provides antibodies that specifically bind to or pair with free light chains, wherein the antibody has a heavy chain variable region comprising any one of SEQ ID NO: 16 to 26, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0011] In a further aspect of the invention, isolated polynucleotides encoding the antibodies, cloning vectors or expression vectors, host cells, methods for producing the antibodies, pharmaceutical compositions comprising the antibodies, and their use in treatment and / or diagnosis are covered. Invention Details

[0013] This disclosure will now be described with reference to certain non-limiting aspects and embodiments thereof, and with reference to certain accompanying drawings and examples.

[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 subject belongs. As used in the specification and claims, the following definitions are provided to facilitate understanding of the invention.

[0015] When the term “and / or” is used in phrases such as “A and / or B” in this document, it is intended to include “A and B”, “A or B”, “A”, and “B”.

[0016] Unless the content explicitly indicates otherwise, the forms “a,” “an,” and “the” include both the singular and plural referents. Thus, for example, references to “an antibody” include both “an antibody” and “a plurality of antibodies.”

[0017] The term "comprising" does not exclude other elements. For the purposes of this disclosure, the term "consisting of" is considered a preferred embodiment of the term "comprising of".

[0018] The term "free light chain," as used herein, refers to an immunoglobulin light chain that is not accompanied by a pair of immunoglobulin heavy chains. In the context of this invention as a whole, "free light chain" does not exclude the possibility of the light chain in monomeric, dimer, trimer, or other oligomeric / polymeric forms. It should be understood that "free light chain" and "light chain" are used interchangeably herein, as this invention is used as a whole in the context of the formation of light chain amyloid fibrils and the purpose of using the antibodies of this invention to prevent their formation.

[0019] The term "amyloidosis light chain" refers to immunoglobulin light chain polypeptides involved in amyloid formation.

[0020] The terms “SMA,” “LEN,” and “REC,” as used herein, refer to the three most studied light chain proteins within the framework of understanding AL amyloidosis. LEN and REC were isolated from the urine of patients with multiple myeloma and AL amyloidosis, respectively. Both are 114 amino acid residues in length and belong to the κ4 subtype. LEN is a non-amyloidogenic immunoglobulin light chain protein (i.e., it does not form amyloid fibrils) that differs from the germline κ4 sequence at a single location (see [link to relevant documentation]). Figure 9 See SEQ ID NO: 45 (Solomon, 1985). SMA is an immunoglobulin light chain protein that causes amyloidosis, also containing 114 amino acid residues and belonging to the κ4 subtype. It is isolated from amyloid plaques derived from lymph nodes of patients with AL amyloidosis (Pras et al., 1968). SMA contains a total of eight amino acid mutations relative to the germline κ4 sequence (see SEQ ID NO: 45) (Solomon, 1985). Figure 9 (See SEQ ID NO: 46). REC is also an amyloidogenic protein, and its sequence differs from germline κ4 at 14 positions (see...). Figure 9 (See SEQ ID NO: 47) (Stevens et al., 1995).

[0021] The term "antibody" refers to both the complete antibody and its functionally active fragments (i.e., molecules containing antigen-binding domains that specifically bind to an antigen, also known as antigen-binding fragments). Unless the context otherwise indicates, the characteristics described herein for antibodies also apply to antibody fragments. Complete antibodies, also known as "immunoglobulins (Ig)," typically refer to intact or full-length antibodies, which are elements comprising two heavy chains and two light chains linked together by disulfide bonds, assembled to define a characteristic Y-shaped three-dimensional structure. Classical, naturally occurring complete antibodies are monospecific because they bind to only one type of antigen, and bivalent because they possess two separate antigen-binding domains.

[0022] The terms “intact antibody,” “full-length antibody,” and “all antibody” are used interchangeably to refer to monospecific bivalent antibodies with a structure similar to that of natural antibodies, containing an Fc region as defined herein. In an intact antibody, each light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The light chain can be κ (kappa) or λ (lambda). The typical κ to λ ratio in normal individuals is between 0.26 and 1.65 (Rajkumar et al., 2005). Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH), which consists of three constant domains CH1, CH2, and CH3, or four constant domains CH1, CH2, CH3, and CH4, depending on the Ig class. The “class” of an Ig or antibody refers to the type of constant region, including IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses, such as IgG1, IgG2, IgG3, and IgG4. The constant regions of an antibody mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions of the antibody according to the invention can be further subdivided into highly variable regions (or “hypervariable regions”, or HVRs) that determine antigen recognition, called complementarity-determining regions (CDRs). Scattered among these hypervariable regions are structurally more conserved regions called frame regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs, together with the FRs, form the variable regions. By convention, the CDRs in the heavy chain variable region of an antibody or its antigen-binding fragment are called CDR-H1, CDR-H2, and CDR-H3, while the CDRs in the light chain variable region are called CDR-L1, CDR-L2, and CDR-L3. They are numbered sequentially from the N-terminus to the C-terminus of each chain.

[0023] CDRs are conventionally numbered according to a system designed by Kabat et al., described in Kabat et al., 1991. Unless otherwise stated, this specification adopts this numbering system.

[0024] According to the Kabat numbering system, the CDR of the heavy chain variable domain typically comprises residues 31-35 (CDR-H1), residues 50-65 (CDR-H2), and residues 95-102 (CDR-H3). However, according to Chothia (1987), the loop equivalent to CDR-H1 extends from residue 26 to residue 32. Therefore, unless otherwise stated, “CDR-H1” as used herein is intended to refer to residues 26 to 35, as described by the combination of the Kabat numbering system and Chothia’s topological loop definition. According to the Kabat numbering system, the CDR of the light chain variable domain typically comprises residues 24-34 (CDR-L1), residues 50-56 (CDR-L2), and residues 89-97 (CDR-L3). In addition to the CDR ring, there is a fourth ring between CDR-2 (CDR-L2 or CDR-H2) and CDR-3 (CDR-L3 or CDR-H3), which is formed by frame 3 (FR3). The Kabat numbering system defines frame 3 as positions 66-94 in the heavy chain and positions 57-88 in the light chain.

[0025] The terms “constant domain” or “constant region” are used interchangeably as used herein to refer to one or more domains in an antibody that are outside the variable region. Constant domains are identical across all antibodies of the same isotype, but differ from one isotype to another. Typically, the constant region of the heavy chain, from the N-terminus to the C-terminus, is formed by a CH1-hinge-CH2-CH3-optional CH4 and contains three or four constant domains.

[0026] The intended function of the antibody molecule of the present invention, and in particular the effector functions that may be required, can be considered when selecting the constant region domain (if present) of the antibody molecule. For example, the constant region domain may be a human IgA, IgD, IgE, IgG, or IgM domain. In particular, the human IgG constant region domain may be used, especially when the antibody molecule is intended for therapeutic use and antibody effector functions are required, using the IgG1 and IgG3 isotypes. Alternatively, when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required, the IgG2 and IgG4 isotypes may be used. It should be understood that sequence variants of these constant region domains may also be used. For example, as described in Angal et al. (Angal et al., 1993), an IgG4 molecule in which the serine at position 241 (numbered according to the Kabat numbering system) has been changed to proline.

[0027] The terms "Fc," "Fc fragment," and "Fc region" are used interchangeably to refer to the C-terminal region of an antibody, which contains the antibody's constant regions but excludes the first constant region, the immunoglobulin domain. Therefore, Fc refers to the last two constant domains of IgA, IgD, and IgG, C-terminus. H2 and C H3 This refers to the last three constant domains of IgE and IgM, and the flexible hinges at the N-termini of these domains. The Fc region of the human IgG1 heavy chain is defined herein as containing residues C226 to its carboxyl terminus, numbered according to the EU index as in Kabat. In the context of human IgG1, according to the EU index as in Kabat, the lower hinge refers to positions 226-236, the CH2 domain to positions 237-340, and the CH3 domain to positions 341-447. The corresponding Fc regions of other immunoglobulins can be identified by sequence alignment. In the context of this disclosure, when present, constant regions or Fc regions can be native as defined above, or can be modified in various ways, provided they contain a functional FcR-binding domain, and preferably a functional FcRn-binding domain.

[0028] The term "antibody" encompasses both monovalent antibodies, which contain only one antigen-binding domain (e.g., a single-arm antibody consisting of an interconnected full-length heavy chain and a full-length light chain, also known as a "half-antibody"), and multivalent antibodies, which contain more than one antigen-binding domain.

[0029] The term "antibody" according to the present invention also covers the antigen-binding fragment of an antibody. Antigen-binding fragments of antibodies include single-chain antibodies (e.g., scFv and dsscfv), Fab, Fab', F(ab')2, Fv, single-domain antibodies, also known as nanobodies (e.g., VH or VL, or VHH or VNAR).

[0030] The term "Fab fragment," as used herein, refers to an antibody fragment comprising: a light chain fragment containing a VL (variable light chain) domain and a constant domain (CL) of the light chain, and a VH (variable heavy chain) domain and a first constant domain (CH1) of the heavy chain. A typical "Fab' fragment" comprises a heavy chain and a light chain pair, wherein the heavy chain contains the variable region VH, the constant domain CH1, and a native or modified hinge region, and the light chain contains the variable region VL and the constant domain CL. The dimer of Fab' according to this disclosure is used to construct F(ab')2, wherein dimerization, for example, can occur via the hinge.

[0031] The term "single-domain antibody," as used herein, refers to an antibody fragment consisting of a single monomeric variable antibody domain. Examples of single-domain antibodies include VH, VL, VHH, or VNAR.

[0032] The term "Fv" refers to two variable domains, such as cooperative variable domains, like cognate pairs, or affinity-mature variable domains, i.e., VH and VL pairs.

[0033] The term “single-chain variable fragment” or “scFv”, as used herein, refers to a single-chain variable fragment that is stabilized by a peptide linker between the VH and VL variable domains.

[0034] The term "multispecific" or "multispecific antibody," as used herein, refers to an antibody having at least two binding domains, i.e., two or more binding domains, such as two or three binding domains, as described herein, wherein the at least two binding domains independently bind two different antigens or two different epitopes on the same antigen. Multispecific antibodies are typically monovalent for each specificity (antigen). The multispecific antibodies described herein encompass both monovalent and multivalent, such as bivalent, trivalent, and tetravalent multispecific antibodies.

[0035] The term "antigen-binding domain," as used herein, refers to a portion of an antibody that specifically interacts with a target antigen, comprising part or all of one or more variable domains, such as part or all of a pair of variable domains VH and VL. Binding domains may comprise monovalent antibodies. In one embodiment, each binding domain is monovalent. Preferably, each binding domain comprises no more than one VH and one VL.

[0036] The term "chimeric" antibody refers to an antibody in which the variable domains (or at least a portion thereof) of the heavy and / or light chains are derived from a specific source or species, such as mice, rats, rabbits, or similar species, while the remaining portion of the heavy and / or light chains (i.e., constant regions) is derived from another species, such as humans. Chimeric antibodies consist of elements derived from two different species, such that said elements retain the characteristics of their respective species of origin. A subcategory of "chimeric antibodies" is "humanized antibodies." Humanized antibodies (including CDR-transplanted antibodies) are antibody molecules having one or more complementarity-determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule. It should be understood that only the specific determining residues of the CDR need to be transferred, rather than the entire CDR. Humanized antibodies may optionally further include one or more framework residues derived from the non-human species from which the CDR originates.

[0037] The term "fully human" antibody refers to an antibody in which the variable regions of both the heavy and light chains, as well as the constant regions (when present), are entirely of human origin or substantially identical to human-derived sequences, but not necessarily derived from the same antibody. Examples of fully human antibodies may include, for example, antibodies produced by the phage display method described above, and antibodies produced by mice in which the murine immunoglobulin variable region genes and optionally the constant region genes have been replaced by their human counterparts.

[0038] Within this invention, the term "epitope" is used interchangeably for both conformational epitopes and linear epitopes. A conformational epitope consists of a discontinuous segment of the primary amino acid sequence of an antigen, while a linear epitope is formed by a sequence of continuous amino acids.

[0039] The term "isolated" antibody refers to an antibody that has been separated from its components in its natural environment (e.g., through purification).

[0040] The term "isolated" polynucleotide refers to a polynucleotide that exists in a physical environment different from the physical environment in which it can occur in nature.

[0041] Term "K" D ", as used in this article, refers to the equilibrium dissociation constant, which is determined by K d With K a The ratio (i.e., K) d / K a K is obtained and expressed as molar concentration (M). d and K a These refer to the dissociation rate and association rate of a specific antigen-antibody (or its antigen-binding fragment) interaction, respectively. The Ka of the antibody... D The value can be determined using methods well established in the art, such as (but not limited to) those used in the embodiments section.

[0042] The terms “blocking,” “blocks,” etc., in the context of antibodies describe antibodies that are able to inhibit or attenuate at least one biological activity of their target (in this case, a light chain or an amyloidogenic light chain). Alternatively, the terms “neutralizing” or “neutralizes” may be used.

[0043] The term "specific binding" refers to an antibody that preferentially or with high affinity binds to the target protein (e.g., light chains or amyloidogenic light chains) but substantially does not bind to other proteins. In other words, the antibody binds to the target protein but shows no significant cross-reactivity to any other molecules. The specificity of an antibody can be further investigated by determining whether the antibody binds to other relevant proteins discussed above, or whether it differentiates between them.

[0044] Antibody-antigen binding is mediated by amino acid interactions at the paratope-epitope interface of the antibody-antigen complex. Conventional immunoglobulin antigen-binding sites are formed by variable regions of the heavy and light chains; binding to epitopes is primarily formed by three hypervariable regions within each chain, known as complementarity-determining regions (CDRs). The number of CDR loop residues directly involved in target binding varies depending on the antibody and the epitope. Recombinant DNA technology and library-based engineering have enabled the direct optimization of antibody complementarities to modulate binding affinity, specificity, and introduce novel functions such as bispecificity. The third hypervariable loop, CDR-L3 (light chain) and, more commonly, CDR-H3 (heavy chain) due to its greater diversity during library development, are the most critical hypervariable loops determining antigen-binding specificity and affinity. Located in the middle of the complementarity region on the antibody, they typically participate in delivering fine-grained binding to the target. Importantly, in conventional double-chain antibodies, the stacked regions of the heavy and light chains are excluded from the solvent and, although they may be crucial for antibody folding, do not directly participate in binding to the target antigen.

[0045] In camelid libraries of single-domain antibodies, differential RNA splicing at the 3' end of V(H)H means that the CH1 region of IgG1 is skipped, the heavy chain is expressed autonomously independent of the light chain, and the variable heavy chain is expressed as a fusion with alternative IgG2 / 3 serum immunoglobulins. Due to the lack of light chain stacking at the light-heavy chain interface, the complementary sites of the V(H)H library can adopt conformations different from those of conventional antibodies. In particular, exposed CDR-3 is conformationally more free than conventional antibodies to explore different complementary site shapes. Complementary residues that directly interact with epitopes can be present within CDRs 1-3, but also within frames, most notably within frame 2 (the interface directly stacked or paired with light chains).

[0046] As used herein, the terms “treatment,” “treatment,” etc., refer to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of the disease or its symptoms, and / or therapeutic in terms of partial or complete cure of the disease and / or adverse effects attributable to the disease. “Treatment” therefore covers any treatment of diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of the disease in subjects who are susceptible to the disease but have not yet been diagnosed with it; (b) suppressing the disease, i.e., preventing its development; and (c) alleviating the disease, i.e., causing its remission.

[0047] The term "therapeutic effective amount" refers to the amount of an active ingredient (e.g., an antibody according to the invention) that, when administered to a mammal or other subject to treat a disease, is sufficient to produce such a treatment for said disease.

[0048] The term "biological sample" encompasses a wide range of sample types obtained from an individual and that may be used for diagnostic or surveillance assays. This definition includes cerebrospinal fluid, blood such as plasma and serum, and other liquid samples of biological origin such as urine and saliva, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny. The definition also includes samples that have been processed in any way after their acquisition, such as by treatment with reagents, solubilization, or enrichment for certain components such as polynucleotides.

[0049] As will be described in more detail below, this invention is based on the identification of specific molecules containing or composed of variable heavy chain (VH) domains, such as CO3 or CO6, using a phage display screening method that target at least three well-known FLC forms, namely SMA, LEN, and REC. The binding and kinetic stabilization of FLCs by the VH were characterized using gel filtration, differential scanning fluorometry, circular dichroism, and surface plasmon resonance. These molecules (e.g., CO3 and CO6) not only bind to or pair with and stabilize (free) LCs, but more importantly, they inhibit the formation of LC dimers and thus inhibit LC fibrils, thereby reducing aggregation to baseline levels even at 10:1 LC:VH concentrations. Atomic-resolution X-ray crystallography data of the LC:VH complex revealed a 1:1 binding ratio that mimics both the antigen-binding site of the dimer of the natural Ig molecule and the structure of the natural LC homodimer. The inventors were able to identify conjugates targeting specific types of LCs as well as pan-LC conjugates. From a therapeutic point of view, such findings are important, for example, by providing physicians with options. Indeed, on the one hand, using pan-LC conjugates to treat any type of disease / condition involving the formation of LC dimers and / or LC fibrils can be beneficial. On the other hand, being able to specifically target a particular form of LC for a given patient can also be beneficial.

[0050] The present invention also provides evidence that the anti-light chain (LC) antibody of the present invention, preferably the VH-based antibody of the present invention, interacts (or pairs) significantly with LC through its frame 2 region, which mimics the antigen-binding site of the dimerization of the natural immunoglobulin molecule, rather than the classic “epitope binding” of the antibody to its target.

[0051] The primary objective of this invention is an antibody that specifically binds to or pairs with a free light chain, such as a free κ light chain and / or (κ) amyloidogenic light chain, wherein the antibody comprises a heavy chain variable region, the heavy chain variable region comprising:

[0052] i. CDR-H1 consisting of SEQ ID NO: 1, 4, 7, 10 or 13, CDR-H2 consisting of SEQ ID NO: 2, 5, 8, 11 or 14, and CDR-H3 consisting of SEQ ID NO: 3, 6, 9, 12 or 15; or

[0053] ii. CDR-H1, CDR-H2 and CDR-H3 sequences that have at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to any of the sequences listed in i.

[0054] As a non-limiting example, the antibody according to the invention may be: a) an antibody comprising CDR-H1 composed of SEQ ID NO: 1, CDR-H2 composed of SEQ ID NO: 2, and CDR-H3 composed of SEQ ID NO: 3 (e.g., such as in antibody "C03"), b) an antibody comprising CDR-H1 composed of SEQ ID NO: 4, CDR-H2 composed of SEQ ID NO: 5, and CDR-H3 composed of SEQ ID NO: 6 (e.g., such as in antibody "C06"), c) an antibody comprising CDR-H1 composed of SEQ ID NO: 7, CDR-H2 composed of SEQ ID NO: 8, and CDR-H3 composed of SEQ ID NO: 9 (e.g., such as in antibody "A03"), d) an antibody comprising CDR-H1 composed of SEQ ID NO: 10, CDR-H2 composed of SEQ ID NO: 11, and CDR-H3 composed of SEQ ID NO: 10, and CDR-H3 composed of SEQ ID NO: 11, and CDR-H3 composed of SEQ ID NO: 11, and CDR-H3 composed of SEQ ID NO: 12, and CDR-H3 composed of SEQ ID NO: 13, and CDR-H3 composed of SEQ ID NO: 14, and CDR-H3 composed of SEQ ID NO: 15, and CDR-H3 composed of SEQ ID NO: 16, and CDR-H3 composed of SEQ ID NO: 17, and CDR-H3 composed of SEQ ID NO: 18, and CDR-H3 composed of SEQ ID NO: 19, and CDR-H3 composed of SEQ ID NO: 10, and CDR-H2 composed of SEQ ID NO: 19, and CDR-H3 composed of SEQ ID NO: 10, and CDR-H3 composed of SEQ ID NO: 19, and CDR An antibody comprising CDR-H3 of SEQ ID NO: 12 (e.g., such as in antibody "D07"), or an antibody comprising CDR-H1 of SEQ ID NO: 13, CDR-H2 of SEQ ID NO: 14, and CDR-H3 of SEQ ID NO: 15 (e.g., such as in antibody "D09"), or an antibody comprising CDR-H1, CDR-H2, and CDR-H3 sequences having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to any of the sequences listed in any of a) through e).

[0055] The anti-light chain antibodies according to the invention (i.e., any combination comprising the CDR sequences described above) are particularly inventive because they provide antibodies with high affinity for free light chains (e.g., κ light chains and / or κ amyloidosis-inducing light chains). As an example, without any limitation, such (free) light chains can be LEN, SMA, and / or REC, and / or such amyloidosis-inducing light chains can be SMA and / or REC.

[0056] In one embodiment of the invention, the anti-light chain antibody further comprises a frame region (FR), the frame region comprising (or consisting of):

[0057] i. FR1 containing SEQ ID NO: 27, 31, 36, 38 or 42, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it;

[0058] ii. FR2 containing SEQ ID NO: 28, 32, 33 or 43, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it;

[0059] iii. FR3 containing SEQ ID NO: 29, 34, 37, 39, 40 or 44, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; and / or

[0060] iv. FR4 containing SEQ ID NO: 30, 35 or 41, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it.

[0061] In another embodiment, the present invention provides an antibody that specifically binds to or pairs with a free light chain, such as a free κ light chain and / or (κ) amyloidogenic light chain, wherein the antibody has a heavy chain variable region comprising any one of SEQ ID NO: 16 to 20, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0062] The present invention also covers antibodies against light chains (optionally, against free light chains), such as anti-κ light chain antibodies and / or anti-(κ) amyloidosis-inducing light chain antibodies, said antibodies comprising a heavy chain variable region comprising a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence given in any one of SEQ ID NO: 21 to 26.

[0063] In another embodiment, the present invention provides an antibody that specifically binds to or pairs with a free light chain, such as a κ light chain and / or (κ) amyloidogenic light chain, wherein the antibody further comprises an Fc domain. Non-limiting examples of the Fc domain include any one of SEQ ID NO: 48-52, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. The heavy chain variable region may be connected to the Fc domain in such a manner, or may include a linker (alternatively called a hinge).

[0064] The antibody according to the invention, as a whole, is preferably selected from the group consisting of: i) antibody portions containing a VH domain but not a VL domain, and ii) nanobodies (which, by definition, are not associated with any light chain). In other words, the antibody according to the invention, as a whole, preferably consists of only one heavy chain variable domain and its active fragment, or only one full-length heavy chain and its active fragment. The antibodies described herein may be chimeric or humanized. If the antibody is humanized, the suitable framework region of the heavy chain for the humanized antibody according to the invention may be derived from human lineage. Standard humanization methods may be used, such as those described by Adair (e.g., WO9109967).

[0065] While the VH according to the invention is preferably in the form of a VH-only or nanobody, alternatively, the VH according to the invention can associate with a VL containing a CDR that specifically binds to a free light chain (e.g., the κ light chain and / or (κ) amyloidogenic light chain). In this case, the resulting antibody is likely to bind to the targeted free light chain, such as the κ light chain and / or (κ) amyloidogenic light chain, as conventional antibodies do, i.e., via its CDR, rather than pairing between the VH and VL. Alternatively, they can associate with a VL containing a CDR that specifically binds to or pairs with one or more other targets to obtain bispecific or multispecific antibodies.

[0066] The anti-light chain antibodies according to the invention neutralize / inhibit the formation of light chain fibrils as a whole. In particular, they primarily bind (or pair with) κ light chains and / or κ amyloidosis-inducing light chains; this binding between the antibodies according to the invention and the light chains prevents / neutralizes / inhibits the formation of light chain fibrils. In some non-limiting examples, the antibodies of the invention may bind to LEN, SMA, and / or REC light chains.

[0067] The anti-light chain antibody according to the invention, as a whole, preferably has an equilibrium dissociation constant (KD) of 1 µM or less for at least one light chain (e.g., the κ light chain or the κ amyloidosis-inducing light chain), for example, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 100 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, or 20 nM or less. D This is as measured in an assay where the anti-light chain antibody is the immobilized portion (an example of such an assay is provided in Example 1). In some embodiments, when using such an assay, the kD is as low as 16 nM or less. KD can be measured / determined by any standard method. For example, the dissociation constant can be determined by surface plasmon resonance (SPR) at a temperature of 25°C between the antibody of the present invention and light chains (e.g., κ light chains and / or (κ) amyloidogenic light chains).

[0068] In one embodiment of the invention, antibodies are provided herein that target binding to or pairing with light chains (e.g., κ light chains and / or (κ) amyloidogenic light chains) and cross-competitive with antibodies comprising a heavy chain variable region, the heavy chain variable region comprising:

[0069] i. CDR-H1 consisting of SEQ ID NO: 1, 4, 7, 10 or 13, CDR-H2 consisting of SEQ ID NO: 2, 5, 8, 11 or 14, and CDR-H3 consisting of SEQ ID NO: 3, 6, 9, 12 or 15;

[0070] ii. CDR-H1 consisting of SEQ ID NO: 1; CDR-H2 consisting of SEQ ID NO: 2; and CDR-H3 consisting of SEQ ID NO: 3;

[0071] iii. CDR-H1 consisting of SEQ ID NO: 4; CDR-H2 consisting of SEQ ID NO: 5; and CDR-H3 consisting of SEQ ID NO: 6;

[0072] iv. CDR-H1 consisting of SEQ ID NO: 7; CDR-H2 consisting of SEQ ID NO: 8; and CDR-H3 consisting of SEQ ID NO: 9;

[0073] v. CDR-H1 consisting of SEQ ID NO: 10; CDR-H2 consisting of SEQ ID NO: 11; and CDR-H3 consisting of SEQ ID NO: 12;

[0074] vi. CDR-H1 consisting of SEQ ID NO: 13; CDR-H2 consisting of SEQ ID NO: 14; and CDR-H3 consisting of SEQ ID NO: 15; or

[0075] vii. A sequence based on any one of SEQ ID NO: 16-26;

[0076] viii. CDR-H1, CDR-H2, and CDR-H3 that have at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity with any of the sequences listed in any of i. through vi.; or

[0077] Any sequence listed in either ix. or vii. has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity.

[0078] To determine whether a particular antibody competes with a reference antibody for binding, the binding method described above is performed in two different experimental settings. In the first setting, the reference antibody is allowed to bind to the antigen under saturation conditions, and then the binding of the test antibody to the antigen is evaluated. In the second setting, the test antibody is allowed to bind to the antigen under saturation conditions, and then the binding of the reference antibody to the protein / peptide is evaluated. If, in both experimental settings, only the first (saturated) antibody is able to bind to the protein / peptide, it is concluded that the test antibody competes with the reference antibody for binding to the antigen. As those skilled in the art will understand, an antibody that competes with the reference antibody for binding may not necessarily bind to the same epitope as the reference antibody, but may spatially block the binding of the reference antibody by binding to overlapping or adjacent epitopes, or induce a conformational change that results in a lack of binding.

[0079] If two antibodies competitively inhibit (block) the binding / pairing of the other with the antigen, then the two antibodies bind to the same or overlapping epitopes / pairing sites. Alternatively, if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody, then the two antibodies have the same epitopes / pairing sites. If some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody, then the two antibodies have overlapping epitopes / pairing sites.

[0080] Further routine experiments (such as peptide mutation and binding assays) can then be performed to confirm whether the observed lack of binding to the test antibody is indeed due to binding to a portion of the same antigen as the reference antibody, or whether steric hindrance (or another phenomenon) is causing the observed lack of binding. Such experiments can be performed using ELISA, RIA, SPR, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0081] The epitopes / pairing sites can be identified by any suitable epitope mapping method known in the art, combined with any of the antibodies provided by the present invention. Examples of such methods include screening peptides of varying lengths derived from full-length light chains (e.g., κ light chains and / or (κ) amyloidogenic light chains) for binding and pairing with the antibodies of the present invention or fragments thereof, and identifying the smallest fragment of a sequence containing an epitope recognized by the antibody that can specifically bind or pair with the antibody. Light chain peptides can be generated synthetically or by proteolytic digestion of the light chain. Antibody-bound peptides can be identified, for example, by mass spectrometry. Methodologies such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, hydrogen-deuterium exchange mass spectrometry (HDX-MS), or cryo-EM can be used to identify antibody-bound epitopes. Typically, when epitope determination is performed by X-ray crystallography, amino acid residues within 4 Å of the CDR in the antigen are considered to be part of the epitope. Once identified, the epitope can be used to prepare fragments that bind to antibodies of the present invention, and, if desired, as an immunogen to obtain additional antibodies that bind to the same epitope.

[0082] Those skilled in the art will also understand that antibodies can undergo a variety of post-translational modifications. The type and extent of these modifications typically depend on the host cell line used to express the antibody and the culture conditions. Such modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. A common modification is the loss of a carboxyl-terminal basic residue (e.g., lysine or arginine) due to the action of carboxypeptidase. Therefore, the C-terminal lysine of the antibody heavy chain may be absent. In one embodiment, the C-terminal amino acid of the antibody is cleaved during post-translational modification. In another embodiment, the N-terminal amino acid of the antibody is cleaved during post-translational modification. In some further embodiments, antibody variants with one or more amino acid substitutions, insertions, and / or deletions are provided. Target sites for mutagenesis used for substitution include CDRs and frame regions. Amino acid substitutions can be introduced into the target antibody, and the product can be screened for desired activities, such as preserved / improved antigen binding and / or reduced immunogenicity.

[0083] In some embodiments, amino acid sequence variants of the antibodies described herein are included. For example, improved binding / pairing affinity and / or other biological properties of the antibody may be desired. Amino acid sequence variants of anti-light chain antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the protein, or by peptide synthesis. Such modifications include, for example, deletions, and / or insertions, and / or substitutions of residues within the amino acid sequence of the anti-light chain antibody (e.g., within one or more CDR sequences and / or framework sequences in the VH domain). Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics.

[0084] In some embodiments of the variant VH sequence provided herein, each CDR (or HVR) is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0085] It should be understood that one or more amino acid substitutions, additions, and / or deletions can be made to the CDR provided by this invention without significantly altering the antibody's ability to bind to or pair with light chains (e.g., κ light chains and / or (κ) amyloidogenic light chains) and to neutralize fibrillation. The effects of any amino acid substitution, addition, and / or deletion can be readily tested by those skilled in the art, for example by using the methods described herein, particularly those exemplified in the examples, to determine light chain binding and inhibition of fibrillation.

[0086] Therefore, in some embodiments of the variant VH sequence, each CDR contains no more than one, two, or three amino acid substitutions, wherein such amino acid substitutions are conserved, and wherein the antibody retains its binding properties to light chains (e.g., κ light chains and / or (κ) amyloidogenic light chains).

[0087] Therefore, this document provides antibody variants that specifically bind to or pair with free light chains, such as free κ light chains and / or (κ) amyloidogenic light chains, wherein the antibody variants comprise a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 sequences having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to any of the sequences listed in any of the following i. to vi.: i) CDR-H1 consisting of SEQ ID NO: 1, 4, 7, 10, or 13; CDR-H2 consisting of SEQ ID NO: 2, 5, 8, 11, or 14; and CDR-H3 consisting of SEQ ID NO: 3, 6, 9, 12, or 15; ii) CDR-H1 consisting of SEQ ID NO: 1; CDR-H2 consisting of SEQ ID NO: 2; and CDR-H3 consisting of SEQ ID NO: 1, 4, 7, 10, or 13; iii) CDR-H3 composed of SEQ ID NO: 4; CDR-H2 composed of SEQ ID NO: 5; and CDR-H3 composed of SEQ ID NO: 6; iv) CDR-H1 composed of SEQ ID NO: 7; CDR-H2 composed of SEQ ID NO: 8; and CDR-H3 composed of SEQ ID NO: 9; v) CDR-H1 composed of SEQ ID NO: 10; CDR-H2 composed of SEQ ID NO: 11; and CDR-H3 composed of SEQ ID NO: 12; vi) CDR-H1 composed of SEQ ID NO: 13; CDR-H2 composed of SEQ ID NO: 14; and CDR-H3 composed of SEQ ID NO: 15.

[0088] The following antibody variants are also provided, which further comprise a frame region (FR) containing or consisting of FR1, FR2, FR3, and FR4 sequences, wherein the FR1, FR2, FR3, and FR4 sequences have at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to any of the sequences listed in any of the following i. to iv.: i. FR1 containing SEQ ID NO: 27, 31, 36, 38, or 42; ii. FR2 containing SEQ ID NO: 28, 32, 33, or 43; iii. FR3 containing SEQ ID NO: 29, 34, 37, 39, 40, or 44; and / or iv. FR4 containing SEQ ID NO: 30, 35, or 41.

[0089] As an example of an antibody variant according to the invention (i.e., comprising CDR and FR as described herein), such an antibody may comprise a heavy chain variable region comprising a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence given in any one of SEQ ID NO: 16 to 20.

[0090] The present invention also covers anti-light chain antibody variants (e.g., anti-κ light chain antibody variants and / or anti-(κ) amyloidosis light chain antibody variants) comprising a heavy chain variable region comprising a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence given in any one of SEQ ID NO: 21 to 26.

[0091] In a further alternative embodiment, the anti-light chain antibody variant of the present invention (e.g., anti-κ light chain antibody variant and / or anti-(κ) amyloidosis light chain antibody variant) comprises an Fc domain containing a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence given in any of SEQ ID NO:48-52.

[0092] The anti-light chain antibody variants provided herein by the present invention retain the advantageous properties of the parent antibody (i.e., the unmodified antibody), namely the functional properties described herein. In one example, the anti-light chain antibody variants provided by the present invention have a dissociation constant (Ki) of 1 µM or less for at least one light chain (e.g., the κ light chain or the κ amyloidosis-inducing light chain). DFor example, the dissociation constants (K) of 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 100 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, and 20 nM or less than 20 nM. D ), as measured in an assay in which the anti-light chain antibody is the immobilized portion (an example of such an assay is provided in Example 1). In some embodiments, when using such an assay, K D As low as 16 nM or lower. K D It can be measured / determined by any standard method. For example, the dissociation constant can be determined by SPR at a temperature of 25°C between the antibody of the present invention and the light chain (e.g., the κ light chain and / or the amyloidogenic light chain).

[0093] The degree of identity and similarity between sequences can be easily calculated. “% sequence identity” (or “% sequence similarity”) is calculated by: (1) comparing two best-aligned sequences in a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.), (2) determining the number of positions containing the same (or similar) amino acids (e.g., the same amino acid appears in both sequences, similar amino acids appear in both sequences) to produce the number of matching positions, (3) dividing the number of matching positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to obtain the % sequence identity or sequence similarity percentage.

[0094] The sequence alignment methods used for comparison are well known in the art. Preferred examples of algorithms suitable for determining the percentage of sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms. Peptide sequences can also be compared using FASTA with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides the alignment of the best overlapping region between the query sequence and the search sequence, as well as the percentage of sequence identity.

[0095] In some implementations, substitution, insertion, or deletion can occur within one or more CDRs, as long as such changes do not substantially reduce the antibody’s ability to bind to the target.

[0096] For example, conserved alterations that do not substantially reduce binding affinity can be made in the CDR. Such alterations can be made outside the antigen contact residues in the CDR.

[0097] Conservative substitutions are shown together with more substantial “exemplary substitutions” in Table 1.

[0098] Table 1: Examples of amino acid substitution

[0099]

[0100] Substantial modifications to the biological properties of antibody variants can be achieved by selecting substitutions that have significantly different effects in the following aspects: maintaining the structure of the polypeptide backbone in the substitution region, the charge or hydrophobicity of the molecule at the target site, or the volume of the side chain.

[0101] One class of substitution variants involves replacing one or more CDR region residues of a parent antibody (humanized antibody or human antibody). Typically, the resulting one or more variants selected for further research will have altered certain biological properties relative to the parent antibody (e.g., increased affinity, decreased immunogenicity), and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques. In short, one or more CDR residues are mutated, the variant antibody is displayed on a phage, and screened for specific biological activities (e.g., binding affinity).

[0102] Alterations (e.g., substitutions) can be made in the CDR to improve antibody affinity, for example. Such alterations can be made in hypervariable region (HVR) “hotspots,” which are residues encoded by codons undergoing high-frequency mutations during somatic maturation, and / or residues that contact the antigen, and binding affinity can be tested on the resulting variant VH. Affinity maturation by constructing a secondary library and reselecting from it has been well described in the literature. In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation using any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. This library is then screened to identify any antibody variants with the desired affinity. One method that can be used to identify residues or regions in an antibody that can be targeted for mutagenesis is alanine scan mutagenesis. Alternatively or additionally, the X-ray structure of the antigen-antibody complex can be used to identify the contact points between the antibody and its antigen. Variants can be screened to determine if they contain the desired properties.

[0103] Antibodies against light chains, such as the κ light chain and / or (κ) amyloidogenic light chains, can be obtained by immunizing animals (preferably non-human animals) with the light chain or a portion thereof using well-known and conventional protocols. Many animals (e.g., rabbits, mice, rats, sheep, cattle, llama, camels, or pigs) can be immunized.

[0104] Monoclonal antibodies can be prepared using a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of the human immunoglobulin gene locus. A non-limiting method for preparing antibodies is described in the Examples section.

[0105] This document also describes a method for identifying antibodies according to the present invention, the method comprising:

[0106] a) Immunize non-human animals with a light chain immunogenic composition;

[0107] b) Recover B cells from the non-human mammal;

[0108] c) Select antibodies produced by the B cells that have at least one or more of the following properties:

[0109] i. Using the dissociation constant K D An affinity of 500 nM or less indicates the binding or pairing of free light chains; and / or

[0110] ii. To block, prevent, or inhibit the formation of free light chain fibrils.

[0111] The present invention also provides isolated polynucleotides encoding antibodies according to the invention. Therefore, this document provides isolated polynucleotides encoding antibodies comprising a heavy chain variable region, the heavy chain variable region comprising:

[0112] i. CDR-H1 consisting of SEQ ID NO: 1, 4, 7, 10 or 13, CDR-H2 consisting of SEQ ID NO: 2, 5, 8, 11 or 14, and CDR-H3 consisting of SEQ ID NO: 3, 6, 9, 12 or 15;

[0113] ii. CDR-H1 consisting of SEQ ID NO: 1; CDR-H2 consisting of SEQ ID NO: 2; and CDR-H3 consisting of SEQ ID NO: 3;

[0114] iii. CDR-H1 consisting of SEQ ID NO: 4; CDR-H2 consisting of SEQ ID NO: 5; and CDR-H3 consisting of SEQ ID NO: 6;

[0115] iv. CDR-H1 consisting of SEQ ID NO: 7; CDR-H2 consisting of SEQ ID NO: 8; and CDR-H3 consisting of SEQ ID NO: 9;

[0116] v. CDR-H1 consisting of SEQ ID NO: 10; CDR-H2 consisting of SEQ ID NO: 11; and CDR-H3 consisting of SEQ ID NO: 12;

[0117] vi. CDR-H1 consisting of SEQ ID NO: 13; CDR-H2 consisting of SEQ ID NO: 14; and CDR-H3 consisting of SEQ ID NO: 15; or

[0118] vii. A sequence based on any one of SEQ ID NO: 16-26;

[0119] viii. CDR-H1, CDR-H2, and CDR-H3 that have at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity with any of the sequences listed in any of i. through vi.; or

[0120] Any sequence listed in either ix. or vii. has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity.

[0121] The isolated polynucleotide according to the invention may further include a sequence encoding an Fc domain, for example, an Fc domain comprising any one of SEQ ID NO: 48-52, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0122] The isolated polynucleotides according to the present invention may comprise synthetic DNA (e.g., synthetic DNA produced by chemical processing), cDNA, genomic DNA, or mRNA, or any combination thereof.

[0123] The present invention also provides cloning vectors or expression vectors comprising one or more of the polynucleotides described herein. In one example, the cloning vector or expression vector according to the present invention comprises one or more isolated polynucleotides as described above.

[0124] The DNA sequence encoding the antibody of this invention can be prepared using standard molecular biology techniques. The desired DNA sequence can be synthesized completely or partially using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate.

[0125] The general methods, transfection methods and culture methods that can be used to construct vectors are well known to those skilled in the art.

[0126] A host cell is also provided, which contains one or more isolated polynucleotide sequences according to the invention, or the host cell contains one or more cloning vectors or expression vectors containing one or more isolated polynucleotide sequences encoding the antibodies of the invention. Any suitable host cell / vector system can be used to express the polynucleotide sequences encoding the antibodies of the invention. Bacteria (e.g., *Escherichia coli*) can be used. E. coli Other microbial systems. Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been “humanized,” thereby producing antibodies with partial or complete human glycosylation patterns. Other host cells that can be used are any eukaryotic cells, such as mammalian cells. Suitable mammalian host cells include Chinese hamster ovary (CHO) cells, human embryonic cells (HEK cells), or lymphoid cells (e.g., Y0, NSO, Sp20 cells). Suitable types of CHO cells according to the invention may include CHO cells and CHO-K1 cells (including dhfr-CHO cells), such as CHO-DG44 cells and CHO-DXB11 cells, which can be used with DHFR select markers, or CHOK1-SV cells, which can be used with glutamine synthase select markers. Other cell types used for antibody expression include lymphocyte lines such as NSO myeloma cells and SP2 cells, COS cells; or human embryonic cells such as HEK293, HEK293F, HEK293S, or EK293T. The host cells can be stably transformed or transfected using the isolated polynucleotide sequences or expression vectors according to the present invention.

[0127] The present invention also provides a method for generating antibodies according to the invention, comprising culturing host cells according to the invention under conditions suitable for generating antibodies according to the invention, and isolating said antibodies.

[0128] The present invention also provides a method for producing a pharmaceutical composition comprising an antibody according to the invention, comprising culturing a host cell according to the invention under conditions suitable for producing an antibody according to the invention, isolating the antibody, and formulating the antibody into a pharmaceutical composition.

[0129] The antibodies of the present invention preferably contain only heavy chain polypeptides, in which case only the heavy chain polypeptide coding sequence needs to be used to transfect host cells (in a single vector). To generate antibodies containing both heavy and light chains, cell lines can be transfected using two vectors: a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector containing sequences encoding both the light and heavy chain polypeptides can be used.

[0130] Therefore, this document provides methods for culturing host cells and expressing antibodies according to the invention, isolating said antibodies, and optionally purifying said antibodies to provide isolated antibodies.

[0131] The present invention also provides a method for generating antibodies according to the invention, comprising culturing a host cell containing a vector of the invention under conditions suitable for causing expression of a protein encoded by DNA encoding an antibody molecule of the invention, and isolating the antibody molecule.

[0132] In one embodiment, purified antibodies, such as humanized antibodies, particularly those according to the invention, are provided in a substantially purified form, especially free of or substantially free of endotoxins and / or host cell proteins or DNA.

[0133] Antibodies according to the invention can be provided in pharmaceutical or diagnostic compositions. Therefore, the invention also provides pharmaceutical or diagnostic compositions comprising an antibody according to the invention or a polynucleotide encoding an antibody according to the invention, in combination with one or more acceptable carriers, excipients, and / or diluents (e.g., pharmaceutically acceptable carriers, excipients, and / or diluents). Preferably, the pharmaceutical or diagnostic composition comprises an antibody that specifically binds to free light chains (e.g., free κ light chains and / or (κ) amyloidogenic light chains), or a polynucleotide encoding such antibodies, and one or more pharmaceutically acceptable carriers, excipients, and / or diluents, wherein the antibody comprises a heavy chain variable region comprising:

[0134] i. CDR-H1 consisting of SEQ ID NO: 1, 4, 7, 10 or 13, CDR-H2 consisting of SEQ ID NO: 2, 5, 8, 11 or 14, and CDR-H3 consisting of SEQ ID NO: 3, 6, 9, 12 or 15;

[0135] ii. CDR-H1 consisting of SEQ ID NO: 1; CDR-H2 consisting of SEQ ID NO: 2; and CDR-H3 consisting of SEQ ID NO: 3;

[0136] iii. CDR-H1 consisting of SEQ ID NO: 4; CDR-H2 consisting of SEQ ID NO: 5; and CDR-H3 consisting of SEQ ID NO: 6;

[0137] iv. CDR-H1 consisting of SEQ ID NO: 7; CDR-H2 consisting of SEQ ID NO: 8; and CDR-H3 consisting of SEQ ID NO: 9;

[0138] v. CDR-H1 consisting of SEQ ID NO: 10; CDR-H2 consisting of SEQ ID NO: 11; and CDR-H3 consisting of SEQ ID NO: 12;

[0139] vi. CDR-H1 consisting of SEQ ID NO: 13; CDR-H2 consisting of SEQ ID NO: 14; and CDR-H3 consisting of SEQ ID NO: 15; or

[0140] vii. A sequence based on any one of SEQ ID NO: 16-26;

[0141] viii. CDR-H1, CDR-H2, and CDR-H3 that have at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity with any of the sequences listed in any of i. through vi.; or

[0142] Any sequence listed in either ix. or vii. has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity.

[0143] The pharmaceutical or diagnostic compositions according to the invention can be used for treatment and / or diagnosis, respectively. The pharmaceutical compositions can be appropriately administered to a patient to determine the required therapeutically effective amount. For any antibody, the therapeutically effective amount can be initially estimated in cell culture assays or in animal models (typically rodents, rabbits, dogs, pigs, or primates). The animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine the useful dose and route of administration in humans.

[0144] For human subjects, the precise therapeutically effective dose will depend on the severity of the disease state, the subject's overall health, age, weight and sex, diet, timing and frequency of administration, combination of one or more drugs, sensitivity to response, and tolerance / response to treatment. Typically, the therapeutically effective dose will be from 0.01 mg / kg to 500 mg / kg, for example from 0.1 mg / kg to 200 mg / kg, for example 100 mg / kg. The pharmaceutical composition can be conveniently presented in unit dosage forms containing a predetermined amount of the active agent of the invention per dose.

[0145] The pharmaceutical or diagnostic compositions according to the invention preferably comprise not only the antibody or polynucleotide encoding such an antibody according to the invention, but also one or more pharmaceutically acceptable carriers, excipients (e.g., buffers, stabilizers, suspending agents, preservatives and / or dispersants or other materials well known to those skilled in the art) and / or diluents. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. Examples of carriers, excipients and / or diluents, as well as methods for preparing pharmaceutical compositions, can be found in Remington's Pharmaceutical Sciences, 20th edition, 2000, pub. Lippincott, Williams & Wilkins.

[0146] Suitable forms of administration include those suitable for parenteral administration, such as by injection or infusion, for example by bolus injection or continuous infusion, in intravenous, aspirable, or subcutaneous form. When the product is intended for injection or infusion, it may be in the form of a suspension, solution, or emulsion in an oily or aqueous medium. Alternatively, the antibody according to the invention may be in a dry form for reconstitution with a suitable sterile liquid prior to use. It may also be prepared in a solid form suitable for dissolving or suspending in a liquid medium prior to injection.

[0147] Once formulated, the compositions of the present invention can be administered directly to a subject, or, depending on their form, after reconstitution. Alternatively, they can be used for diagnostic purposes.

[0148] This document also provides the use of antibodies or polynucleotides encoding such antibodies according to the invention for the preparation of pharmaceuticals.

[0149] Preferably, the pharmaceutical composition according to the invention is suitable for administration to primates, such as human subjects or non-human subjects.

[0150] This article also covers pharmaceuticals comprising antibodies that specifically bind to light chains (e.g., κ light chains and / or (κ) amyloidogenic light chains), or polynucleotides encoding such antibodies, and one or more pharmaceutically acceptable carriers, excipients, and / or diluents for therapeutic and / or diagnostic purposes, wherein said antibody comprises a heavy chain variable region comprising:

[0151] i. CDR-H1 consisting of SEQ ID NO: 1, 4, 7, 10 or 13, CDR-H2 consisting of SEQ ID NO: 2, 5, 8, 11 or 14, and CDR-H3 consisting of SEQ ID NO: 3, 6, 9, 12 or 15;

[0152] ii. CDR-H1 consisting of SEQ ID NO: 1; CDR-H2 consisting of SEQ ID NO: 2; and CDR-H3 consisting of SEQ ID NO: 3;

[0153] iii. CDR-H1 consisting of SEQ ID NO: 4; CDR-H2 consisting of SEQ ID NO: 5; and CDR-H3 consisting of SEQ ID NO: 6;

[0154] iv. CDR-H1 consisting of SEQ ID NO: 7; CDR-H2 consisting of SEQ ID NO: 8; and CDR-H3 consisting of SEQ ID NO: 9;

[0155] v. CDR-H1 consisting of SEQ ID NO: 10; CDR-H2 consisting of SEQ ID NO: 11; and CDR-H3 consisting of SEQ ID NO: 12;

[0156] vi. CDR-H1 consisting of SEQ ID NO: 13; CDR-H2 consisting of SEQ ID NO: 14; and CDR-H3 consisting of SEQ ID NO: 15; or

[0157] vii. A sequence based on any one of SEQ ID NO: 16-26;

[0158] viii. CDR-H1, CDR-H2, and CDR-H3 that have at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity with any of the sequences listed in any of i. through vi.; or

[0159] A sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to any sequence listed in either ix. or vii.

[0160] This document also covers antibodies, polynucleotides, or pharmaceutical compositions according to the invention for treatment, particularly for treating conditions or illnesses as described herein. Such antibodies (or polynucleotides or pharmaceutical compositions) are administered in therapeutically effective amounts.

[0161] This invention provides a method for treating a condition or illness as described herein in a subject with such need, the method comprising administering to the subject an antibody, polynucleotide, or pharmaceutical composition according to the invention. Such antibodies are administered in a therapeutically effective amount.

[0162] The present invention also provides the use of the antibodies, polynucleotides or pharmaceutical compositions of the present invention for the preparation of medicines, particularly for the treatment of diseases or conditions as described herein.

[0163] In the context of this invention as a whole, the disease or condition is characterized by the overexpression of light chains, aggregation of light chains, and / or the formation and accumulation of light chain fibrils. Preferably, the disease or condition is selected from the group consisting of: light chain (AL) amyloidosis, light chain deposition disease (LCDD), light and heavy chain deposition disease (LHCDD), crystal-storing histiocytosis, asthma, inflammatory diseases and autoimmune diseases (e.g., rheumatoid arthritis, Sjogren's disease, idiopathic urticaria, multiple sclerosis, diabetes, systemic lupus erythematosus, lupus nephritis and inflammatory bowel disease), and some classes of cancers such as multiple myeloma (including λ myeloma and / or κ myeloma).

[0164] As mentioned above, the inventors were able to identify pan-LC conjugates and more specific LC conjugates. From a therapeutic point of view, such findings are important. Indeed, on the one hand, it could be beneficial to use pan-LC conjugates to treat all patients with amyloid light chains without requiring prior diagnosis of those who need it. Alternatively, a combination of two or more specific LC conjugates could be an option to avoid the need for prior diagnosis of those who require it. On the other hand, it could also be beneficial to be able to specifically target the form of LC for a given patient. In this case, the patient would need to have their light chains sequenced, and high-affinity therapeutic agents could subsequently be designed on the VH scaffold (personalized medicine).

[0165] This document also covers the use of anti-light chain antibodies (e.g., anti-κ light chain antibodies and / or anti-(κ)-amyloidosis light chain antibodies) or their variable domain fragments as diagnostic agents or for diagnostic assays, such as for diagnosing conditions or illnesses as described herein. The diagnosis may preferably be performed on biological samples, such as human biological samples (e.g., urine, blood, plasma, serum, erythrocytes, tissue, saliva, placental tissue, bone marrow, breast milk, bronchoalveolar lavage fluid, feces, pleural effusion, synovial fluid, and semen). The diagnostic test may preferably be performed in vitro.

[0166] As specified above, the anti-light chain (LC) antibody of the present invention, and more particularly the heavy chain domain of the present invention, interacts (or pairs) significantly with the FLC through its frame 2 region, mimicking the antigen-binding site of the dimerization of the natural immunoglobulin molecule, rather than the classic “epitope binding” of the antibody to its target. Therefore, throughout this document, the terms / concepts “anti-light chain (LC) antibody that specifically binds to the free light chain” (and any equivalent expressions thereof) and “heavy chain domain that specifically interacts with or pairs with the free light chain” are interchangeable. Thus, for example:

[0167] - This invention provides heavy chain domains that specifically interact with or pair with free light chains, such as free κ light chains and / or (κ) amyloidogenic light chains, wherein the heavy chain domains comprise heavy chain variable regions, the heavy chain variable regions comprising:

[0168] i. CDR-H1 consisting of SEQ ID NO: 1, 4, 7, 10 or 13, CDR-H2 consisting of SEQ ID NO: 2, 5, 8, 11 or 14, and CDR-H3 consisting of SEQ ID NO: 3, 6, 9, 12 or 15; or

[0169] ii. Combinations of CDR-H1, CDR-H2, and CDR-H3 sequences that have at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to any of the sequences listed in i.

[0170] - The heavy chain structural domain according to the present invention may further include a heavy chain variable region, the heavy chain variable region comprising:

[0171] i. CDR-H1 consisting of SEQ ID NO: 1; CDR-H2 consisting of SEQ ID NO: 2; and CDR-H3 consisting of SEQ ID NO: 3;

[0172] ii. CDR-H1 consisting of SEQ ID NO: 4; CDR-H2 consisting of SEQ ID NO: 5; and CDR-H3 consisting of SEQ ID NO: 6;

[0173] iii. CDR-H1 consisting of SEQ ID NO: 7; CDR-H2 consisting of SEQ ID NO: 8; and CDR-H3 consisting of SEQ ID NO: 9;

[0174] iv. CDR-H1 consisting of SEQ ID NO: 10; CDR-H2 consisting of SEQ ID NO: 11; and CDR-H3 consisting of SEQ ID NO: 12;

[0175] v. CDR-H1 consisting of SEQ ID NO: 13; CDR-H2 consisting of SEQ ID NO: 14; and CDR-H3 consisting of SEQ ID NO: 15; or

[0176] vi. and any of the sequences listed in any of i. to v. have at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to the CDR-H1, CDR-H2 and CDR-H3 sequences.

[0177] - The present invention provides a heavy chain domain that specifically interacts with or pairs with a free light chain, such as a κ light chain and / or a (κ) amyloidogenic light chain, wherein the heavy chain domain has a heavy chain variable region comprising any one of SEQ ID NO: 16 to 26, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0178] - The heavy chain domain of the present invention is selected from the group consisting of: i) a heavy chain domain containing a VH domain but not a VL domain, or ii) a nanobody.

[0179] - The heavy chain structural domain of the present invention is selected from the group consisting of: chimeric heavy chain structural domain or humanized heavy chain structural domain.

[0180] - The heavy chain domain of the present invention may further include an Fc domain selected from the group consisting of any one of SEQ ID NO: 48 to 52, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0181] - The heavy chain domain of the present invention preferably neutralizes or inhibits the formation of light chain dimers and / or light chain fibrils.

[0182] - The heavy chain domain of the present invention preferably has an equilibrium dissociation constant (K0) of less than 500 nM for the light chain. D ).

[0183] This article also provides:

[0184] ○ Isolated polynucleotides, said isolated polynucleotides encoding the heavy chain domain of the present invention,

[0185] ○ A cloning vector or expression vector comprising a separated polynucleotide encoding the heavy chain domain of the present invention.

[0186] ○ Host cell, the host cell comprising a separate polynucleotide encoding the heavy chain domain of the present invention, or comprising an expression vector comprising a separate polynucleotide encoding the heavy chain domain of the present invention.

[0187] ○ A method for generating the heavy chain domain of the present invention, comprising culturing host cells under suitable conditions for generating the heavy chain domain and isolating the heavy chain domain.

[0188] ○ A pharmaceutical composition comprising the heavy chain domain of the present invention or a polynucleotide encoding the heavy chain domain, and one or more pharmaceutically acceptable carriers, excipients and / or diluents.

[0189] ○ The heavy chain domain of the present invention, the polynucleotide of the present invention, or the pharmaceutical composition of the present invention are used for the treatment and / or diagnosis of, for example, for the treatment of diseases or conditions selected from the group consisting of: light chain (AL) amyloidosis, light chain deposition disease (LCDD), light chain and heavy chain deposition disease (LHCDD), crystal storage histiocytosis, asthma, inflammatory diseases and autoimmune diseases (e.g., rheumatoid arthritis, Sjögren's syndrome, idiopathic urticaria, multiple sclerosis, diabetes, systemic lupus erythematosus, lupus nephritis and inflammatory bowel disease), and some classes of cancers such as multiple myeloma (including λ myeloma and κ myeloma).

[0190] Brief description of the attached figures

[0191] Figure 1 Analytical gel filtration spectra of LEN (A), SMA (B), and REC (C) in their individual forms and when they form complexes with CO3.

[0192] Figure 2Biacore SPR analysis of CO3 binding with LEN (A), SMA (B), and REC (C). Light chains were injected at different concentrations through captured CO3. Exemplary binding curves (shown in light gray) are presented for each ligand and fitted using both a 1:1 binding model (i) and a heterogeneous ligand model (ii) (black line).

[0193] Figure 3 Thermal denaturation. Average normalized fluorescence (%) measured by DSF for SMA (A), REC (C), and LEN (E) individually and in complex with CO3 (i). Plot the recorded Tm values ​​and label the SEM error bars (ii). This indicates that the Tm of the LC:CO3 complex was statistically significantly higher than that of LC alone (unpaired t-test, P < 0.0001). N = 3 for each DSF experiment. A single transition was observed in all proteins except LEN and the LEN:CO3 complex. Here, the two transitions showed different unfolding synergies, indicating the presence of two distinct conformations. The two transitions were analyzed and plotted separately (E, iii). Thermal transition curves of LC measured by circular dichroism (CD) at 202 nm between 30–70 °C were plotted, and Boltzmann curves were fitted for SMA (B), REC (D), and LEN (F) in their individual presence and when they formed complexes with CO3. The calculated Tm values ​​are presented as a bar graph (G) in Table 6.

[0194] Figure 4 The presence of CO3 inhibits amyloid formation in SMA. A) 1:1 SMA:CO3, B) 2:1 SMA:CO3, C) 10:1 SMA:CO3. D) Transmission electron microscopy image of SMA (1 mg / mL), E) Transmission electron microscopy image of SMA (1 mg / mL) at higher resolution compared to D), F) 1:1 SMA:CO3, G) 2:1 SMA:CO3, H) 10:1 SMA:CO3, I) CO3 (1 mg / mL) after 18 days of incubation. Scale bar 500 nm.

[0195] Figure 5 Structural analysis of the SMA homodimer crystal structure. The following magnified images show: A) the hydrogen bond network involving the residues Gln44, Gln48, and Tyr93 of each halfmer (the upper and lower half), and B) the salt bridge interaction between His100 and Glu61 of each halfmer.

[0196] Figure 6Structural analysis of the SMA:CO3 heterodimer crystal structure. The following magnified images show: A) the hydrogen bond network involving SMA residues (Gln44, Tyr42, Tyr93, and Gln106) and CO3 residues (Gln39, Lys43, Tyr95, and Tyr105), and B) the salt bridge interaction between His100(S) / Asp59(V) and Glu61(S) / Lys106(V).

[0197] Figure 7 Multiple sequence alignments of SMA with fifty randomly selected (amyloidogenic) κ LC sequences identified by searching an AL-type amyloidosis database. Any sequences longer than 120 residues were truncated. Residues proven essential for SMA:CO3 binding are highlighted by boxes.

[0198] Figure 8 Multiple sequence alignments of SMA with fifty randomly selected (amyloidogenic) λLC sequences identified by searching an AL-type amyloidosis database. Any sequences longer than 120 residues were truncated. Residues proven essential for SMA:CO3 binding are highlighted by boxes.

[0199] Figure 9 Structural overview and multiple sequence alignment of LEN, SMA, and REC. Crystal structures of the classic dimer LEN (left) and the non-classical dimer REC (right). LEN has two monomeric subunits oriented in the same direction, while each subunit of the REC dimer is oriented 180° relative to each other. Structural information for SMA is currently unavailable. Multiple sequence alignments of the germline κIV sequence (below) with LEN, SMA, and REC are shown, with residue variations underlined. LEN differs from the germline by only one residue (N29S). SMA differs from LEN by a total of 8 residues, and REC by 14 residues (highlighted along the edge of the central triangle).

[0200] Figure 10 The full-length sequences of eleven hits selected for further analysis were aligned. Conventional VH (FR2) residues and three CDR residues were identified.

[0201] Figure 11 BLI analysis of CO3 (as VH) binding to LEN (A), SMA (B), and REC (C). Different concentrations of CO3 were used. Binding curves using a 1:1 binding model are provided for each ligand.

[0202] Sequence description:

[0203]

[0204]

[0205]

[0206] Note: In the table above, "VH" in the right column represents the variable heavy chain, "CDR" represents the complementarity determination region, and "FR" represents the frame region.

[0207] Example

[0208] method

[0209] Expression and purification of light chain variable domains (i.e., targets) The coding regions of three light chains, SMA, LEN, and REC, were cloned into pET11a(+), which carries a C-terminal Avi tag and an N-terminal hexahistidine tag containing a SUMO cleavage site. Simultaneously, the pET11a(+) construct was cloned into the pOPINS vector, which also carries an N-terminal hexahistidine tag containing a SUMO cleavage site. The light chain (LC) construct was transformed into SHuffle cells (New England Bioscience) and grown at 37°C. Protein expression was induced for 16 h by adding 0.5 mMIPTG at 16°C. After harvesting, cells were lysed using a cell disruptor (Constant Systems), and the clarified lysate (in 20 mM Na2HPO4, 500 mM NaCl, 20 mM imidazole, pH 7.4) was loaded onto a 5 mL His-Trap FF column (GE Healthcare). The bound protein was eluted in 250 mM imidazole for 5 column volumes. The His6-SUMO tag was removed by cleavage with SUMO protease at 4°C, followed by dialyzing into PBS (Sigma). The sample was then reloaded onto a His-Trap column and the flow-through was collected. The purified LC was dialyzed into 10% v / v PBS:ddH2O, lyophilized, and reconstituted in 10% ddH2O of the original volume.

[0210] Protein biotinylation Site-specific biotinylation of Avi-tagged LEN, SMA, and REC was performed using the BirA-500 kit (Avidity) following the manufacturer's instructions.

[0211] Phage library enrichment for LC Enrichment of the native llama VHH phage library for monomeric / dimeric LCs was performed as follows: 500 nM biotinylated LCs were pre-immobilized onto Dynabeads M-280 Streptavidin-(Invitrogen) and Sera-Mag SpeedBeads Neutravidin-coated magnetic beads (Cytiva) for rounds 1 and 2 / 3, respectively. The magnetic beads were then incubated with the phage population. After incubation, unbound phages were removed by washing 5 or 10 times with PBS-T buffer (PBS, pH 7.4, and 0.1% (v / v) Tween-20) for rounds 1 / 2 and 3, respectively. Bound phages were eluted from the magnetic beads by adding Tris-buffered saline / calcium chloride (TBSC) buffer containing 100 μg / ml trypsin, and the mixture was incubated at room temperature for 30 minutes. Infecting Escherichia coli with bacteriophages ( E. coli ), and carry out the rescue as previously described (Wilkes et al., 2020).

[0212] Isolation of anti-LC nanobodies Monoclonal rescue of single colonies from phages in rounds 2 and 3 of panning, as previously described (Wilkes et al., 2020). Binding / pairing with three LCs (LEN, SMA, and REC, here) was assessed using ELISA assays (using standard protocols).

[0213] VH Fc fusion protein construct design, mammalian expression, and purification The selected VH sequence was synthesized and cloned into a mammalian Fc fusion protein expression plasmid (Twist Bioscience), and expressed using the Expi-293 expression system kit according to the manufacturer's instructions. Purification of the VH fusion protein was achieved using the PhyNexus MEA2 system with the protein A tip. The eluted fractions were combined, and the buffer was replaced with PBS using a Zeba Spin 7K MWCO column (Thermofisher).

[0214] VH antibody fragment expression and purification The selected VH coding region was cloned into pET11a(+), which carries an N-terminal His6 tag containing a TEV cleavage site. The VH construct was transformed into SHuffle cells (New England Bioscience) and grown at 37°C. Protein expression was induced for 16 h at 16°C by adding 0.5 mM IPTG. After harvesting, cells were lysed using a cell disruptor (Constant Systems), and the clarified lysate (in 20 mM Na2HPO4, 500 mM NaCl, 20 mM imidazole, pH 7.4) was loaded onto a 5 mL His-Trap FF column (GE Healthcare) and eluted with 250 mM imidazole for 5 column volumes. CO3 was further purified using a HiLoad 16 / 600 Superdex 200 pg gel filtration column (Cytiva) equilibrated in PBS.

[0215] VH-LC Interactions – Analytical Gel Filtration A 1:1 LC-VH complex (total concentration of 1 mg / mL, i.e., 0.5 mg / mL for each protein) was loaded via a superloop onto a HiLoad 10 / 300 Superdex 75 pg size size size size column. The column was pre-equilibrated with PBS, and chromatograms were recorded at a flow rate of 0.5 mL / min. Controls of LC-only and VH-only (500 µL of 0.5 mg / mL) were also recorded.

[0216] VH-LC binding dynamics—surface plasmon resonance (SPR) (heavy chain immobilization) Kinetic experiments were performed automatically using a Biacore system (Cytiva) in PBS at pH 7.4. Series S Sensor Chip NTAs (GE Healthcare) were prepared via a capture-coupling method for initial nickel injection and activated using a mixture of N-hydroxysuccinimide (NHS) and N-ethyl-N-(3-diethylaminopropyl)carbodiimide (EDC), followed by immobilization with 0.2 μM His-tagged VH (flow rate 30 μL / min; contact time 180 s) (according to the manufacturer's instructions). Control flow cells without immobilized ligands were prepared and used to correct for refractive index changes and nonspecific interactions in the experimental data. Each sensor chip contained one reference flow cell channel and three sample flow cells. To derive the binding kinetics of LEN, SMA, and REC, five-point, three-fold serial dilutions (range 1–0.012 µM) were prepared and sequentially injected into functional and reference flow cells (flow rate 5 μL / min; contact time 120 s). Dissociation was then performed with running buffer (flow rate 5 μL / min; contact time 80 s). A total of n=3 biological replicates (9 technical replicates) were recorded for SMA, and n=2 replicates (6 technical replicates) were recorded for LEN and REC. Light chains were injected at different concentrations into captured CO3: 0.012 µM, 0.037 µM, 0.11 µM, 0.33 µM, and 1 µM.

[0217] VH-LC binding kinetics via biolayer interferometry (light chain immobilization) The streptavidin-coated biosensor tip (ForteBio, Inc.) was pre-wetted with 0.05% Tween 20 PBS buffer for 1 minute to establish a baseline before LC immobilization. Biotinylated LC was loaded at a concentration of 500 nM and held for 5 minutes, then associated with various concentrations of VH1 (4 µM–31.3 nM for LEN and SMA, and 8 µM–250 nM for REC) until saturation at the highest concentration was reached. The sensor was then placed back into the baseline buffer for 5 minutes to detect dissociation.

[0218] Differential scanning fluorescence determination Thermal stability assays were performed using a StepOnePlus real-time PCR system (Applied Biosystems) within a temperature range of 25–95 °C using Sypro-Orange protein gel dye (Invitrogen). 10 µM of individual SMA and VH, and 20 µM of the SMA:VH complex, were prepared in PBS (20 mM Na₂HPO₄, 150 mM NaCl, pH 7.4). Data were processed using the Boltzmann equation to generate thermal transition curves. The melting temperature (Tm) corresponding to the unfolded equilibrium point was recorded as the V₅₀ value of the sigmoid melting curve.

[0219] Circular dichroism spectroscopy Protein samples were dialyzed into 20 mM Na₂HPO₄, 150 mM KF, pH 7.4 to overcome interference from salt ions at lower wavelengths. Proteins were analyzed at 0.05 mg / mL in the 180–260 nm range using a 1 mm path length cuvette on a J-100 series CD circular dichroism spectrometer (JASCO). The scan rate was set to 50 nm / min, the digital integration time (DIT) to 1 second, and a total of 3 cumulative scans were performed. Spectra were recorded at 2-degree intervals between 20–80 °C. Each temperature increment was held for 120 seconds before recording the spectra.

[0220] Light chain amyloid fibril formation and thioflavin T detection Each LC protein was incubated at 1 mg / mL (~80 μM) for 18 days at 37°C with agitation. Samples were taken at specific time points (every 2 days apart), flash-frozen in liquid nitrogen, and stored at -80°C. For analysis, these samples were diluted to 20 μM to occupy 50 μL per well in a clear-bottom / black-walled microplate (Greiner), and 1 µL of ThT solution was added to a final concentration of 20 μM. Three replicate wells were used for each LC at each time point. The contents of the plate were mixed, and ThT fluorescence was recorded using an excitation wavelength of 440 nm and an emission wavelength of 490 nm (Flexstation 3, Molecular devices). Each experiment was performed in triplicate.

[0221] Transmission electron microscopy The endpoint sample from the ThT experiment was analyzed by transmission electron microscopy. 5 μL was loaded onto a carbon-coated copper grid for 2 minutes, blotted dry, stained with 4% (w / v) uranyl acetate for 30 seconds, and then allowed to dry. Images were acquired using a 120 kV Tecnai G2 Spirit BioTWIN electron microscope (FEI) equipped with a SIS Megaview III camera.

[0222] crystallization SMA:VH complex crystals were grown in 48-well Hampton Research crystallization plates using a hanging drop method. Seeding was used to promote growth by mixing 1.5 µL of 8 mg / mL protein with 1.5 µL of a stock solution consisting of pH 5.5 Bis-Tris buffer and 20% PEG3350, and equilibrating above wells containing 100 µL of the stock solution. For pure SMA, crystals were grown at similar concentrations in 96-well crystallization plates using a seated drop vapor-phase diffusion method under JCSG+ F11 conditions (0.1 M HEPES buffer, pH 7.0, 1 M succinate, and 1% w / v PEG2000 MME). For SMA:VH complex crystals, they were transferred to a stock solution supplemented with 20% ethylene glycol, while for pure SMA, a 20% glycerol supplement was used before flash freezing in liquid nitrogen.

[0223] Data collection, refinement, and model building Data were collected using an Eiger2 XE 16M detector at the IO4 beamline (wavelength = 0.95373 Å) of the Diamond Source synchrotron. Data were processed using DIALS (Winter et al., 2018) and scaled using AIMLESS (Evans and Murshudov, 2013). Both structures were resolved using MOLREP (Vagin and Teplyakov, 1997) with the LEN structure (PDB ID: 1LVE) as the starting model and refined using REFMAC5 (Murshudov et al., 1997) in the CCP4i2 suite. Model building between refinement rounds was performed using COOT (Emsley and Cowtan, 2004). Protein structure images were created using Pymol. Crystal structures were analyzed using the PISA (Protein Interfaces, Surfaces, and Assemblies) server (https: / / www.ebi.ac.uk / msd-srv / prot_int / cgi-bin / piserver) (Krissinel and Henrick, 2007). Statistics on crystallographic data collection and refinement are shown in Table 2.

[0224] Table 2 - Data collection and refinement statistics of SMA homodimer and SMA:CO3 heterodimer (Note: Vh1 is an alternative name for CO3)

[0225]

[0226] Example 1 – Identification of Five Promising Candidates

[0227] Identification of promising conjugates Surprisingly, despite representing less than 10% of the initial naïve llama library, only routine VH-like sequences were identified during the panning process. After three rounds of screening, the hit rate was >90%, and the ELISA absorbance reading was >0.15. 141 clones (47 from each of the separate LEN, SMA, and RCE titration plates) were selected for sequencing and output analysis. Of these, only 11 conjugates were considered more promising (see Table 3; each name is based on the original panning target and plate-well number (e.g., LEN A01), however, for simplicity, they are generally referred to by their well number (e.g., A01)). Some of these eleven hits contained repetitive CDR3 sequences. This convergence indicated binding superiority, highlighting them as hits of particular interest. Selection criteria included: selecting representative sequences from each convergent CDR3 group, and selecting multiple sequences from those groups where differential binding patterns were observed.

[0228] Table 3 – CDR3 of the 11 VH proteins selected after screening

[0229]

[0230] The selected VH forms a stable complex with LC. Analytical gel filtration was performed to determine whether the interaction between the VH candidates identified during the initial screening and the LC was transient or led to the formation of a stable complex. For example, for CO3, an elution peak was observed at 15.8 mL. Figure 1 Despite being very similar in size, LEN eluted at approximately 18 mL. Figure 1 A), while SMA eluted at 16.9 mL ( Figure 1 B). When LEN or SMA was combined with CO3 in a 1:1 ratio, a strong elution peak was observed at 13.4 mL for both (see [link to relevant documentation]). Figure 1 These shifts in elution volume confirmed the formation of a stable CO3:LC complex, with almost no / no visible CO3 or LC peaks remaining in the sample, indicating that both were incorporated into the stable complex in a 1:1 ratio. LEN and SMA behaved as monomers at the concentration of 0.5 mg / mL used throughout the experiment, corresponding to their reported KD values ​​of approximately 40 μM and 70 μM, respectively (Stevens et al., 1995). In contrast, REC eluted at a volume of 13.4 mL ( Figure 1C indicates that its molecular weight is comparable to that of the dimerized complex, consistent with the low KD (approximately 0.2 μM) reported for REC (Stevens et al., 1995). Although dimerized at this concentration, REC is still able to form a stable complex with CO3, as indicated by the increased peak size at 13.4 mL and the decrease in peak size at 15.7 mL. Figure 1 C).

[0231] The binding of eleven hits was evaluated not only against their original target but also against two additional targets (Table 4). ELISA results showed that CO3, B04, H02, C10, D09, and D01 were able to bind (or pair) with all three light chain LCs. A03 and D04 were also able to bind, but appeared to be less efficient at binding SMA. The same was true for G02, although REC binding was less efficient in this case. C06 was shown not to bind REC, and D07 was specific for SMA. CO3 is of particular interest because its protein expression level was significantly lower than the other VHs (data not shown), yet it still exhibited very strong binding. The binding of some candidates to the three LCs suggests that these LCs share similar conformations, thus allowing pairing with a variety of VHs.

[0232] Table 4 – Binding profiles of 11 VH proteins as determined by ELISA – A630 readings for each well (streptavidin control excluded), along with the specified binding category (Y = yes, N = no, Y(-) = weak binding). The specified criteria are: values ​​<0.5 are considered no binding, 0.5–2 are weak binding, and >2 are strong binding.

[0233]

[0234] SPR was used to evaluate binding kinetics. CO3 was selected for this experiment. The sensor plot showed that the SPR signal increased proportionally with the injection of LC, indicating a specific interaction between the increased amount of LC and CO3. Figure 2 CO3 appears to bind to LEN and SMA with a higher affinity than to REC. To determine the approximate LC:CO3 binding kinetics, SPR data were analyzed using two different kinetic models: i) a 1:1 model and ii) a heterogeneous ligand model (respectively). Figure 2 i, ii (black lines). Based on this analysis, it becomes clear that the heterogeneous ligand model fits the experimental data better than the 1:1 model. Gel filtration data indicate that the interaction between LC and CO3 occurs via 1:1 binding, and the heterogeneity of ligand sites can be explained by the lack of controlled amine coupling with CO3.

[0235] The equilibrium constants (KD) calculated using Kd / Ka for 1:1 models of LEN, SMA, and REC are 5.6 nM, 16 nM, and 499 nM, respectively. These values, though approximate due to poorly fitted models, indicate high affinity binding of CO3 to LEN and SMA, and significantly lower binding to REC. This is further supported by the average Rmax values, which are 55.05 RU for REC, compared to 195.62 RU for LEN and 145.13 RU for SMA. The equilibrium constants for the heterogeneous model are: LEN:K D 1 = 1.19 nM and K D 2 = 26.6 nM, SMA:K D 1 = 131 nM, K D 2 = 17.8 nM, and REC: K D 1 = 14.2 µM, K D 2:382 nM. The equilibrium constants recorded for each LC describe two different K values. D This indicates that binding sites are not uniformly available in every case. This supports the hypothesis that the random orientation of CO3 is the cause of the epigenetic ligand model, resulting from partial blockage of binding sites. Alternatively, there may exist different pools of LC species with variable aggregation states / orientations, exhibiting different affinities for CO3.

[0236] Table 5 - Binding kinetics parameters of LEN, SMA, and REC with CO3 recorded by SPR, using 1:1 model and heterogeneous ligand model.

[0237]

[0238] 1 Association rate (Ka); 2 Dissociation rate (Kd); 3 Equilibrium dissociation constant (KD)

[0239] 4 Maximum total response (Rma); n = 6 repetitions; n=9 repetitions

[0240] Final choice Based on the accumulated data on eleven candidates, five conjugates (C03, C06, A03, D07, and D09) were ultimately selected for further consideration. This decision was based not only on their specific binding profiles but also on whether they consistently yielded reliable binding via both ELISA and BLI. For example, C03, A03, and D09 bound all three LCs with high affinity, while C06 and D07 appeared to show specificity for LEN / SMA and SMA, respectively. These pan-LC conjugates suggest a common / similar conformation among LEN, SMA, and REC. This is potentially beneficial because if this is confirmed as a stabilizing interaction, the binding could slow the development of SMA and REC into amyloid fibrils, showing promise for therapeutic applications.

[0241] VH vs. VHH As mentioned above, only VH candidates were identified in the VHH library used. This specific type of selection can be explained by the inherent framework-native interactions that can occur between the heavy and light chains. The typically hydrophobic interface within the FR2 region of VH proteins allows for natural pairing with VL (van der Linden et al., 1999). This region, between approximately amino acids 36-49, typically contains the sequence “WVRQAAGKGLEWV”, which can vary slightly due to conserved mutations (see [link to relevant documentation]). Figure 10 This sequence feature was observed in the eleven selected VHs. Conserved residues V37, G44, L45, and W47 tend to form tight associations with VLs via hydrophobic bonds within the intact IgG structure. Therefore, this binding may have a higher affinity than that achievable by the VHH clones present in the panning library. The VHH proteins lack these hydrophobic residues and have long, frame-stacking CDR3 loops. While these additional features do confer beneficial properties such as improved thermal stability and solubility, they prevent this type of frame interaction between VHs and VLs and provide further evidence why they were not selected during panning.

[0242] Conclusion of Example 1 141 clones were screened for binding, and 115 binding compounds were identified and sequenced. Eleven of these showed promising binding results. Subsequently, based on their specific binding profiles and the consistent and reliable binding results obtained by both ELISA and BLI, five more binding compounds were selected.

[0243] Example 2 – The VH can stabilize LC

[0244] result Stabilization studies have been conducted on CO3, one of the VH candidates. Differential scanning fluorescence and circular dichroism were used to investigate how the binding of VH candidates to LCs affects their thermal stability. Obtaining thermal transition curves allowed for the determination of the melting temperature using the equilibrium point between the folded and unfolded states. CD spectra at the initial (20°C) and final (80°C) temperatures confirmed the transition from a β-sheet structure to a random coil, indicating complete unfolding under all conditions. All LCs showed increased stability after forming a complex with CO3. Figure 3 The most significant thermal stability displacement >5.5℃ was observed in SMA (Surveyed Mathematics and Microscopy). Figure 3 A, 3B, Table 6). SMA and REC exhibit a single transition, occurring between 40-60℃ ( Figure 3 However, LEN appears to have undergone a two-phase unfolding step as observed by DSF (AD). Figure 3 E, i), demonstrated by multiple transformations. This biphase unfolding was also observed in the LEN-CO3 composite sample. To more effectively assess the effect of CO3 on the thermal stability of LEN, the first and second transformations of LEN and the LEN:CO3 composite were analyzed separately (E, i). Figure 3 (E, ii, and iii). Comparable Tm values ​​were obtained using both methods, showing an increase in LC stability in the presence of CO3 (Table 6). CD analysis of LEN showed a single transition, contrasting with the dual transition observed by DSF. CD measures changes in secondary structure upon unfolding, while DSF monitors the exposure of hydrophobic surfaces, suggesting that DSF may be more sensitive to local conformational changes that can differ in LEN compared to SMA and REC.

[0245] Conclusion of Example 2 The VH candidate (as shown with CO3) increased the stability of LEN, SMA, and REC. The most significant displacement was observed with the binding of CO3 to SMA. An increase in Tm at 8°C was observed, which appears to confer some degree of resistance to fibril formation, as detected by ThT measurement (see Example 3).

[0246] Table 6 - Tm values ​​calculated from DSF and CD data. Values ​​for individual LC and LC forming a complex with CO3 are presented, in °C, with standard error enclosed in parentheses (Note: VH1 is an alternative name for CO3).

[0247]

[0248] Example 3 – The VH inhibits SMA fibril formation

[0249] result Using CO3 as a model, the VH candidate was further investigated. It is known that SMA aggregates into fibrils during incubation at 37°C with agitation (Raffen et al., 1999). To investigate how the interaction between CO3 and SMA affects its aggregation tendency, thioflavin-T (ThT) assays were performed. Here, SMA and CO3 were incubated at ratios of 1:1, 2:1, and 10:1 for 18 days, with time points taken at two-day intervals. SMA showed a significant increase in ThT fluorescence signal after day 12, accompanied by observation of fibrils by transmission electron microscopy (TEM). Figure 4 AE). However, no increase in fluorescence was observed in the presence of CO3 at SMA:CO3 ratios of 1:1, 2:1, and 10:1, suggesting a reduction in fibril formation (AE). Figure 4 AC), which was confirmed by electron microscopy. Figure 4 Interestingly, CO3 alone, incubated at 1 mg / mL, showed an increase in ThT fluorescence after day 10. Figure 4 A) indicates that CO3 also aggregates at these concentrations, but does not aggregate at lower concentrations. Figure 4 B and 4C). For incubation with 1 mg / mL CO3 alone, fibrils were observed on day 18. Figure 4 I), but not observed at lower concentrations (data not shown).

[0250] Conclusion of Example 3 Significant reductions in fibril formation were observed through CO3 binding at SMA:VH1 concentrations of 1:1, 2:1, and 10:1. The very few fibrils observed by TEM after incubation in the presence of VH1 exhibited markedly altered morphologies compared to SMA alone, such as appearing as “broken” fibrils.

[0251] Example 4 – Further Characterization

[0252] Crystallography It was confirmed that CO3 binds / pairs with SMA to form a complex, in which analytical GF, DSF, and CD revealed that this interaction is both stable and highly affinity, promoting increased thermal stability. This data leads to the hypothesis that CO3 binds in a 1:1 ratio, disrupting the LC dimer and stabilizing the monomeric subunits. Using X-ray protein crystallography, we identified the structure of the CO3-SMA binding complex and were able to compare it with the newly resolved SMA dimer structure.

[0253] Structural characterization of SMA homodimer The crystal structure of a single SMA was resolved to a resolution of 2.2 Å. Figure 5The crystallographic data collection and refinement statistics are shown in Table 2. As expected, the SMA forms a dimer structure in which the two subunits are oriented in the same direction. This is referred to as a classical dimer, which is similar to the dimer LEN structure (PDB ID: 1LVE). Like other LCs, the SMA monomeric subunit consists of nine β-chains forming a 4:5 Greek key β-sandwich motif. This motif is stabilized by a disulfide bond (data not shown) formed between Cys23 and Cys94 residues, which holds the two β-sheets in this conformation.

[0254] PISA analysis of the SMA crystal structure identified 18 residues at the SMA dimer interface, forming 12 hydrogen bonds and 4 salt bridges. The interacting SMA residues, along with the identified hydrogen bonds and salt bridges, were mapped onto the structure of the dimer, as shown below. Figure 5 As shown. Here, the residues involved in the binding appear to be primarily located in the ring region, where interactions cluster towards the top and bottom of the interface. Gln44, Gln48, and Tyr93 on each SMA chain form a network of hydrogen bonds at the base of the interface. Figure 5 A). The bond distances range from 2.85 Å (between Gln44 and Gln44) to 3.67 Å (between Tyr93 and Gln44). The top region of the dimer interface is formed by a total of four salt bridges between residues Glu61 and His100 on each chain. Figure 5 B), and the two hydrogen bonds between Glu61 and Gln102 (B ...). Figure 5 B) is stable.

[0255] Structural characterization of the SMA:CO3 complex The crystal structure of the SMA:CO3 complex was resolved to a resolution of 1.18 Å. Figure 6 As predicted, the SMA:CO3 complex forms in a 1:1 ratio and adopts a dimer structure comparable to that determined for the SMA homodimer, whereby both the SMA and CO3 subunits are oriented in the same direction. CO3 appears to bind at a position very similar to that observed between the subunits in the native SMA dimer; the difference lies in a slight rotation of CO3 compared to the SMA. As in the homodimer, the heterodimer SMA monomer forms a β-sandwich motif, stabilized by an intramolecular disulfide bond formed between residues Cys23 and Cys94 (not shown in the image). Like the SMA, the CO3 monomer structure also consists of nine β-chains arranged in a 4:5 β-sandwich motif. In this case, disulfide bonds between the β-sheets form between residues Cys22 and Cys96 (not shown in the image).

[0256] PISA analysis of the SMA:CO3 crystal structure identified 24 SMA residues and 23 CO3 residues at the interface of the SMA:CO3 heterodimer, forming 9 hydrogen bonds and 3 salt bridges. The interacting SMA and CO3 residues, along with the identified hydrogen bonds and salt bridges, were mapped onto the structure of this dimer. Figure 6 Similar to SMA dimers, the residues involved in the binding appear to cluster towards the top and bottom of the interface. SMA residues Gln44 and Tyr93 again participate in hydrogen bonding towards the base of the interface, forming a network of bonds with CO3 residues Gln39 / Tyr95 and Gln39 / Lys43, respectively. Figure 6 A). The identified hydrogen bond distances range from 2.91 Å (between Gln44 and Gln39) to 3.71 Å (between Gln44 and Tyr95). The top region of the dimer interface is stabilized by a total of three salt bridges formed between Glu61 and His100 of SMA and Lys106 and Asp59 of CO3, respectively. Figure 6 B).

[0257] Comparison of the interfaces of SMA homodimer and SMA:CO3 heterodimer The preceding sections have described the crystal structures of SMA homodimers and SMA:CO3 heterodimers and provided in-depth insights into the residues responsible for stabilizing interfacial interactions. PISA analysis showed that the heterodimer complex interface was more favorable than the homodimer, with a complex formation significance score (CSS) of 1.000 compared to 0.829 for the homodimer. The increased likelihood of SMA forming a complex with CO3 is further supported by the calculated free energy gain (ΔiG) of both complexes (data not shown).

[0258] Although the heterodimer has a significantly increased number of SMA residues at the interface compared to the homodimer (18 SMA residues, 24), the homodimer was predicted to have more hydrogen bonds and salt bridges at the interface. Interestingly, this is not correlated with the obtained binding and stability data. The binding affinity of SMA:CO3, estimated by SPR, is an order of magnitude lower than the KD (approximately 70 µM) of the SMA dimer in the low nanomolar range, and an increase in the thermal stability of SMA >5.5 °C was observed after CO3 binding. Together with the PISA results described above, the increased affinity and stability of the SMA:CO3 complex can be further explained by the increased side-chain stacking observed at the interface compared to the SMA homodimer. Surface views of the homodimer highlight the clear separation of the subunits and the pocket of space between the two SMA subunits. In contrast, the heterodimer shows clear merging at the interface, indicating that the side chains are in close proximity and efficiently stacked. The bond distances between the homodimers and heterodimers do not provide an explanation for why the increased distance between the homodimer subunits was observed. Therefore, it is likely that additional residues present at or near the interface caused some degree of repulsion or steric hindrance.

[0259] VH1 LC binding kinetics To further evaluate the binding dynamics, biolayer interferometry (BLI) was used. For example... Figure 11 As shown, a proportional increase in signal was observed with the injection of CO3, confirming the specific interaction between the increased amount of CO3 and LC. CO3 appears to bind to LEN and SMA with a higher affinity than REC, as evidenced by the fact that higher concentrations of CO3 are required to achieve the same level of response when REC is immobilized, and by the observed increased dissociation. Figure 11 The results confirmed the 1:1 model (see Example 1). Kinetic parameters are reported in Table 7. Under the experimental setup, the KD values, given by Kd / Ka, for the 1:1 models of LEN, SMA, and REC were 0.64 µM, 1.69 µM, and 43.5 µM, respectively. These values ​​confirm the high affinity of CO3 for LEN and SMA, and lower affinity for REC. However, as in Example 1 / Figure 1 As shown in C, REC appears to exist as a dimer, which means that direct comparisons of Kd values ​​obtained with REC should be approached with caution, given the differences in monomer-dimer states between REC and SMA and LEN.

[0260] Table 7 The binding kinetic parameters of VH1 with LEN, SMA and REC, as well as the standard error of the mean (SEM), were recorded by BLI using a 1:1 model.

[0261]

[0262] 1 The rate of association (Ka); 2 Dissociation rate (Kd); 3 Equilibrium dissociation constant (KD)

[0263] Overall conclusion:

[0264] As described herein, inhibiting amyloid formation by stabilizing LC monomers is a promising potential therapeutic mechanism. Although there is uncertainty regarding the exact non-native LC structure that causes organ toxicity, it has been previously hypothesized that stabilizing native and correctly folded LCs leads to a reduction in misfolding and aggregation (Morgan et al., 2021). This kinetic stabilization pattern has been proven successful in the treatment of different types of amyloidosis, specifically TTR amyloidosis, where the stabilizing interaction between the drug tafamadis and TTR tetramers has been shown to be particularly effective in reducing aggregation and slowing disease progression (Bulawa et al., 2012). In the embodiments presented herein, the selective binding of CO3, particularly to the native states of SMA and LEN, likely increases the unfolding free energy barrier, resulting in the observed slowing of their unfolding and aggregation. The pharmacologically and kinetically stabilization described herein may prove beneficial in patients with AL amyloidosis because it will increase renal excretion of native LCs. The inventors have shown that substoichiometric concentrations of CO3 are sufficient to reduce LC aggregation. This strongly suggests that removing subspecies of LC that readily enhance seeding or elongation of LC fibrils is sufficient to prevent fibril formation. This explanation is consistent with the heterogeneous binding observed during SPR, indicating the existence of multiple LC species with different binding preferences for CO3.

[0265] In summary, this invention describes novel VH-based antibodies and their active fragments that can bind / pair and disrupt the formation of light chain-based fibrils. It has been shown that stabilizing the native structure of the light chain with VH can potentially prevent misfolding of LC, thereby halting the initiation of aggregation pathways characterized by fibril formation. SPR analysis confirmed that this interaction is of high affinity, with the equilibrium constant of the LC:CO3 complex estimated in the low nanomolar range, compared to approximately 200 nM for the native VH:VL interaction (Jäger and Plückthun, 1999), confirming an efficient panning process.

[0266] Therefore, these VH-based antibodies and their active fragments can be reliably used to treat conditions involving light chain-based fibrils, such as those described in this specification as a whole.

[0267] References

[0268] 1) Gertz, MA, 2018. Blood Cancer Journal; 8(5), p.44.

[0269] 2) Fotiou, D. et al., 2020. HemaSphere; 4(4), p.12.

[0270] 3) Merlini et al., 2013, haematologica, 98(10):1492-1495

[0271] 4) Gurung and Tingting, 2022, The American Journal of Medicine, 135:S38-S43

[0272] 5) Weiss, BM et al., 2016. Blood; 128(22), p.4448.

[0273] 6) Edwards, CV et al., 2021. Blood; 138(25), pp.2632–2641.

[0274] 7) Gertz, MA et al., 2022a. Blood; 140 (Supplement 1), pp. 1839–1840

[0275] 8) Broggini, L. et al., 2023. Journal of Molecular Biology; 435(24), p.168320

[0276] 9) Solomon, A., 1985. Methods Enzymol.: 116: 101-121

[0277] 10) Pras et al., 1968. J Clin Invest 47(4): 924-933.

[0278] 11) Stevens et al., 1995. Protein Sci 4(3):421-432.

[0279] 12) Rajkumar et al., 2005, Blood. 106(3):812-817

[0280] 13) Kabat et al., 1991. NIH publication No. 91-3242

[0281] 14) Chothia, C. and Lesk, AM, 1987, Journal of Molecular Biology; 196:901-917

[0282] 15) Angal et al., 1993, Mol Immunol, 30:105-108

[0283] 16) WO9109967

[0284] 17) Wilkes, J. et al., 2020. ACS Synthetic Biology; 9(10):2828–2839

[0285] 18) Winter, G. et al., 2018. Research papers Acta Cryst; 74:85–97.

[0286] 19) Evans, PR and Murshudov, GN, 2013. urn:issn:0907-4449. 69(7):1204–1214]

[0287] 20) Vagin, A. and Teplyakov, A, 1997. J. Appl. Cryst.; 30:1022–1025

[0288] 21) Murshudov, G. et al., 1997. Acta Crystallogr D Biol Crystallogr.; 53(3):240–255.

[0289] 22) Emsley, P. and Cowtan, K., 2004. Acta Crystallogr D BiolCrystallogr.; 60(12):2126–2132

[0290] 23) Krissinel, E. and Henrick, K., 2007. Journal of Molecular Biology;372(3):774–797

[0291] 24) Raffen, R. et al., 1999. Protein Science. 8(3):509–517.

[0292] 25) van der Linden et al., 1999

Claims

1. An antibody that specifically binds to a free light chain, such as a free κ light chain and / or (κ) amyloidogenic light chain, wherein the antibody comprises a heavy chain variable region, the heavy chain variable region comprising: i. CDR-H1 consisting of SEQ ID NO: 1, 4, 7, 10 or 13, CDR-H2 consisting of SEQ ID NO: 2, 5, 8, 11 or 14, and CDR-H3 consisting of SEQ ID NO: 3, 6, 9, 12 or 15; or ii. Combinations of CDR-H1, CDR-H2, and CDR-H3 sequences that have at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to any of the sequences listed in i.

2. The antibody of claim 1, wherein the antibody comprises a heavy chain variable region, the heavy chain variable region comprising: i. CDR-H1 consisting of SEQ ID NO: 1; CDR-H2 consisting of SEQ ID NO: 2; and CDR-H3 consisting of SEQ ID NO: 3; ii. CDR-H1 consisting of SEQ ID NO: 4; CDR-H2 consisting of SEQ ID NO: 5; and CDR-H3 consisting of SEQ ID NO: 6; iii. CDR-H1 consisting of SEQ ID NO: 7; CDR-H2 consisting of SEQ ID NO: 8; and CDR-H3 consisting of SEQ ID NO: 9; iv. CDR-H1 consisting of SEQ ID NO: 10; CDR-H2 consisting of SEQ ID NO: 11; and CDR-H3 consisting of SEQ ID NO: 12; v. CDR-H1 consisting of SEQ ID NO: 13; CDR-H2 consisting of SEQ ID NO: 14; and CDR-H3 consisting of SEQ ID NO: 15; or vi. and any of the sequences listed in any of i. to v. have at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to the CDR-H1, CDR-H2 and CDR-H3 sequences.

3. An antibody that specifically binds to a light chain, such as a κ light chain and / or (κ) amyloidogenic light chain, wherein the antibody has a heavy chain variable region comprising any one of SEQ ID NO: 16 to 26, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

4. The antibody according to any one of the preceding claims, wherein the antibody is selected from the group consisting of: i) an antibody moiety containing a VH domain but not a VL domain, or ii) a nanobody.

5. The antibody according to any one of the preceding claims, wherein the antibody is a nanobody.

6. The antibody according to any one of the preceding claims, wherein the antibody is chimeric or humanized.

7. The antibody according to any one of the preceding claims, wherein the antibody comprises an Fc domain selected from the group consisting of any one of SEQ ID NO: 48 to 52 or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it.

8. The antibody according to any one of the preceding claims, wherein the antibody neutralizes or inhibits the formation of light chain dimers and / or light chain fibrils.

9. The antibody according to any one of the preceding claims, wherein the antibody has an equilibrium dissociation constant (K0) of less than 500 nM for the light chain. D ).

10. An isolated polynucleotide encoding an antibody according to any one of claims 1 to 9.

11. A cloning vector or expression vector comprising the polynucleotide according to claim 10.

12. A host cell comprising the polynucleotide of claim 10 or the expression vector of claim 11.

13. A method for producing an antibody according to any one of claims 1 to 9, the method comprising culturing a host cell according to claim 12 under suitable conditions for producing the antibody, and isolating the antibody.

14. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 9 or a polynucleotide according to claim 10, and one or more pharmaceutically acceptable carriers, excipients and / or diluents.

15. The antibody according to any one of claims 1 to 9, the polynucleotide according to claim 10, or the pharmaceutical composition according to claim 14, for treatment and / or diagnosis.

16. An antibody according to any one of claims 1 to 9 and 15, a polynucleotide according to claim 10 or 15, or a pharmaceutical composition according to claims 14 and 15, for the treatment of a disease or condition selected from the group consisting of: light chain (AL) amyloidosis, light chain deposition disease (LCDD), light chain and heavy chain deposition disease (LHCDD), crystal storage histiocytosis, asthma, inflammatory diseases and autoimmune diseases (e.g., rheumatoid arthritis, Sjögren's syndrome, idiopathic urticaria, multiple sclerosis, diabetes, systemic lupus erythematosus, lupus nephritis and inflammatory bowel disease), and certain classes of cancers such as multiple myeloma (including λ myeloma and κ myeloma).

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