Method of sequence independent quantification of proteolytically labile plasma lambda free light chain proteins for AL amyloidosis diagnosis

Abstract

A method for combining restrictive proteolysis and immunoassays to specifically detect and quantify amyloid-genic XFLC in a biological fluid. This method is accomplished by the use of mAbs that enable the detection and quantification of neoepitopes on dLCCD biomarkers produced after restricted proteolysis of ZFLC, which is very advantageous for kinetically unstable or amyloid-genic LFLC. The methods do not depend on FLC primary sequences in variable and constant domains, and can be used to detect and quantify up to 99% of human XFLC. The clinical utility of the assay includes early detection in individuals suspected of having plasma cell disorders (MGUS, SMM, MM, IgM-AL and AL), differentiation from other amyloidosis such as ATTR, use in companion diagnosis, proving of target conjugation, assessment of treatment response, detection of MRD, detection of recurrence. Assays, such as MSD, lateral flow, mass spectrometry, are also implemented that quantify the resulting dLCCD biomarkers and / or can further increase assay sensitivity.

Description

[0001] This application claims priority to U.S. Provisional Application No. 63 / 486,587, filed February 23, 2023, U.S. Provisional Application No. 63 / 594,556, filed October 31, 2023, U.S. Provisional Application No. 63 / 607,763, filed December 8, 2023, which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates generally to the field of biomarkers and their use in healthcare. BACKGROUND

[0003] Amyloid light chain amyloidosis (AL) is a rare, progressive, and often fatal degenerative disease that affects the heart, kidneys, and liver. AL is caused by the abnormal clonal proliferation of plasma cells that produce excess amounts of amyloidogenic monoclonal free light chains (FLCs) and secrete them into the circulation. Of all AL patients, lambda FLCs are implicated in about 80% of cases. Amyloidogenic FLCs are kinetically unstable, leading to protein misfolding and the formation of pathogenic conformations including soluble aggregates and amyloid. Specific quantification of amyloidogenic FLCs can provide critical guidance in clinical practice, but is hindered by the “sequence diversity” of immunoglobulin light chains.

[0004] Immunoglobulin light and heavy chain proteins are produced by plasma cells, a type of white blood cell found in the bone marrow, in the process of making various immunoglobulins such as IgG, IgA, IgM, etc. In some cases, clonally proliferating plasma cells that are oncogenically transformed secrete excess amounts of amyloidogenic free light chains (FLCs) into the blood circulation. Hematologically, high levels of FLCs can be detected in the blood and urine, which leads to abnormal serum kappa / lambda FLC ratios. Amyloidogenic monoclonal FLCs differ from normal polyclonal FLCs produced as part of the adaptive immune response in terms of the amount of circulating FLCs and their amyloidogenicity. Amyloidogenic FLCs are less stable and prone to misfolding, proteolysis, and aggregation. Misfolded conformations (soluble aggregates and fibrils) as well as fragments of FLCs are considered toxic and can ultimately be deposited as amyloid on various organs. Organ toxicity manifests as cardiomyopathy, nephrotic syndrome, and / or end-stage renal failure, collectively classified as light chain amyloidosis (AL). Both amyloidogenic lambda and kappa FLCs can cause AL, although lambda FLCs are often identified as the responsible or implicated FLCs in AL. The estimated incidence of AL is 10 per million population worldwide.

[0005] Lambda FLC monomers are approximately 227 amino acids and are composed of an N-terminal variable domain (V L), a joining junction region (J), and a C-terminal constant domain (C L ) typically exists as a homodimer connected by an intermonomer disulfide bond at the C-terminal constant domain of the protein. Variations in FLC sequences are the result of incorporation of germline genes and somatic hypermutations. While each AL patient typically has a single clonal population of plasma cells, and thus one unique monoclonal amyloidogenic FLC sequence, it exists against the background of a highly diverse polyclonal FLC repertoire that exists in any human. This sequence diversity and lack of methods to distinguish amyloidogenic FLCs presents unique challenges to the diagnosis and targeted treatment of AL.

[0006] The current standard of care (SOC) for AL patients is similar to that for multiple myeloma, by targeting the proliferating clonal plasma cells, and typically employs a combination of anti-CD38 immunotherapy and a chemotherapy cocktail (often containing a proteasome inhibitor), or a chemotherapy cocktail alone, or, if possible, stem cell transplant. While therapeutic agents that reduce clonal plasma cell burden have improved overall survival of AL patients, there remains a significant unmet medical need in the clinical management of AL due to lack of early diagnosis, treatment resistance, persistent minimal residual disease (MRD), low organ response despite hematologic response, and disease relapse. It is believed that patients diagnosed and treated at an early stage of disease progression, especially prior to significant organ involvement, have a much higher chance of survival and even remission.

[0007] Because the signs and symptoms of AL are similar to those of other common diseases, AL is often diagnosed late or not until late. Clinical symptoms of specific organ involvement are the most common triggers to suspect the disease. Confirmation of AL diagnosis often takes 6 to 12 months, with 3 to 4 physician visits, and mortality at 6 months after AL diagnosis is close to 25%, with a median survival of about 4 years. Laboratory tests of blood and urine M-protein and FLC, such as immunofixation and FreeLite nephelometry assays (Binding Site. UK), are often used to assess FLC levels, dFLC levels (the difference between lambda and kappa FLC levels), and kappa / lambda ratios; followed by tissue biopsy, where the presence of amyloid is examined and sometimes the protein identity. Imaging tests such as echocardiogram and magnetic resonance imaging are also prescribed in the case of organ involvement. These tests are useful tools, but are still plagued by low sensitivity, lack of specificity, and inability to distinguish amyloidogenic FLCs from non-amyloidogenic FLCs. Recent advances in mass spectrometry-based clonal LC detection have significantly improved sensitivity, but are only available in a limited number of clinics, and are not suitable for quantification or indication of amyloidogenicity.

[0008] There is a large unmet need for a test that is sensitive, specific, easy to obtain, inexpensive, and can assess amyloidogenicity of monoclonal FLC. Such a test would enable early diagnosis of AL, as well as monitoring response to therapeutic agents and MRD. SUMMARY

[0009] One aspect of the application is a method of generating a novel biomarker and using a monoclonal antibody to detect a novel epitope on the biomarker formed by amyloidogenic lambda free light chain (λFLC) protein, the method comprising the steps of: performing a limited proteolysis of lambda free light chain (λFLC) protein by proteinase K, wherein the limited proteolysis generates a novel epitope exposed on a fragment of amyloidogenic λFLC protein, and wherein the resulting fragment is an intact light chain constant domain dimer and monomer ("LCCD" or "dLCCD" or "dLCCD biomarker" or "LCCD biomarker" or "LCCD fragment" or "LCCD domain"), wherein the dLCCD biomarker has a representative sequence as in (SEQ ID NO: 1) and can exist as a monomer or a dimer formed via disulfide bonding through the C-terminal cysteine, and wherein the novel epitope is xQP, representing the N-terminus of a particular cleavage product (LCCD or dLCCD) starting with the amino acid sequence xQPKA, wherein x is one of G (glycine), R (arginine), or S (serine); generating a monoclonal antibody to the novel epitope on the N-terminus of the LCCD fragment of λFLC, wherein the generated monoclonal antibody binds to the novel epitope xQP at the N-terminus of the peptide chain.

[0010] Another aspect of the application entails optimizing the proteolysis conditions for a target sample such that the LCCD biomarker dimer or monomer is specifically generated from amyloidogenic λFLC protein compared to other λ light chain containing proteins present in the target sample or in a different sample. The λFLC can be in the form of recombinant human FLC in a buffer or in the form of recombinant and / or endogenous λFLC in an ex vivo biological matrix such as plasma, serum, urine, etc. Other λ light chain containing proteins include any immunoglobulin (IgA, IgG, IgM, IgE, etc.) and non-amyloidogenic λFLC. And the optimization includes testing one or more of the following conditions: varying the source of proteinase K used, varying the concentration of proteinase K used, varying the time of incubation, varying the composition of the matrix containing the test subject, and varying the temperature of incubation. Specificity is achieved when the LCCD fragment is generated from a representative amyloidogenic λFLC such as WIL (SEQ.ID.No.: 2) at least twice the level of the fragment from a representative non-amyloidogenic λFLC such as JTO (SEQ.ID.No.: 3) under a particular condition.

[0011] In certain embodiments, the neoepitope is N-terminal GQP, GQPK, or GQPKA. In certain embodiments, the neoepitope is N-terminal RQP, RQPK, or RQPKA. In certain embodiments, the neoepitope is N-terminal SQP, SQPK, or SQPKA. In certain embodiments, the produced antibody (LCCD-G) binds to the neoepitope GQP, GQPK, or GQPKA. In certain embodiments, the produced antibody (LCCD-R) binds to the neoepitope RQP, RQPK, or RQPKA. In certain embodiments, the produced antibody (LCCD-S) binds to the neoepitope SQP, SQPK, or SQPKA. In certain embodiments, the LCCD fragment of lambda FLC is about 23 kDa fragment under non-denaturing conditions. In certain embodiments, the LCCD fragment of lambda FLC is about 11.5 kDa fragment under denaturing conditions. In certain embodiments, the restricted proteolysis by proteinase K comprises the steps of treating recombinant amyloidogenic lambda FLC in a matrix with optimized proteolysis conditions to produce the LCCD fragment. In certain embodiments, the restricted proteolysis by proteinase K comprises the steps of treating an ex vivo AL plasma sample with optimized proteolysis conditions to produce the LCCD fragment. In certain embodiments, the optimized proteolysis conditions are 0.02 μΜ to 30 μΜ proteinase K at 15 °C to 70 °C for 30 seconds to 20 hours. In particular embodiments, the conditions are for 1 minute to 2 hours.

[0012] Another aspect of the application is an antibody for detecting a neoepitope formed by amyloidogenic lambda free light chain (lambda FLC) protein, wherein the antibody is encoded by a nucleotide sequence encoding the heavy chain and light chain of LCCD-G as described herein.

[0013] Another aspect of the application is an antibody for detecting a neoepitope formed by amyloidogenic lambda free light chain (lambda FLC) protein, wherein the antibody is encoded by a nucleotide sequence encoding the heavy chain and light chain of LCCD-R as described herein.

[0014] Another aspect of the application is an antibody for detecting a neoepitope formed by amyloidogenic lambda free light chain (lambda FLC) protein, wherein the antibody is encoded by a nucleotide sequence encoding the heavy chain and light chain of LCCD-S as described herein.

[0015] It will be understood by one of ordinary skill in the art that, for any of the described nucleotide sequences of LCCD-G, LCCD-R, and LCCD-S, variations in the nucleotide sequence (which are changes in the genetic triplet code of the sequence, but do not change the encoded amino acid) are also encompassed within the present application.

[0016] Another aspect of the present application is a method of assessing target engagement or compound efficacy in the discovery of small molecule FLC stabilizers, the method comprising the steps of: performing a restricted proteolysis on proteins from a sample containing a lambda FLC protein, wherein the sample contains a recombinant lambda FLC protein or is an ex vivo biological sample, such as serum, plasma, or urine, and wherein the restricted proteolysis generates a neoepitope exposed on the N-terminus of the LCCD fragment of the lambda FLC protein; detecting the presence of the neoepitope on the LCCD fragment of the lambda FLC protein; and quantifying the amount of the LCCD fragment of the lambda FLC protein generated by the restricted proteolysis based on the detected presence of the neoepitope, wherein target engagement is assessed by comparing a first level of the LCCD fragment before a small molecule has been introduced into the test sample to a second level of the LCCD fragment after the small molecule has been introduced into the test sample, wherein a decrease in the level of the LCCD fragment or the LCCD biomarker normalized to total lambda FLC or dFLC in the second quantified level is indicative of target engagement.

[0017] Another aspect of the present application is a method of assessing target engagement of a FLC stabilizer for treating light chain amyloidosis, the method comprising the steps of: performing a restricted proteolysis on proteins from a biological sample, wherein the biological sample is from a subject receiving treatment by a targeted therapeutic molecule for light chain amyloidosis, and wherein the restricted proteolysis generates a neoepitope exposed on the N-terminus of the LCCD fragment of the lambda FLC protein; detecting the presence of the neoepitope on the LCCD fragment of the lambda FLC protein; and quantifying the amount of the LCCD fragment of the lambda FLC protein generated by the restricted proteolysis based on the detected presence of the neoepitope, wherein target engagement is assessed by comparing a first level of the LCCD fragment before the targeted therapeutic molecule has been introduced into the subject or ex vivo into a sample from the subject to a second level of the LCCD fragment after the targeted therapeutic molecule has been introduced into the subject or ex vivo into a sample from the subject, wherein a decrease in the level of the LCCD fragment or the LCCD fragment normalized to total lambda FLC or dFLC in the second quantified level is indicative of target engagement.

[0018] Another aspect of this application is a method for evaluating target binding of FLC stabilizers or pharmacological molecular chaperones for the treatment of light chain amyloidosis, the method comprising the steps of: restrictive proteolysis of a protein from a biological sample, wherein the biological sample is derived from a subject treated with a targeted therapeutic molecule against light chain amyloidosis, and wherein the restrictive proteolysis produces a novel epitope exposed on an LCCD fragment of a λFLC protein, and wherein the novel epitope is an N-terminal xQP, where x is one of G, R, or S; and detecting the presence of the LCCD fragment of the λFLC protein by binding of a monoclonal antibody to the novel epitope, wherein the monoclonal antibody... The binding to the novel epitope xQP; and the quantification of the level of the LCCD fragment generated by restriction proteolysis based on the signal detected by antibody binding, wherein target binding is assessed by comparing a first level of the amount of antibody binding to the novel epitope on the LCCD present before the targeted therapeutic molecule has been introduced into the subject or ex vivo into a sample from the subject with a second level of the amount of antibody binding to the novel epitope on the LCCD present after the targeted therapeutic molecule has been introduced into the subject or ex vivo into a sample from the subject, wherein a decrease in the LCCD level or the LCCD level normalized to total λFLC or dFLC at the second quantification level indicates target binding.

[0019] In some implementations, target binding of the therapeutic molecule is considered to have occurred when a first level of the LCCD fragment present before the therapeutic molecule has been introduced into the subject is greater than a second level of the LCCD fragment present after the therapeutic molecule has been introduced into the subject.

[0020] In some embodiments, target binding to the therapeutic molecule has not occurred when a first level of the LCCD fragment present before the therapeutic molecule has been introduced into the subject is equal to or less than a second level of the LCCD fragment present after the therapeutic molecule has been introduced into the subject. In some embodiments, the biological sample is one of plasma, urine, serum, cerebrospinal fluid (CSF), or a tissue biopsy section.

[0021] Another aspect of this application is a method for evaluating the therapeutic response of a therapeutic agent for treating light chain amyloidosis, which is carried out by quantifying the level of LCCD fragments in a biological sample subjected to restriction proteinase K hydrolysis at two or more different time points before and after the therapeutic molecule has been introduced into the subject or during treatment, wherein a decrease in the LCCD fragment level at the time point after treatment or later indicates a favorable response to the therapeutic molecule, regardless of the total level of λFLC.

[0022] Another aspect of this application is a method for assessing whether AL patients will benefit from a light chain stabilizer as a therapy by quantifying the level of LCCD fragments in biological samples from subjects believed to have received treatment with light chain stabilizer molecules targeting light chain amyloidosis. This method involves incubating an ex vivo plasma sample with either the stabilizer or a solvent control before subjecting the sample to restriction protein K hydrolysis, and quantifying the resulting LCCD level. A decrease in the LCCD biomarker level in the sample incubated with the stabilizer relative to the solvent control sample indicates that the subject may benefit from treatment with the light chain stabilizer.

[0023] Another aspect of this application is a method for detecting the presence of amyloidogenic λ free light chain (λFLC) protein in a subject, the method comprising the steps of: performing restrictive proteolysis on a protein from a biological sample, wherein the restrictive proteolysis produces a new epitope exposed on the N-terminus of an LCCD fragment of the λFLC protein; detecting the presence of the new epitope on the fragment of the λFLC protein; quantifying the level of the LCCD fragment of the λFLC protein by measuring the new epitope via an LCCD mAb; and comparing the LCCD fragment levels of the λFLC protein before and after restrictive proteolysis, wherein amyloidogenic λFLC protein is detected when the LCCD fragment level is higher after restrictive proteolysis.

[0024] Another aspect of this application is a method for detecting the presence of amyloid-derived λ free light chain (λFLC) protein in a subject, the method comprising the steps of: restrictive proteolysis of a protein from a biological sample by proteinase K, wherein the restrictive proteolysis produces a novel epitope exposed at the N-terminus of an LCCD fragment of the λFLC protein, and wherein the novel epitope is xQP, where x is one of G, R, or S; detecting the presence of the LCCD fragment of the λFLC protein by binding a monoclonal antibody to the novel epitope xQP. The LCCD fragment level is quantified by measuring the amount of antibody bound to the new epitope; and a first level of the amount of LCCD fragment present after restriction proteolysis in a biosample taken from a healthy subject without light chain amyloidosis (AL) is compared with a second level of the amount of LCCD fragment after restriction proteolysis in a biosample taken from a person suspected of having AL, wherein the first level is a reference level; and wherein amyloidogenic λFLC protein is detected when the second level of LCCD fragment after restriction proteolysis in the biosample is greater than the reference level. In some embodiments, mass spectrometry is used to detect and quantify the LCCD fragment as a 23 kDa homodimer or an 11.5 kDa monomer. In some embodiments, light chain amyloidosis is present in the subject when the level of LCCD fragment from λFLC is detected after restriction proteolysis by proteinase K and the level is elevated above the reference level. In some embodiments, the elevated LCCD level in the subject leads to a clinical decision, wherein the clinical decision is to treat and manage light chain amyloidosis (AL). In some implementations, the subject has one of the following: clinical suspicion of amyloidosis, multiple myeloma (MM), light chain amyloidosis (AL), smoldering multiple myeloma (SMM), IgM light chain amyloidosis, monoclonal gammopathy of undetermined significance (MGUS), minimal residual disease (MRD) in AL, and relapse of AL. In some implementations, the subject receives plasma cell therapy, and elevated LCCD levels are used to detect residual levels of amyloid-derived FLC, indicating minimal residual disease (MRD). In some implementations, elevated LCCD levels in subjects with stable disease indicate disease relapse. In some implementations, the biological sample is one of plasma, urine, serum, CSF, or tissue biopsy sections.

[0025] In some implementations, the absence of elevated LCCD levels in the subject leads to a clinical decision to rule out an AL diagnosis and receive treatment for other systemic amyloidosis, which may include thyroxine transporter amyloidosis (ATTR).

[0026] Another aspect of this application is a kit for detecting the presence of amyloid-derived λ free light chain (λFLC) protein in a subject, the kit comprising one or more of antibodies LCCD-G, LCCD-R, and LCCD-S; and instructions for use of the kit according to the method described herein.

[0027] In some embodiments, the kit includes a protein having a novel N-terminal epitope as a reference standard. In some embodiments, this protein is dLCCD. In some embodiments, the novel epitope is xQP, where x is one of G, R, or S. In some embodiments, the kit includes one or more antibodies targeting the novel epitope described herein. In some embodiments, the antibody is LCCD-G, LCCD-R, or LCCD-S. In some embodiments, the kit includes instructions for generating LCCD fragments from λFLC via restrictive proteolysis via proteinase K. Unless otherwise stated, the terms fragment LCCD and dLCCD are used interchangeably in this application. In some embodiments, the dLCCD level of a particular sample is a parameter calculated based on one or more readings of one or more dLCCD signals generated by said sample under restrictive proteolysis conditions. Attached Figure Description

[0028] Figure 1A An exemplary SDS-PAGE gel illustrating proteinase K (PK) digestion of recombinant λ light chains is shown, comprising recombinant amyloid-genic (based on sequences from AL patients) and non-amyloid-genic (based on sequences from normal healthy individuals) λFLC proteins generated from randomly selected 293F cells, compared with and without PK treatment. PK digestion results in proteolytic fragments producing homodimers corresponding to disulfide-linked LC constant domains (LC-C domains or dLCCD).

[0029] Figure 1B The LC structure is highlighted in the hinge region. PK cleaves in the hinge region between LC-V and LC-C, producing the dLCCD fragment. PK treatment exposes novel epitopes that are highly conserved across all LC sequences. Exposure depends on the dynamic / amyloid-genic nature of λFLC.

[0030] Figure 1C The λLC sequences surrounding the cleavage site indicate highly conserved PK cleavage sites exposing novel xQP epitopes. Based on analysis of public databases, the N-terminal sequences of dLCCD fragments after PK cleavage in AL λFLC occur at approximately: 84% GQP, 11% SQP, and 5% RQP.

[0031] Figure 2A and Figure 2BThe binding of LCCD-G mAb to LCCD, and the hydrolysis of IgG+PK in buffered protein in (a) full-length WIL or (b) buffered protein by restriction PK protein hydrolysis, demonstrate that 1) LCCD mAb specifically binds to the cleaved and exposed xQP epitope, rather than xQP in the protein sequence; and 2) the specific generation of the LCCD-G epitope occurs specifically in amyloid-derived FLC, but not in full-length immunoglobulin.

[0032] Figure 2C LCCD mAbs exhibiting high specificity for novel epitopes exposed by protein hydrolysis are shown. LCCD-G, LCCD-R, and LCCD-S mAbs (green signal, approximately 25 kDa) are used for specific detection of recombinant FLC proteins containing GQP, RQP, and SQP at the cleavage site after protein hydrolysis with proteinase K. Anti-λFLC polyclonal antibody (red signal, approximately 50 kDa) is used to indicate all full-length and LCCD fragments from the sample.

[0033] Figure 3A LCCD-G mAb assay of LC protein hydrolysis in 10 untreated AL plasma samples in Western blot.

[0034] Figure 3B This demonstrates the quantification of dLCCD fragment levels in pre-treatment AL patients (post-PK treatment) as determined by Western blot.

[0035] Figure 4 This study showed the levels of dLCCD fragments generated under restricted proteolysis, as determined by Western blot, in plasma of patients with AL (N=32), normal plasma (N=23), and patients diagnosed with multiple myeloma (MM) (N=19). Statistical analysis: one-way ANOVA. AL samples showed dLCCD signals ranging from 10x to 200x of normal values ​​under the same conditions in Western blotting and after restricted PK proteolysis (p=0.0007). AL samples also differed significantly from MM samples under restricted PK proteolysis, as quantified by LCCD mAb (p=0.0008).

[0036] Figure 5 shows the MSD dLCCD immunoassay. Figure 5A The level of dLCCDs generated under restrictive proteolysis was determined on the MesoScale Discovery (MSD) platform using a mixture of LCCD-X as the capture antibody. LCCD-X is an equimolar mixture of three LCCD mAbs: LCCD-G, LCCD-S, and LCCD-R. Figure 5BdLCCD levels differentiated λAL from patients with normal λAL (N=40), multiple myeloma (MM) (N=19), and TTR amyloidosis (ATTR, including cardiomyopathy and polyneuropathy phenotypes) (N=35). Recombinant dLCCD was used as a reference standard.

[0037] Figure 6 shows that baseline dLCCD levels are associated with overall survival in λAL patients. Figure 6A Log-rank survival test for patients with high (solid line) and low (shaded line) baseline dFLC levels, separated by a median of 18 mg / dL. P = 0.1474. Figure 6B Log-rank survival test for patients with high (shaded) and low (solid) baseline dLCCD levels, separated by a median level of 300 nM. P = 0.0479. dLCCD levels in untreated plasma samples from 50 confirmed λAL patients were quantified using the MSD LCCD immunoassay. dFLC levels in the same samples were determined by FreeLite assay. OS = 1 month. Note: The 300 nM median level is a coarse grouping method. This may not be the final cutoff value.

[0038] Figure 7 The concordance between dFLC and dLCCD predicts overall survival. Log-rank survival test compared patients with low baseline levels of both biomarkers (solid lines; downward sloping background shading in the lower figure) with patients with high levels of both biomarkers (dashed lines; dotted background shading in the lower figure) and patients with inconsistent dFLC and dLCCD (dotted-dashed lines; upward sloping shading in the background figure). High and low levels of dFLC and dLCCD were defined by the median baseline dFLC of 18 mg / dL and dLCCD of 300 nM. P = 0.0159. MSD LCCD immunoassay was performed on untreated plasma samples from 50 confirmed λAL patients. OS = 1 month. Note: The median level of 300 nM is a coarse grouping method. This may not be the final cutoff value.

[0039] Figure 8A Western blots of restricted proteolytic samples were displayed. Anti-λFLC monoclonal antibody (50 kDa band) and dLCCD mAb (23 kDa solid band) were used. Figure 8B Quantification of bands detected by antibodies on the LiCor system and fitting to a variable slope (four parameters) reaction curve using Prism. EC2 using two antibodies. 50S They are similar. Figure 8CThe protective effect of PTG-1412 against limiting proteolysis, quantified by dLCCD levels after proteolysis, in 13 ex vivo λAL plasma samples with and without PTG-1412. Detailed Implementation

[0040] Reference will be made in detail to certain aspects and exemplary embodiments of this application, with examples illustrated in the appended structures and figures. Various aspects of this application, including methods, materials, and embodiments, will be described in conjunction with exemplary embodiments. This description is non-limiting, and the scope of this application is intended to cover all equivalents, alternatives, and modifications generally known or incorporated herein. 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 application pertains. Those skilled in the art will recognize many techniques and materials similar to or equivalent to those described herein, which can be used in the practice of various aspects and embodiments of this application. The aspects and embodiments described in this application are not limited to the described methods and materials.

[0041] As used in this specification and the appended claims, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” include plural references.

[0042] A range herein may be expressed as from “about” a particular value and / or to “about” another particular value. When such a range is expressed, another embodiment includes from that one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation using the antecedent “about,” it should be understood that the particular value forms another embodiment. It should also be understood that each endpoint of a range is related to and independent of the other endpoint, both of which are important. It should also be understood that multiple values ​​are disclosed herein, and each value is also disclosed herein as “about” that particular value, in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It should also be understood that when a value is disclosed, “less than or equal to” the value, “greater than or equal to” the value, and possible ranges between values ​​are also disclosed, as properly understood by those skilled in the art. For example, if the value “10” is disclosed, then “less than or equal to 10” and “greater than or equal to 10” are also disclosed.

[0043] It should be understood that the exact concentration and units of the reagents can vary depending on the source of the enzyme reagents, and therefore customized methods can be used to optimize and achieve the same limiting protein hydrolysis results.

[0044] Unless otherwise defined, all terms used herein (including technical and scientific terms) shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that unless expressly defined herein, terms, such as those defined in common dictionaries, shall be interpreted as having the meaning consistent with their meaning in the context of the relevant technology and this disclosure, and shall not be interpreted in an idealized or overly formal sense.

[0045] In describing this invention, it should be understood that numerous techniques and steps are disclosed. Each of these techniques and steps has its own individual benefit, and each technique and step can be used in conjunction with one or more, or in some cases with, all other disclosed techniques. Therefore, for clarity, this specification will avoid unnecessarily repeating every possible combination of individual steps. However, upon reading the specification and claims, it should be understood that such combinations are fully within the scope of this invention and the claims.

[0046] In the following description, numerous specific details are set forth for purposes of explanation in order to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention may be practiced without these specific details.

[0047] Using restrictive proteolysis, this application describes a method for generating novel epitopes at the N-terminus of λFLC cleavage products and a monoclonal antibody (mAb) that specifically recognizes such epitopes. Under optimized proteolysis conditions, the generation of these novel epitopes is directly related to the lack of kinetic stability of amyloid-derived FLC, which can be corrected for by small molecule stabilizers binding to dimerized λFLC. The level of amyloid-derived FLC can also be reduced by other therapeutic interventions. Immunoassays using these mAbs, combined with restrictive proteolysis via proteinase K, have shown the ability to detect and quantify amyloid-derived λFLC, particularly in plasma samples from AL patients, regardless of the patient's λFLC sequence. The sequence-independent quantification of proteolytically unstable plasma λFLC described herein should provide a specific, sensitive, and low-cost diagnostic method for λAL amyloidosis, as well as a method for tracking trace residual disease and monitoring therapeutic responses. In addition to monoclonal antibodies, those skilled in the art will understand that the methods described herein can be performed using other means targeting specific epitopes, such as aptamers, nanobodies, etc. The use of monoclonal antibodies herein is not limiting of the invention.

[0048] Those skilled in the art will understand that the novel epitopes described herein can be recognized by other epitope recognition agents, such as, but not limited to, nanobodies, aptamers, and affinity molecules. Those skilled in the art will also understand that methods for detecting novel epitopes or biomarkers as described herein are not limiting of the invention and may include, but are not limited to, immunoassays (e.g., ELISA, Simoa, Olink), lateral flow assays, mass spectrometry, etc.

[0049] Definitions

[0050] As used herein, the term "antibody" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen via one or more immunoglobulin variable regions. An antibody can be a complete antibody, its antigen-binding fragment, or a single chain. The term "antibody" encompasses a wide range of polypeptides that can be distinguished biochemically. Those skilled in the art will understand that heavy chains are classified as alpha, delta, epsilon, gamma, and mu, or α, δ, ε, γ, and μ, among which there are several subclasses (e.g., γ1 to γ4). The nature of this chain determines the "class" of the antibody as IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, etc., have been well characterized and are known to confer functional specialization. Given this disclosure, modifications of each of these classes and isotypes are readily identifiable to those skilled in the art and are therefore all within the scope of this disclosure. All immunoglobulin classes are within the scope of this disclosure, and the following discussion will broadly refer to immunoglobulin molecules of the IgG class.

[0051] The antibodies or antibody antagonists of this application may include, but are not limited to, polyclonal, monoclonal, multispecific, bispecific, trispecific, human, humanized, primate-derived, chimeric, and single-chain antibodies. The antibodies disclosed herein may be derived from any animal source, including birds and mammals. In one embodiment, the antibody is a human, mouse, rat, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibody. In some embodiments, the variable region may be derived from cartilaginous fish (e.g., from sharks).

[0052] The term "antibody fragment" or "antigen-binding fragment" is used to refer to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, single-chain Fv (scFv), single-chain antibody, disulfide-linked Fv (sdFv), fragments containing VL or VH domains, fragments generated from Fab expression libraries, and anti-idiotypic (anti-Id) antibodies. Regardless of structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes DART and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein containing the variable region of an immunoglobulin, which acts like an antibody by binding to a specific antigen to form a complex. "Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable region of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In some aspects, said region is linked to a short linker peptide of 10 to about 25 amino acids. The linker can be enriched with glycine for flexibility and serine or threonine for solubility, and can link the N-terminus of VH to the C-terminus of VL, and vice versa. Despite the removal of the constant region and the introduction of the linker, the protein retains the specificity of the original immunoglobulin. Regarding IgG, the standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with molecular weights ranging from 53,000 to 70,000. The four chains are typically linked in a "Y" configuration by disulfide bonds, with the light chain starting at the opening of the "Y" and continuing to enclose the heavy chain together through the variable region.

[0053] Both the light and heavy chains are divided into structurally and functionally homologous regions. The terms "constant" and "variable" are used functionally. In this respect, it should be understood that the variable domains of both the light chain (VL) and heavy chain (VH) portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of constant domains in conventional antibodies increases as they move further away from the antibody's antigen-binding site or N-terminus. In conventional antibodies, the N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CL domains actually contain the carboxyl terms of the heavy and light chains, respectively.

[0054] As used herein, a “variant” of an antibody, antibody fragment, or antibody domain means (1) sharing at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the original antibody, antibody fragment, or antibody domain, and (2) specifically binding to the same target to which the original antibody, antibody fragment, or antibody domain specifically binds. It should be understood that when the measure of sequence identity is presented in the form of the phrase “at least x% identical” or “at least x% identity,” such embodiments include any and all integer percentages equal to or higher than the lower limit.

[0055] Furthermore, it should be understood that when an amino acid sequence is presented in this application, it should be interpreted as additionally disclosing or covering "variant" amino acid sequences that have sequence identity with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid sequence.

[0056] It should be understood that when this document presents a range of sequence homology, such as in, for example, the phrase “about 80% to about 100%”, such an implementation includes any and all subranges therein, where the lower number can be any integer between 80 and 100.

[0057] As used herein, the phrase “humanized antibody” refers to an antibody derived from a non-human antibody (typically a mouse monoclonal antibody). Alternatively, a humanized antibody may be derived from a chimeric antibody that retains or substantially retains the antigen-binding properties of the parental non-human antibody, but exhibits reduced immunogenicity compared to the parental antibody when administered to humans.

[0058] As used herein, the phrase "chimeric antibody" refers to an antibody in which the immunoreactive region or site is derived from or derived from a first species, and the constant region (which may be complete, partial, or modified according to this disclosure) is derived from a second species. In some embodiments, the target binding region or site will be derived from a non-human source (e.g., mouse or primate), and the constant region is human.

[0059] The range of antibodies mentioned in this article includes various compositions and methods, including asymmetric IgG-like antibodies (e.g., triomab / quadroma, Trion Pharma / Fresenius Biotech); mortar and pestle antibodies (Genentech); CrossMAb (Roche); electrostatically matched antibodies (AMGEN); LUZ-Y (Genentech); chain exchange engineered domain (SEED) antibodies (EMD Serono; Biolonic, Merus); Fab exchange antibodies (Genmab); symmetric IgG-like antibodies (e.g., dual-targeting (DT)-Ig (GSK / Domantis); dual-antibody (Genentech); cross-linked mAb (Karmanos Cancer Center), mAb2 (F-star); Cov X-body (Cov X / Pfizer); dual variable domain (DVD)-Ig fusion (Abbott); IgG-like bispecific antibodies (Eli Lilly); Ts2Ab (Medimmune / AZ); BsAb (ZymoGenetics); HERCULES (BiogenIdec, TvAb, Roche); scFv / Fc fusion; SCORPION (Emergent BioSolutions / Trubion, ZymoGenetics / BMS); Fc-DART (bi-affinity retargeting technology), MacroGenics; Bis(scFv)2-Fab (National Research Center for Antibody Medicine); F(ab)2 fusion (Medarex / AMGEN); Dual-acting or Bis-Fab (Genentech); Dock-and-Lock (DNL, ImmunoMedics); Fab-Fv (UCB-Celltech); Antibodies based on scFv and biantibodies (e.g., bispecific T-cell connectives (BiTE, Micromet)); Tandem biantibodies (Tandab, Affimed); DART (MacroGenics); Single-chain biantibodies; TCR-like antibodies (AIT, Receptor Logics); Human serum albumin scFv fusion (Merrimack); COMBODIES (Epigen Biotech); and IgG / non-IgG fusions (e.g., immune cytokines (EMDSerono, Philogen, ImmunGene, ImmunoMedics).

[0060] The term "specific binding" or "specific to" generally refers to an antibody binding to an epitope via its antigen-binding domain, and this binding requires a certain complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to the epitope more easily via its antigen-binding domain than to a random, unrelated epitope. The term "specific" is used herein to define the relative affinity of a particular antibody to a particular epitope. For example, antibody "A" may be considered to have higher specificity to a given epitope than antibody "B," or antibody "A" may be considered to bind to epitope "C" with higher specificity than to its associated epitope "D." In some embodiments, if the antibody or antibody fragment forms a 10-1 affinity with the antigen... -6 M or smaller, 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M or smaller or 10 -10 For complexes with a dissociation constant (Kd) of M or smaller, the antibody or antibody fragment is "specific" to that antigen.

[0061] Methods of generating monoclonal antibodies for detecting neoepitopes formed by amyloidogenic lambda free light chain (λFLC) proteins Methods of detecting the presence of amyloidogenic lambda free light chain (λFLC) proteins in a subject

[0062] One aspect of this application is a method for generating a monoclonal antibody for detecting novel epitopes formed from amyloid-derived λ free light chain (λFLC) protein, the method comprising the steps of: (a) hydrolyzing a sample containing λ free light chain (λFLC) protein with proteinase K, wherein the protein hydrolysis generates a novel epitope at the N-terminus of a fragment of about 23 kDa exposed to at least a portion of the constant domain of λFLC, or, when λFLC is a monomer, generating an N-terminus of a fragment of about 11.5 kDa exposed to at least a portion of the constant domain of λFLC protein. (a) A novel epitope on the terminal; and wherein the novel epitope is xQP, where x is one of G, R, or S (G = glycine, R = arginine, S = serine); (b) generating a monoclonal antibody against the novel epitope on the λFLC fragment, wherein the generated monoclonal antibody (LCCD) specifically binds to the novel epitope xQP, and wherein the proteolysis conditions are optimized such that the intensity of the LCCD signal obtained from an approximately 23 kDa fragment or an approximately 11.5 kDa fragment after proteolysis from amyloid-derived λFLC is at least twice that of non-amyloid-derived λFLC. Those skilled in the art will understand that the method for determining the LCCD signal is not limiting.

[0063] In some implementations, the new tabletop is GQP. In some implementations, the new tabletop is RQP. In some implementations, the new tabletop is SQP.

[0064] In some embodiments, the generated antibody (LCCD or dLCCD-G) binds to the novel epitope GQP. In some embodiments, the generated antibody (LCCD-R or dLCCD-R) binds to the novel epitope RQP. In some embodiments, the generated antibody (LCCD-S or dLCCD-S) binds to the novel epitope SQP.

[0065] In some embodiments, the λFLC fragment is a homodimeric fragment, which is approximately 23 kDa under non-denaturing conditions. In some embodiments, the λFLC fragment is a monomeric fragment, which is approximately 11.5 kDa under denaturing conditions.

[0066] In some embodiments, the restricted proteolysis via proteinase K includes the following steps: treating a liquid sample with optimized proteolysis conditions to generate LCCD domains. In some embodiments, the optimized proteolysis conditions are dependent on the specific activity of the protease, with 0.02 μM to 20.0 μM proteinase K applied at 37°C for 30 seconds to 20 hours. Those skilled in the art will understand that the specific activity of the protease can be determined independently and is not limiting.

[0067] One aspect of this application is an antibody for detecting novel epitopes formed by amyloid-derived λ free light chain (λFLC) protein, wherein the antibody comprises a heavy chain (HC3) encoded by the nucleotide sequence of LCCD-G SEQ.ID.No.: 4 and a light chain (LC7) encoded by the nucleotide sequence of LCCD-G SEQ.ID.No.: 5.

[0068] One aspect of this application is an antibody for detecting novel epitopes formed by amyloid-derived λ free light chain (λFLC) protein, wherein the antibody comprises a heavy chain (HC2) encoded by the nucleotide sequence of LCCD-R SEQ.ID.No.: 6 and a light chain (LC9) encoded by the nucleotide sequence of LCCD-R SEQ ID No.: 7.

[0069] One aspect of this application is an antibody for detecting novel epitopes formed by amyloid-derived λ free light chain (λFLC) protein, wherein the antibody comprises a heavy chain (HC2) encoded by the nucleotide sequence of LCCD-S SEQ.ID.No.: 8 and a light chain (LC5) encoded by the nucleotide sequence of LCCD-S SEQ ID No: 9.

[0070] One aspect of this application is a novel epitope on λFLC prepared by restrictive proteolysis of amyloid-derived λ free light chain (λFLC) protein using proteinase K. In some embodiments, the novel epitope is xQP, where x is one of G, R, or S. In some embodiments, an antibody against the novel epitope described herein is generated. In some embodiments, the antibody is LCCD-G, LCCD-R, or LCCD-S.

[0071] One aspect of this application is a method for optimizing the limiting proteolysis conditions using proteinase K, the method comprising the steps of optimizing one or more of the following factors: (1) the source of proteinase K; (2) the composition of the test sample; (3) the concentration of proteinase K; (4) the time of the proteolysis reaction; and (5) the temperature of the proteolysis reaction; wherein the proteolysis products of LCCD fragments are obtained differentially from amyloid-derived λFLC and non-amyloid-derived λFLC.

[0072] Key features achieved by the immunoassays described herein include: (1) biomarkers associated with the underlying cause of AL amyloidosis (i.e., unstable variant LC protein associated with amyloidogenicity); (2) applicability to most (if not all) AL variants (λFLC AL as the current focus); (3) ease of use with biofluids (serum, plasma, and urine) for immunoassays; (4) sensitive, specific, and quantitative assays; (5) early diagnosis or screening based on amyloidogenicity of each λFLC variant (personalized medicine), regardless of the FLC primary sequence or the exact λFLC level; (6) assessment of treatment response; and (7) tracking MRD and predicting relapse.

[0073] To develop diagnostic methods capable of achieving the aforementioned desired characteristics, a key principle in comparing amyloid-derived FLC with non-amyloid-derived FLC is that amyloid-derived FLC proteins are thermodynamically and / or kinetically less stable, making them more prone to protein misfolding, proteolysis, and aggregation. With increased stress, amyloid-derived FLC readily produces LCCD fragments and exposes novel, detectable cryptic epitopes, including those detected by antibodies. Therefore, this application discloses specific stress (i.e., restrictive proteolysis via proteinase K) to expose such novel epitopes on LCCD fragments in plasma samples from AL patients and mAbs specific for these novel epitopes.

[0074] Considering that proteinase K from different sources may have different enzyme activities, those skilled in the art can optimize the restriction protein hydrolysis by changing the concentration, specific activity, matrix containing the test subject, concentration of the test subject, reaction temperature, and reaction time, so as to achieve the condition that the LCCD domain is mainly generated in the AL sample relative to the normal or MM sample.

[0075] In addition to AL diagnosis, this assay is expected to detect residual amyloid-derived FLC after treatment and at the onset of relapse due to its sensitivity.

[0076] Methods of assessing target engagement of FLC stabilizers or therapeutic response of light chain amyloidosis

[0077] One aspect of this application is a method for detecting the presence of amyloidogenic λ free light chain (λFLC) protein in a subject, the method comprising the steps of: performing restrictive proteolysis on a protein from a biological sample, wherein the restrictive proteolysis produces a new epitope exposed on an LCCD fragment of the λFLC protein; detecting the presence of the new epitope at the N-terminus of the LCCD fragment of the λFLC protein; quantifying the presence of the LCCD fragment from the λFLC protein based on measuring the level of the new epitope on the fragment using LCCD mAb; and comparing the levels of the LCCD fragment before and after restrictive proteolysis, wherein amyloidogenic λFLC protein is detected when the level of the LCCD fragment from the λFLC protein is higher after restrictive proteolysis. Those skilled in the art will understand that the method for determining the level (e.g., amount) of the LCCD fragment is not limiting.

[0078] In some embodiments, the method further includes the steps of: measuring a first level of the LCCD fragment of λFLC protein present after restriction proteolysis in a biological sample taken from a healthy subject without light chain amyloidosis (AL), wherein the first level is a reference level; measuring a second level of the fragment of λFLC protein present after restriction proteolysis in a biological sample from a person suspected of having AL; and wherein amyloidogenic λFLC protein is detected when the second level of the LCCD fragment of λFLC protein after restriction proteolysis of the biological sample is greater than the reference level.

[0079] In some implementations, light chain amyloidosis is present in a subject when the level of LCCD fragments from λFLC is detected after restriction proteolysis by proteinase K and the level is elevated above a reference level.

[0080] In some implementations, elevated LCCD fragment levels in subjects lead to clinical decisions regarding the treatment and management of light chain amyloidosis (AL).

[0081] In some implementations, the absence of elevated LCCD fragment levels in the subject leads to a clinical decision to rule out an AL diagnosis and receive treatment for other systemic amyloidosis, which may include thyroxine transporter amyloidosis (ATTR).

[0082] In some implementations, the LCCD fragment is a 23 kDa homodimer and is detected and quantified using mass spectrometry.

[0083] In some implementations, the LCCD fragment is an 11.5 kDa monomer and is detected and quantified using mass spectrometry.

[0084] In some implementations, the subject has one of the following: clinical suspicion of amyloidosis, monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM), light chain amyloidosis (AL), smoldering multiple myeloma (SMM), IgM light chain amyloidosis, minimal residual disease (MRD) of AL, and relapse of AL.

[0085] In some implementations, the biological sample is one of plasma, urine, serum, CSF, or tissue biopsy section.

[0086] One aspect of this application is a kit for detecting the presence of amyloid-derived λ free light chain (λFLC) protein in a subject, the kit comprising: antibodies LCCD-G, LCCD-R, and LCCD-S; and instructions for use of the kit according to the methods described herein.

[0087] In some implementations, the method for detecting the presence of amyloid-derived λ free light chain (λFLC) protein in a subject as described herein further includes obtaining one or more readings from a dLCCD signal obtained by measuring the level of a new epitope on the fragment using an LCCD mAb.

[0088] In some implementations, the method for detecting the presence of amyloid-derived λ free light chain (λFLC) protein in a subject, as described herein, further includes obtaining a reading of the dLCCD signal using an algorithm in which the dLCCD level is a component.

[0089] Methods for detecting LCCD fragments generated by the hydrolysis of restriction PK proteins may include mass spectrometry, which requires taking into account a finite number of sequence variations in the constant domains of λFLC.

[0090] Other methods may include immunoassays using antibodies that recognize LCCD fragments. For example, gel electrophoresis combined with Western blotting using anti-λFLC antibodies. All commercial anti-λFLC antibodies (>20) tested in this study recognized not only λFLC but also the λ light chain associated with immunoglobulins. To address this issue, as reported herein, this study developed a rabbit mAb that specifically recognizes one of three unique xQP epitopes at the N-terminus of LCCD fragments exposed by restricted PK protein hydrolysis. This application demonstrates that immunoassays based on LCCD-G mAbs (or, in other embodiments, combinations of LCCD-S and LCCD-R mAbs or LCCD mAbs) can sensitively and specifically detect and quantify proteolytically hydrolyzed LCCD domains and serve as an indicator of the kinetic instability of the λFLC protein.

[0091] The LCCD rabbit mAb described herein can be used alone or in a mixture of two or three mAbs for determination. In one embodiment, an equimolar mixture of three mAbs is prepared and used, and this mixture is referred to as LCCD-X.

[0092] The approximately 23 kDa LCCD or dLCCD fragment is a disulfide-linked homodimer. Under reducing conditions, the LCCD exists as a monomer of approximately 11.5 kDa (SEQ ID NO: 2), but still contains an N-terminal xQP epitope. This monomer can also be used for the purposes contemplated in this application.

[0093] Those skilled in the art will understand that variants of the constant domain of the λFLC protein may include novel epitopes; this application also covers antibodies or antibody fragments that identify novel epitopes when they are part of a variant of the constant domain of the λFLC protein.

[0094] Solution-based LCCD immunoassays, such as ELISA, MSD, Simoa, and Olink, can provide higher sensitivity than Western blotting methods. This improved sensitivity is particularly valuable when applied to the early diagnosis and subsequent trace residual disease of AL, where total λFLC levels are much lower.

[0095] By combining restricted PK protein hydrolysis with specific detection and quantification of LCCD fragments, this application greatly simplifies the detection of amyloid-derived λFLC. Furthermore, the ease of using LCCD immunoassays with biofluids such as plasma, serum, and urine samples allows for immediate use of such assays, further expanding their utility.

[0096] LCCD immunoassay can be used to demonstrate target binding for treatments targeting stable amyloid-derived λFLC (as described in, but not limited to, WO2022 / 226166). This method can be used as a companion diagnostic to select AL patients most likely to respond to LC stabilizers in clinical settings, thereby enabling personalized medicine.

[0097] LCCD signals can also be used in combination with other clinical biomarkers and assessments, such as λFLC levels, dFLC, troponin, and NT-proBNP, to guide clinical practice. Levels of LCCD biomarkers measured using ex vivo samples from patients can also be used to assess the prognosis of AL disease. Due to the pathogenic nature of LCCD biomarkers, higher LCCD levels can indicate poorer clinical outcomes.

[0098] The method combining restrictive proteolysis of λFLC and detection of the resulting LCCD fragments can also be used in drug discovery to identify molecules that can affect the stability of λFLC and to find effective drug candidates.

[0099] Other biological samples containing λFLC, such as urine and bone marrow biopsy sections, can also be used in combination with restriction proteinase K protein hydrolysis and LCCD immunoassay or mass spectrometry methods for the diagnosis and treatment response assessment of AL.

[0100] Other reagents, such as aptamers and nanobodies, can also be generated that specifically bind to novel xQP epitopes exposed at the N-terminus and used for the detection and quantification of LCCD biomarkers.

[0101] Clinical utility of limited proteolysis in combination with LCCD mAb

[0102] One aspect of this application is to evaluate target binding of an FLC stabilizer for the treatment of light chain amyloidosis, comprising the steps of: restrictive proteolysis of a protein from a biological sample, wherein the biological sample is a recombinant protein or from a subject treated with a targeted therapeutic molecule against light chain amyloidosis, and wherein the restrictive proteolysis produces a novel epitope exposed on an LCCD fragment of a λFLC protein; detecting the presence of the novel epitope at the N-terminus of the LCCD fragment of the λFLC protein; and quantifying the level of the λFLC protein fragment produced by the restrictive proteolysis based on the detected presence of the novel epitope, wherein target binding is evaluated by comparing a first level of the amount of LCCD fragment present before the targeted therapeutic molecule has been introduced into the subject or ex vivo into a sample from the subject with a second level of the amount of LCCD fragment present after the targeted therapeutic molecule has been introduced into the subject or ex vivo into a sample from the subject, wherein a decrease in the LCCD level or the LCCD level normalized to total λFLC at the second quantification level indicates target binding.

[0103] One aspect of this application is a method for assessing the therapeutic response to a therapeutic agent for treating light chain amyloidosis by quantifying LCCD fragments in a plasma sample at two different time points, before and after the therapeutic molecule has been introduced into the subject or during treatment. The decrease in LCCD levels after treatment or at the second time point indicates a favorable response to the therapeutic molecule, independent of the total λFLC level. The therapeutic agent includes, but is not limited to, therapeutic agents as light chain stabilizers, plasma cell-targeted therapies, gene and cell therapies, stem cell transplants, and chemotherapeutic agents. Assessment based on dLCCD biomarkers can be performed once or multiple times after the therapeutic treatment.

[0104] One aspect of this application is a method for assessing whether AL patients will benefit from a light chain stabilizer as a therapy by quantifying LCCD fragments in plasma samples from subjects believed to have received treatment with light chain stabilizer molecules targeting light chain amyloidosis, wherein the ex vivo plasma sample is incubated with the stabilizer or a solvent control before subjecting the sample to restrictive proteolysis, and the resulting LCCD level is quantified, wherein a decrease in the LCCD level in the sample incubated with the stabilizer relative to the solvent control sample indicates that the subject may benefit from treatment with the light chain stabilizer.

[0105] In some implementations, the targeted therapeutic molecule has achieved target binding or a therapeutic response when a first level of the LCCD fragment present before the targeted therapeutic molecule has been introduced into the subject is greater than a second level of the LCCD fragment present after the targeted therapeutic molecule has been introduced into the subject.

[0106] In some embodiments, target binding or therapeutic response has not occurred when a first level of the LCCD fragment present before the targeted therapeutic molecule has been introduced into the subject is equal to or less than a second level of the LCCD fragment present after the targeted therapeutic molecule has been introduced into the subject. In some embodiments, the biological sample is one of plasma, urine, serum, cerebrospinal fluid (CSF), or a tissue biopsy section.

[0107] Beyond their important utility in AL diagnosis and target binding monitoring, xQP mAbs also have other general uses in biotechnology. xQPs can be the smallest known uncharged N-terminal protein tags. N-terminal or C-terminal tags are widely used in recombinant proteins and cellular systems for target protein detection and / or purification. Antibodies bound to these tags are important tools for studying target proteins and protein complexes. Commonly used tags include His tags, Flag tags, HA tags, and myc tags, all containing 6 to 10 charged amino acids. The three novel mAbs discussed in this application (LCCD-G, LCCD-S, and LCCD-R) specifically bind to small N-terminal tags of 1 to 5 amino acids, with GQP and SQP being uncharged. In some respects, such small tags (epitopes) can be genetically engineered to be placed at the N-terminus of target proteins to enhance this utility.

[0108] Examples

[0109] In some embodiments, the methods and compositions described herein can be used to diagnose light chain amyloidosis (AL) in patients with plasma cell disorders suspected of having multiple myeloma (MM), light chain amyloidosis (AL), smoking multiple myeloma (SMM), monoclonal gammaglobulinosis of undetermined significance (MGUS), and IgM-AL (including those without clinical symptoms).

[0110] In some embodiments, the methods and compositions described herein can be used to demonstrate target binding by measuring the stabilizing effect of a clinical candidate (or drug) of a light chain stabilizer in plasma samples from individual AL patients after treatment, protecting them from proteolytic degradation. In some embodiments, the methods and compositions described herein can be used to monitor the response of AL amyloidosis patients to light chain stabilizer or plasma cell-targeted therapeutic treatments.

[0111] In some embodiments, the methods and compositions described herein can be used for prognostic biomarkers, such as the level of LCCD biomarkers, which can indicate disease severity and clinical outcomes.

[0112] In some embodiments, the methods and compositions described herein can be used to detect minimal residual disease (MRD) and relapse: light chain amyloidosis (AL) MRD and relapse can be detected by measuring the amount of LCCD signal in a patient's plasma sample.

[0113] In some embodiments, the methods and compositions described herein can be used for companion diagnostics, such as testing the ability of a clinical candidate (or drug) of an LC stabilizer to protect against proteolytic degradation in plasma samples from an individual pre-treatment AL patient, and using the results to determine whether the patient will benefit from treatment with the LC stabilizer.

[0114] In some embodiments, the methods and compositions described herein can be used as alternative biomarkers or therapeutic response markers, such as to demonstrate the proteolytic protective effect of LC stabilizer clinical candidates (or drugs) and as an indicator of clinical efficacy.

[0115] For the purposes described above, other biological samples, such as urine, serum, CSF, and tissue biopsy sections, may also be used.

[0116] This application is further illustrated by the following embodiments, which should not be construed as limiting. All references, patents and published patent applications, as well as the contents of drawings and tables cited throughout this application, are incorporated herein by reference.

[0117] ELISA assay using LCCD mAb

[0118] Method of limited proteolysis for generating LCCD from λFLC

[0119] In a sandwich ELISA binding assay, the binding of LCCD mAb to WIL and IgG was tested with and without proteinase K (PK) treatment. Briefly, goat anti-IgG (H+L) antibody from Bethyl was placed at a concentration of 1 μg / mL in pH 9.6 carbonate buffer on a high-binding ELISA plate and incubated overnight at 4°C. After blocking with Superblock, serial dilutions of WILLC and purified human IgG (with and without PK (0.2 μM) digestion) were added to the plate and incubated at room temperature for 1 hour. Unbound proteins were then washed away. 1 μg / mL of LCCD mAb was added to the wells, and the plate was incubated at room temperature for 1 hour. Anti-rabbit HRP was then added to the washed plate for 30 minutes, followed by thorough washing. Tetramethylbenzidine (TMS), the enzyme substrate, was then added to each well. The reaction was stopped by adding 0.5 M sulfuric acid, and the absorbance was read at 450 nm using a plate reader.

[0120] Detection of dLCCD in plasma using western blot

[0121] In one embodiment, a plasma sample is incubated with 1 μM proteinase K at 37°C for 2 hours, and then the reaction is stopped by adding a benzyl sulfonyl fluoride protease inhibitor.

[0122] Example 1: Identification of neoepitopes following limited proteolysis of λFLC

[0123] Plasma samples treated with proteinase K under restrictive proteolysis conditions were run on a non-reducing SDS-PAGE gel and then subjected to a standard Western blot procedure. In one embodiment, the gel was transferred to a nitrocellulose membrane. The membrane was then blocked with a blocking reagent and incubated overnight at 4°C with 0.5 μg / mL LCCD-G mAb. The washed blot was imaged using an anti-rabbit antibody conjugated with 800 dye on a LiCor Odyssey system. The fluorescence intensity of the dLCCD bands at approximately 23 kDa was recorded, and the fluorescence signal was quantified.

[0124] Figure 1A

[0125] Compared to their non-amyloid-derived counterparts, amyloid-derived λFLC tends to be kinetically less stable, making it prone to denaturation. Restriction proteolysis has been widely used to probe protein structural dynamics. In this paper, restriction proteolysis was used to assess the kinetic stability of λFLC, in which the FLC protein was incubated with a protease. Quantification of the remaining intact full-length FLC protein or the proteolysis products served as an indicator of FLC protein stability. Proteinase K (PK) has been previously used to study the stability of λFLC, although it was assumed that λFLC would yield small peptides after PK treatment, and only the intact full-length λFLC was tracked and analyzed. PK is considered a broad-spectrum protease. The primary cleavage site of PK is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with a blocked α-amino group. Proteins can be completely digested by PK if the incubation time is long enough and the PK concentration is high enough. 5 μM LC recombinant protein was treated with 200 nM proteinase K at 37 °C for 2 h. The gel was run under non-reducing conditions, stained with Coomassie dye, and visualized. Under current optimized restricted PK treatment, recombinant amyloid-derived λFLC protein (such as WIL) was extracted. Figure 1B Consistently producing a 23 kDa molecular weight band (under non-reducing conditions), regardless of the primary sequence of the amyloid-derived λLC construct. Under the same conditions, this fragment is not readily produced using non-amyloid-derived λLC proteins (such as JTO). Edman degradation sequencing identified a GQPKA at the N-terminus of this 23 kDa band, referred to herein as dLCCD, LCCD, dLCCD fragment, dLCCD biomarker, or dLCCD domain, corresponding to the disulfide-linked homodimeric constant domain of the protein. Figure 1C Under reducing conditions, a fragment of approximately 11.5 kDa representing the monomeric LCCD will be produced. The cleavage site, composed of the GQPKA sequence, is a novel epitope exposed only through restriction protein hydrolysis via PK.

[0126] Given the undefined digestive properties of proteinase K known in the art, the consistent production of this fragment on λFLC is unexpected.

[0127] The PK recognition site located at the direct N-terminus of this novel epitope is >99% conserved in λFLC, and this was confirmed by analysis of the public databases ABYSIS and AL Bases, which contain >6,000 different λFLC sequences. Example 2: Generation and characterization of LCCD monoclonal antibodies Sequence analysis of the λFLC sequence database shows that Gly, Ser, or Arg at the start of a new epitope account for approximately 84%, 11%, and 5% of all λFLC sequences, respectively. The new epitope is therefore referred to as xQP, where x = G, S, or R in the sequence.

[0128] Figure 2A

[0129] Monoclonal antibodies (mAbs) were generated by immunizing rabbits with a peptide-KLH conjugate as the antigen. The peptide antigen contained the sequence xQPKA, where x = G, S, or R in the sequence. Antiserum titers were monitored using an ELISA against the antigen. Upon detection of high titers, antigen-specific B cells were isolated to specifically recognize the antigen. Cells were expanded, and antibody genes were amplified and cloned. The amplified antibody heavy and light chains were expressed in HEK293 cells, and the supernatant was analyzed by ELISA to confirm antibody specificity. The mAbs derived from the immunization were selected, sequenced, and named LCCD-G, LCCD-S, and LCCD-R, respectively, based on the identity of x in the xQP epitope.

[0130] First, the specificity of LCCD mAb was tested in a sandwich ELISA assay using purified recombinant LC protein, where LCCD mAb was used as the capture antibody and goat polyclonal anti-LC antibody was used as the detection antibody. ELISA assays were performed on recombinant WIL (an amyloid-derived λFLC protein) before and after restriction PK digestion. Data showed that LCCD-G mAb bound strongly only to PK-digested WIL LC and had minimal or no binding with full-length undigested LC. Figure 2B In the human proteome, the same light chain proteins can also heterodimerize with heavy chain proteins to form full-length immunoglobulins. This study investigated whether the light chains in immunoglobulins are stable under restrictive proteolytic conditions. To test this, purified human IgG was treated with PK, and the resulting samples were subjected to LCCD ELISA assays. Example 3: Quantitation of LCCD in AL plasma following limited proteolysis As shown, no signal was detected after PK treatment of IgG.

[0131] Figure 3A

[0132] Standard Western blot (WB) was used to detect and quantify proteolytic products in plasma samples from AL patients using LCCD mAb, and imaging was performed on a LiCor system. Figure 3A LCCD mAb exhibits high specificity for LCCD or dLCCD fragments generated after restriction protein hydrolysis, and no other signals were detected in AL plasma samples. Figure 3B Of the 43 plasma samples from untreated AL patients, 37 samples (86.0%) had detectable LCCD-G signals under restrictive proteolysis conditions.

[0133] A modest correlation was observed between dLCCD fluorescence signal and λFLC as measured by the FreeLite clinical test based on turbidimetry in 20 untreated AL plasma samples. Example 4: LCCD immunoassay distinguishes AL from normal plasma Under current restrictive proteolysis conditions, such a correlation cannot be established for normal or MM plasma samples.

[0134] Under optimized stress conditions, in this application, signal in 32 randomly selected λAL plasma samples was detected using standard Western blot analysis with a few microliters of sample. Under the same stress conditions, the signal was much lower (if not undetectable) in 18 of the 19 normal (N=23) and MM plasma samples. In fact, the mean signal in AL samples was 10x to 200x higher than that in normal or most MM plasma samples, and the results were statistically significant. It is possible that a single MM patient with high signal may have coexisting AL. This result establishes the applicability of this assay as a diagnostic tool for AL.

[0135] Figure 4

[0136] Under restricted proteolysis conditions, using Western blotting, LCCD mAb distinguished AL plasma samples from normal or MM plasma samples. Example 5: LCCD immunoassay for quantitation of dLCCD (p=0007 and 0.0008, respectively). This method can therefore be used to specifically diagnose AL patients or track MRD and recurrence based on dLCCD signals in patient samples.

[0137] The ability to specifically detect amyloid-derived FLC in biological samples by detecting and quantifying LCCD signals through restricted proteolysis forms the basis for the diagnostic utility of LCCD-based assays.

[0138] Figure 5A

[0139] Figure 5BThis demonstrates the steps of an ELISA-based dLCCD immunoassay, which uses ex vivo blood samples to distinguish between AL and MM, normal, and ATTR after restriction proteolysis. The Meso Scale Discovery (MSD) platform was used in this example. The plasma / serum assay steps produce a combination of labeled detection antibodies (anti-LC pAbs) that bind to the analyte (dLCCD), which in turn binds to a capture antibody (dLCCD mAb) loaded on the working electrode. MSD electrochemiluminescence offers high sensitivity and a large dynamic range (>400x); typically, 96-well or 384-well configurations are used, with as little as approximately 2 μl of biofluid used per assay. Readings are obtained within hours. Example 6: High baseline dLCCD is associated with poor overall survival The results of dLCCD assays using plasma samples after restrictive protein hydrolysis are shown. dLCCD levels differentiated λAL from normal (N=40), multiple myeloma (MM) (N=19), and TTR amyloidosis (ATTR, including cardiomyopathy and polyneuropathy phenotypes) (N=35). Recombinant dLCCD (a homodimer of SEQ ID NO: 2 linked by disulfide bonds) was used as a reference standard.

[0140] Figure 6A

[0141] Figure 6 shows the baseline level of dLCCD as correlated with overall survival in λAL patients. dLCCD was measured at the 2-hour time point in the experiment. Figure 6B Log-rank survival test showing patients with high (shaded) (N=25) and low (solid) (N=25) baseline dFLC levels. dFLC levels of the same samples were determined by FreeLite assay. Example 7: dFLC18 and dLCCD300 concordance predicts overall survival Log-rank survival test showing patients with high (solid line) (N=25) and low (shaded line) (N=25) baseline dLCCD levels. dLCCD levels were quantified from untreated plasma samples from 50 confirmed λAL patients using the MSD dLCCD immunoassay described herein.

[0142] Figure 7

[0143] Example 8: dLCCD as a biomarker of LC stabilizer target engagement in biofluids or as a response to therapyThe concordance between dFLC and dLCCD was shown based on the data in Figure 6, which predicted overall survival. This figure shows the log-rank survival test compared to patients with low baseline levels of both biomarkers (red curve) (N=17), patients with high levels of both biomarkers (green curve) (N=17), and patients with inconsistent dFLC and dLCCD (blue curve) (N=16). High and low levels of dFLC and dLCCD were defined by a baseline dFLC of 18 mg / dL and a median dLCCD of 300 nM. Results were based on treatment-naïve plasma samples from 50 confirmed λAL patients using the MSD LCCD immunoassay as described herein. Significant differences in OS were observed; specifically, median OS: dFLC 低 / dLCCD 低 162 months; dFLC 高 / dLCCD 高 36 months; inconsistent, 16 months. dLCCD levels were further correlated with cTnT and NT-proBNP (p=0.05). dLCCD is a biomarker indicating pathogenicity in most λAL patients. Baseline dLCCD is a better predictor of OS than dFLC. Therefore, reducing dLCCD levels with therapeutic agents is important for achieving clinical benefit; for example, by eradicating clonal plasma cells that produce amyloid-producing FLC, or by stabilizing λFLC to minimize its amyloid-producing nature, or a combination of both.

[0144] Biomarker levels Reference to electronic sequence listing

[0145] Small molecules that bind directly to the dimer interface of dimer λFLC protein have been shown to stabilize the protein and reduce its amyloidogenicity (Morgan et al. 2019, PMC6486714). These small molecules are considered light chain (LC) stabilizers. The levels of unstable or amyloidogenic λFLC in biofluids can be quantified and used as an indicator of target binding by comparing the levels of dLCCD generated in ex vivo plasma samples before and after compound treatment and after restrictive proteolysis using proteinase K. In Figure 8, the LC stabilizer PTG-1412 was incubated with ex vivo AL plasma, and the assay showed a dose-dependent protective effect against λFLC in the sample.

[0146] Those skilled in the art can perform the same measurements on biofluids from AL patients before and after therapeutic treatment and use the difference in dLCCD levels as an indicator of response to or lack of response to the therapy.

[0147] While various embodiments have been described above, it should be understood that such disclosure is presented by way of example only and not as limiting. Therefore, the breadth and scope of the compositions and methods of the present invention should not be limited to any of the exemplary embodiments described above, but should be defined solely by the following claims and their equivalents.

[0148] The above description is intended to teach those skilled in the art how to practice the invention and is not intended to detail all obvious modifications and variations of the invention that will become apparent to those skilled in the art upon reading this specification. However, all such obvious modifications and variations are intended to be included within the scope of the invention as defined by the appended claims. Unless the context clearly indicates otherwise, the claims are intended to cover components and steps in any order that effectively satisfies the intended objectives.

[0149] Appendix

[0150] This application includes a sequence list, which is submitted electronically in .XML format and is incorporated herein by reference in its entirety. The .XML copy was created on February 21, 2024, named "PROT 119 WO.xml", and is approximately 19,125 bytes in size. The sequence list contained in this .XML file is part of the specification and is incorporated herein by reference in its entirety.

[0151] Definitions Methods of generating monoclonal antibodies for detecting neoepitopes formed by amyloidogenic lambda free light chain (λFLC) proteins Methods of detecting the presence of amyloidogenic lambda free light chain (λFLC) proteins in a subject Methods of assessing target engagement of FLC stabilizers or therapeutic response of light chain amyloidosis Clinical utility of limited proteolysis in combination with LCCD mAb Examples ELISA assay using LCCD mAb Method of limited proteolysis for generating LCCD from λFLC Detection of dLCCD in plasma using western blot Example 1: Identification of neoepitopes following limited proteolysis of λFLC Figure 1A Figure 1B Figure 1C Example 2: Generation and characterization of LCCD monoclonal antibodies Figure 2A Figure 2B Example 3: Quantitation of LCCD in AL plasma following limited proteolysis Figure 3A Figure 3A Figure 3B Example 4: LCCD immunoassay distinguishes AL from normal plasma Figure 4 Example 5: LCCD immunoassay for quantitation of dLCCD Figure 5A Figure 5B Example 6: High baseline dLCCD is associated with poor overall survival Figure 6A Figure 6B Example 7: dFLC18 and dLCCD300 concordance predicts overall survival Figure 7 Example 8: dLCCD as a biomarker of LC stabilizer target engagement in biofluids or as a response to therapy Biomarker levels Reference to electronic sequence listing Appendix

[0152] Monomeric LCCD sequence (SEQ ID NO: 1):

[0153]

[0154] In the xQP epitope, the N-terminal G can be S or R. Monomeric LCCDs can form dimer LCCD proteins via disulfide bonds through the C-terminal Cys (underlined).

[0155] There are other constant domain sequences that retain N-terminal xQP epitopes. These are collectively referred to as LCCD, dLCCD, dLCCD biomarkers, LCCD fragments, LCCD biomarkers, or LCCD domains.

[0156] amyloid-derived FLC protein WIL (SEQ ID No.: 2):

[0157]

[0158] Non-amyloid-derived FLC protein JTO (SEQ ID No.: 3):

[0159]

[0160] LCCD-G mAb HC3(SEQ ID No.: 4):

[0161]

[0162]

[0163] LCCD-G mAb LC7(SEQ ID.NO: 5)

[0164]

[0165] LCCD-R mAb HC2(SEQ ID No.: 6):

[0166]

[0167] LCCD-R mAb LC9(SEQ ID NO.: 7)

[0168]

[0169] LCCD-S mAb HC2(SEQ ID No.: 8):

[0170]

[0171] LCCD-S mAb LC5(SEQ ID NO.: 9)

[0172]

Claims

1. A method for generating a monoclonal antibody for detecting novel epitopes formed from amyloid-derived λ free light chain (λFLC) protein, the method comprising the steps of: (a) A sample containing a λ free light chain (λFLC) protein is hydrolyzed with proteinase K, wherein the hydrolysis produces a novel epitope on the N-terminus of an approximately 23 kDa fragment exposed to at least a portion of a constant domain containing the λFLC. Alternatively, when λFLC is a monomer, a novel epitope is generated at the N-terminus of an approximately 11.5 kDa fragment exposing at least a portion of the constant domain of the λFLC protein; and The novel epitope is xQP, where x is one of G, R, or S (G = glycine, R = arginine, S = serine); and (b) Generating a monoclonal antibody against the novel epitope on the fragment of said λFLC, wherein the generated monoclonal antibody (LCCD) specifically binds to said novel epitope xQP, and The proteolysis conditions were optimized such that the intensity of the LCCD signal obtained from the approximately 23 kDa fragment or the approximately 11.5 kDa fragment after proteolysis of amyloid-derived λFLC was at least twice that of non-amyloid-derived λFLC.

2. The method of claim 1, wherein the new epitope is a GQP.

3. The method of claim 1, wherein the new epitope is an RQP.

4. The method of claim 1, wherein the new epitope is an SQP.

5. The method of claim 2, wherein the generated antibody (LCCD-G) binds to the novel epitope GQP.

6. The method of claim 3, wherein the generated antibody (LCCD-R) binds to the novel epitope RQP.

7. The method of claim 4, wherein the generated antibody (LCCD-S) binds to the novel epitope SQP.

8. The method according to any one of claims 1 to 7, wherein the λFLC fragment is a homodimeric fragment that is about 23 kDa under non-denaturing conditions.

9. The method according to any one of claims 1 to 7, wherein the λFLC fragment is a monomeric fragment that is about 11.5 kDa under denaturing conditions.

10. The method according to any one of claims 1 to 9, wherein the restricted proteolysis by proteinase K comprises the step of treating a liquid sample with optimized proteolysis conditions to generate LCCD domains.

11. The method according to any one of claims 1 to 10, wherein the optimized proteolysis conditions are dependent on the specific activity of the protease, and 0.02 μM to 20.0 μM proteinase K is administered at 37°C for 30 seconds to 20 hours.

12. An antibody for detecting novel epitopes formed by amyloid-derived λ free light chain (λFLC) protein. The antibody comprises a heavy chain (HC3) encoded by the nucleotide sequence of LCCD-G SEQ.ID.No.: 4 and a light chain (LC7) encoded by the nucleotide sequence of LCCD-G SEQ.ID.No.:

5.

13. An antibody for detecting novel epitopes formed by amyloid-derived λ free light chain (λFLC) protein, wherein the antibody comprises a heavy chain (HC2) encoded by the nucleotide sequence of LCCD-S SEQ.ID.No.: 8 and a light chain (LC5) encoded by the nucleotide sequence of LCCD-S SEQ.ID.No.:

9.

14. An antibody for detecting novel epitopes formed by amyloid-derived λ free light chain (λFLC) protein. The antibody is encoded by nucleotide sequences (SEQ ID No.: 8 (HC2) and SEQ ID No.: 9 (LC5)) that encode LCCD-S.

15. A method for evaluating target binding of an FLC stabilizer for the treatment of light chain amyloidosis, the method comprising the steps of: Restrictive proteolysis of proteins from biological samples. The biological sample mentioned above is a recombinant protein or comes from a subject who has been treated with a targeted therapeutic molecule against light chain amyloidosis. The restriction protein hydrolysis produces new epitopes exposed on the LCCD fragment of the λFLC protein; The presence of the novel epitope on the N-terminus of the LCCD fragment of the λFLC protein was detected; as well as Based on the detected presence of the novel epitope, the level of the fragment of the λFLC protein generated from the restriction protein hydrolysis was quantified. The comparison involves a first level of the amount of the LCCD fragment present before the targeted therapeutic molecule has been introduced into the subject or before it has been introduced into a sample from the subject. Target binding is assessed at a second level by measuring the amount of the LCCD fragment present after the targeted therapeutic molecule has been introduced into the subject or ex vivo into a sample from the subject. The decrease in LCCD level or LCCD level normalized to total λFLC in the second quantitative level indicates target binding.

16. A method for assessing the therapeutic response of a therapeutic agent for treating light chain amyloidosis by quantifying LCCD fragments in a plasma sample before and after the therapeutic molecule has been introduced into a subject or at two different time points during treatment, wherein a decrease in LCCD levels after treatment or at the second time point indicates a favorable response to the therapeutic molecule, independent of the total level of λFLC.

17. A method for assessing whether AL patients will benefit from light chain stabilizers as a therapy by quantifying LCCD fragments in plasma samples from subjects believed to have received treatment with light chain stabilizer molecules targeting light chain amyloidosis. Prior to subjecting the sample to restrictive protein hydrolysis, the ex vivo plasma sample is incubated together with the stabilizer or solvent control, and the resulting LCCD level is quantified. A decrease in the LCCD level in the sample incubated with the stabilizer relative to the solvent control sample indicates that the subject can benefit from treatment with the light chain stabilizer.

18. The method according to any one of claims 15 to 17, wherein the first level of the LCCD fragment present before the targeted therapeutic molecule has been introduced into the subject is greater than At the second level of the LCCD fragment present after the targeted therapeutic molecule has been introduced into the subject. The targeted therapeutic molecule has then undergone target binding.

19. The method according to any one of claims 15 to 17, wherein the first level of the LCCD fragment present before the targeted therapeutic molecule has been introduced into the subject is equal to or less than At the second level of the LCCD fragment present after the targeted therapeutic molecule has been introduced into the subject. The targeted therapeutic molecule has not yet undergone target binding.

20. The method according to any one of claims 15 to 19, wherein the biological sample is one of plasma, urine, serum, cerebrospinal fluid (CSF), or a tissue biopsy section.

21. A method for detecting the presence of amyloid-derived λ free light chain (λFLC) protein in a subject, the method comprising the following steps: Restrictive proteolysis of proteins from biological samples. The restriction protein hydrolysis produces new epitopes exposed on the LCCD fragment of the λFLC protein; The presence of the novel epitope on the N-terminus of the LCCD fragment of the λFLC protein was detected; The presence of the LCCD fragment from the λFLC protein was quantified based on the level of the novel epitope on the fragment measured using LCCD mAb; and The levels of the LCCD fragments before and after the restriction protein hydrolysis were compared, wherein amyloid-derived λFLC protein was detected when the level of the LCCD fragments from λFLC protein was higher after the restriction protein hydrolysis.

22. The method according to claim 21, further comprising the step of: The first level of the LCCD fragment of the λFLC protein present after the restriction protein hydrolysis was measured in a biological sample taken from a healthy subject who did not have light chain amyloidosis (AL), wherein the first level was a reference level; Measure a second level of the fragment of the λFLC protein present after the restriction protein hydrolysis in a biological sample from a person suspected of having AL; and Wherein, after the restriction protein hydrolysis of the biological sample, the second level of the LCCD fragment of the λFLC protein is greater than the reference level. Amyloid-derived λFLC protein was detected.

23. The method according to any one of claims 21 to 22, wherein light chain amyloidosis is present in the subject when a level of LCCD fragments from λFLC is detected after restriction proteolysis by proteinase K and the level is elevated above the reference level.

24. The method of claim 23, wherein elevated LCCD fragment levels in the subject lead to a clinical decision, wherein the clinical decision is for the treatment and management of light chain amyloidosis (AL).

25. The method of claim 23, wherein there is no elevated LCCD fragment level in the subject leading to a clinical decision, wherein the clinical decision is to rule out an AL diagnosis and receive treatment for other systemic amyloidosis, wherein the other systemic amyloidosis may include thyroxine transporter amyloidosis (ATTR).

26. The method according to any one of claims 21 to 25, wherein the LCCD fragment is a 23 kDa homodimer and is detected and quantified using mass spectrometry.

27. The method according to any one of claims 21 to 25, wherein the LCCD fragment is an 11.5 kDa monomer and is detected and quantified using mass spectrometry.

28. The method according to any one of claims 21 to 27, wherein the subject suffers from one of a plurality of the following: clinical suspicion of amyloidosis, monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM), light chain amyloidosis (AL), smoldering multiple myeloma (SMM), IgM light chain amyloidosis, minimal residual disease (MRD) of AL, and relapse of AL.

29. The method according to any one of claims 21 to 28, wherein the biological sample is one of plasma, urine, serum, CSF, or tissue biopsy section.

30. A kit for detecting the presence of amyloid-derived λ free light chain (λFLC) protein in a subject, the kit comprising: Antibodies LCCD-G, LCCD-R, and LCCD-S; The method of claim 21 uses the instructions for the kit.

31. A novel epitope on λFLC prepared by restrictive proteolysis of amyloid-derived λ free light chain (λFLC) protein using proteinase K.

32. The new epitope according to claim 31, wherein the new epitope is xQP, where x is one of G, R or S.

33. An antibody or antibody mixture targeting a novel epitope according to claim 31 or 32.

34. The antibody according to claim 33, wherein the antibody is LCCD-G, LCCD-R or LCCD-S.

35. A method for optimizing restrictive protein hydrolysis conditions using proteinase K, the method comprising the step of optimizing one or more of the following factors: (1) The source of the proteinase K; (2) The composition of the test sample; (3) Concentration of proteinase K; (4) The time of protein hydrolysis reaction; (5) The temperature of the protein hydrolysis reaction; The proteolytic products of the LCCD fragment were obtained by differentiating between amyloid-derived λFLC and non-amyloid-derived λFLC.

36. The method for detecting the presence of amyloid-derived λ free light chain (λFLC) protein in a subject according to any one of claims 21 to 29, the method further comprising: Readings are obtained from the LCCD signal obtained by measuring the level of the new epitope on the segment using the LCCD mAb.

37. The method for detecting the presence of amyloid-derived λ free light chain (λFLC) protein in a subject according to claim 36, the method further comprising: The reading of the LCCD signal is obtained by using an algorithm.

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