WO2024178117A1 - Method of sequence-independent quantification of proteolytically unstable plasma lambda free light chain protein for al amyloidosis diagnosis - Google Patents

Abstract

A method for combining limited proteolysis and immunoassays for the specific detection and quantification of amyloidogenic XFLC in biofluids. The method is enabled by using mAbs that are capable of detecting and quantifying neo-epitopes on the dLCCD biomarker, generated after limited proteolysis of ZFLC. which heavily favors kinetically unstable, or amyloidogenic, LFLC. This method is independent of FLC primary sequences in the variable and constant domains and can be used on the detection and quantification of up to 99% of human XFLC. The clinical utilities of the assay include early detection in individuals suspected of plasma cell disorders (MGUS, SMM, MM, IgM-AL, and AL), differentiation from other amyloidosis such as ATTR, use in companion diagnostics, demonstration of target engagement, assessment of therapeutic response, detection of MRD, detection of relapse. Assays that quantify the resulting dLCCD biomarker and/or that can further improve the assay's sensitivity, such as MSD, lateral flow, mass spectrometry, are also embodied.

Description

TITLE

METHOD OF SEQUENCE-INDEPENDENT QUANTIFICATION OF PROTEOLYTICALLY UNSTABLE PLASMA LAMBDA FREE LIGHT CHAIN PROTEIN FOR AL AMYLOIDOSIS DIAGNOSIS

[0001] The application claims priority from 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.

FIELD

[0002] This application relates generally to the field of biomarkers and use in health care.

BACKGROUND

[0003] Light chain amyloidosis (AL) is a rare, progressive, and often fatal degenerative disease affecting the heart, kidney, and liver. AL is caused by aberrant clonal plasma cell proliferation, where excess amount of amyloidogenic monoclonal free light chain (FLC) was produced and secreted into the circulation. Amongst all AL patients, involved λFLC accounts for -80% of the cases. Amyloidogenic FLC are kinetically unstable, leading to protein misfolding and the formation of pathogenic conformations including soluble aggregates and amyloids. Specific quantification of amyloidogenic FLC can provide critical guidance in clinical practice but has been hampered by the sequence diversity' of immunoglobulin light chains.

[0004] Immunoglobulin light chain and heavy chain proteins are produced by plasma cells, a ty pe of white blood cell found in the bone marrow, in the process of making various ty pes of immunoglobulins, such as IgG, IgA, IgM, etc. In some cases, oncogenically transformed clonal plasma cells proliferate and secret excessive amounts of amyloidogenic free light chain (FLC) into the blood circulation. Hematologically, high levels of FLC can be detected in the blood and urine, leading to abnormal serum κ/λ FLC ratio. Amyloidogenic monoclonal FLC differs from the normal polyclonal FLC generated as part of the adaptive immune response in terms of the amount of circulating FLC and its amyloidogenicity. Amyloidogenic FLC are less stable and prone to misfolding, proteolysis, and aggregation. Misfolded conformations (soluble aggregates and fibrils) as well as fragments of FLC are believed to be toxic and can eventually deposit onto various organs as amyloids. Organ toxicity manifests as cardiomyopathy, nephrotic syndrome and/or end-stage renal failure, collectively categorized as Light Chain Amyloidosis (AL). Both amyloidogenic and K FLC may cause AL, although λFLC is frequently identified as the responsible, or involved, FLC in AL. Estimated AL incidence is 10 in every million population worldwide.

[0005] λFLC monomer is around 227 amino acids and is made up of an N-terminal variable domain (VL), a joint linker region (J), and a C-terminal constant domain (CL). λFLC usually presents as a homodimer linked by an inter-monomer disulfide bond at the C-terminal constant domain of the protein. Variations in FLC sequences are the result of the germline genes and the incorporation of somatic hypermutations. While each AL patient usually possesses a single clonal plasma cell population and therefore one unique monoclonal amyloidogenic FLC sequence, it exists on the background of a highly diverse pool of polyclonal FLC that are present in any human being. Such sequence diversity and the lack of means to differentiate FLC amyloidogenicity pose unique challenges for the diagnosis and targeted treatment of AL.

[0006] Current standard of Care (SOC) for AL patients mirrors treatment for Multiple Myeloma by targeting the proliferating clonal plasma cells and commonly employs a combination of anti-CD38 immunotherapy and chemotherapy cocktails that typically contain proteasome inhibitors, or chemotherapy cocktails alone, or, when possible, stem cell transplants. Although therapeutics reducing clonal plasma cell burden have improved overall survival rate in AL patients, there are still significant unmet medical needs in AL clinical management due to the lack of early diagnosis, treatment resistance, persisting minimal residual disease (MRD), low organ response despite hematological response, and disease relapse. It is believed that patients who are diagnosed and treated early in their disease progression, especially before significant organ involvement, have a much better chance at survival and even recovery.

[0007] AL is often diagnosed late or goes undiagnosed until later stage because the signs and symptoms of AL mimic those of other common diseases. Clinical symptom of specific organ involvement is the most common trigger of suspicion of the disease. Confirmation of AL diagnosis ty pically takes 6-12 months with visits to 3-4 physicians, while the mortality rate at six months after AL diagnosis approaches 25% with mean survival around four years. Laboratory tests of blood and urine M-protein and FLC, such as immunofixation and the FreeLite turbidimetry assays (BindingSite. UK), are commonly used to assess FLC levels, dFLC levels (difference in the levels of λFLC and KFLC) and κ/λ ratio; followed by tissue biopsy where amyloid presence and occasionally protein identity are examined. Imaging tests such as echocardiogram and magnetic resonance imaging are also prescribed in cases of organ involvement. These tests are useful tools but nonetheless are beset by low sensitivity, lack of specificity, and the inability to differentiate amyloidogenic from non-amyloidogenic FLC. The more recent development in mass spectrometr -based clonal LC detection has significantly improved sensitivity yet is only available in limited numbers of clinics and is not poised for quantification nor indication of amyloidogenicity.

[0008] There is a substantial unmet need for a test that is sensitive, specific, easy to access, inexpensive, and can assess amyloidogenicity of the monoclonal FLC. Such a test would enable early diagnosis of AL, as well as monitor response to therapeutics and MRD.

SUMMARY

[0009] An aspect of the application is a method of generating a novel biomarker and using a monoclonal antibody for detection of a neo-epitope on the biomarker formed from amyloidogenic λ free light chain (λFLC) proteins, comprising the steps of: limited proteolysis of λ free light chain (λFLC) proteins by proteinase K, wherein the limited proteolysis results in a neo-epitope exposed on a fragment of the amyloidogenic λFLC proteins, and wherein the resulting fragment is the intact light chain constant domain dimer and monomer (“LCCD,” or “dLCCD,” or “dLCCD biomarkef ’, 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 either a monomer or a dimer formed via disulfide bonding through the C-term Cysteine, and wherein the neo-epitope is xQP, representing the N-terminal of the specific cleavage product (LCCD or dLCCD) that starts with amino acid sequence of xQPKA, where x is one of G (Glycine), R (Arginine) or S (Serine); generating a monoclonal antibody to the neo-epitope on the N-terminal of the LCCD fragment of the λFLC, wherein the generated monoclonal antibody binds to the neo-epitope of xQP at the N-terminal of a peptide chain.

[0010] Another aspect of the application requires optimization of the proteolysis condition for the sample of interest, so that the LCCD biomarker dimer or monomer is specifically generated from amyloidogenic λFLC proteins comparing to other X light chaincontaining proteins exist in the said sample of interest or in a different sample. λFLC may be in the form of recombinant human FLC in buffer, or recombinant and/or endogenous λFLC in an ex vivo biological matrix such as plasma, serum, urine, etc. Other X light chain-containing proteins include any immunoglobulins (IgA, IgG. IgM, IgE, etc.) and non-amyloidogenic λFLC. While optimization includes testing one or more of the following conditions: vary ing the source of the Proteinase K used, vary ing the concentration of the Proteinase K used, varying time of incubation, varying the composition of the matrix containing the test subjects, and varying the temperature of incubation. Specificity is achieved yvhen LCCD fragment is generated from a representative amyloidogenic λFLC such as WIL (SEQ.ID. No.: 2) at a level at least two times more than that from a representative non-amyloidogenic λFLC such as JTO (SEQ.ID. No.: 3) under a specific condition.

[0011] In certain embodiments, the neo-epitope is N-terminal GQP, GQPK, or GQPKA. In certain embodiments, the neo-epitope is N-terminal RQP, RQPK, or RQPKA. In certain embodiments, the neo-epitope is N-terminal SQP, SQPK, or SQPKA. In certain embodiments, the generated antibody (LCCD-G) binds to the neo-epitope of GQP, GQPK, or GQPKA. In certain embodiments, the generated antibody (LCCD-R) binds to the neo-epitope of RQP, RQPK, or RQPKA. In certain embodiments, the generated antibody (LCCD-S) binds to the neo-epitope of SQP, SQPK, or SQPKA. In certain embodiments, the LCCD fragment of the λFLC is a ~23kDa fragment under non-denaturing condition. In certain embodiments, the LCCD fragment of the λFLC is a ~11 ,5kDa fragment under denaturing condition. In certain embodiments, the limited proteolysis by proteinase K comprises the steps of: recombinant amyloidogenic λFLC in a matrix treated yvith an optimized proteolysis condition to produce the LCCD fragment. In certain embodiments, the limited proteolysis by proteinase K comprises the steps of: ex vivo AL plasma sample treated yvith an optimized proteolysis condition to produce the LCCD fragment. In certain embodiments, the optimized proteolysis condition is 0.02-30pM Proteinase K at 15°C - 70°C for 30 seconds to 20 hrs. In particular embodiments, condition is for 1 minute to 2 hours.

[0012] Another aspect of the application is an antibody for detection of a neo-epitope formed from amyloidogenic Z free light chain (λFLC) proteins, wherein the antibody is encoded by a nucleotide sequence encoding heavy and light chains of LCCD-G as described herein.

[0013] Another aspect of the application is an antibody for detection of a neo-epitope formed from amyloidogenic λ free light chain (λFLC) proteins, wherein the antibody is encoded by a nucleotide sequence encoding heavy and light chains of LCCD-R as described herein.

[0014] Another aspect of the application is an antibody for detection of a neo-epitope formed from amyloidogenic Z free light chain (λFLC) proteins, wherein the antibody is encoded by a nucleotide sequence encoding heavy and light chains of LCCD-S as described herein.

[0015] One of ordinary skill will understand that variations in a nucleotide sequence that are alterations in the genetic triplet code of that sequence, but do not alter the amino acid being encoded, are also encompassed within this application for any described nucleotide sequences of LCCD-G, LCCD-R, and LCCD-S.

[0016] Another aspect of the application is a method of assessing target engagement or compound potency in the discovery of a small molecule FLC stabilizer, comprising the steps of: limited proteolysis of proteins from a sample containing λFLC proteins, wherein the sample contains recombinant λFLC proteins or is an ex vivo biological sample such as serum, plasma, or urine, and wherein the limited proteolysis results in a neo-epitope exposed on an N-terminal of an LCCD fragment of the λFLC proteins; detecting the presence of the neoepitope on the LCCD fragment of the λFLC proteins; and quantitating the amount of the LCCD fragment of the λFLC proteins resulting from the limited proteolysis based on the neoepitope presence detected, wherein target engagement is assessed by comparing a first level of LCCD fragment before the small molecule has been introduced to the test sample, to a second level of LCCD fragment after the small molecule has been introduced to the test sample, wherein a reduction in the LCCD fragment level, or the LCCD biomarker level normalized to the total λFLC or dFLC in the second level of quantification indicates target engagement.

[0017] Another aspect of the application is a method of assessing target engagement of a FLC stabilizer for treatment of light chain amyloidosis, comprising the steps of: limited proteolysis of 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 limited proteolysis results in a neo-epitope exposed on an N-terminal of an LCCD fragment of the λFLC proteins; detecting the presence of the neo-epitope on the LCCD fragment of the λFLC proteins; and quantitating the amount of the LCCD fragment of the λFLC proteins resulting from the limited proteolysis based on the neo-epitope presence detected, wherein target engagement is assessed by comparing a first level of LCCD fragment before the targeted therapeutic molecule has been introduced to the subj ect, or to a sample from a subject ex vivo, to a second level of LCCD fragment after the targeted therapeutic molecule has been introduced to the subject, or to a sample from a subject ex vivo, wherein a reduction in the LCCD fragment level, or the LCCD fragment level normalized to the total λFLC or dFLC in the second level of quantification indicates target engagement. [0018] Another aspect of the application is a method of assessing target engagement of a FLC stabilizer or pharmacological chaperone for treatment of light chain amyloidosis, comprising the steps of: limited proteolysis of 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 limited proteolysis results in a neo-epitope exposed on a LCCD fragment of the λFLC proteins, and wherein the neo-epitope is N-terminal xQP, where x is one of G, R or S; detecting the presence of the LCCD fragment of the λFLC proteins by binding of a monoclonal antibody to the neo-epitope, wherein the monoclonal antibody binds to the neo-epitope of xQP; and quantitating the level of LCCD fragment resulting from the limited proteolysis based on the signal detected through antibody binding, wherein target engagement is assessed by comparing a first level of quantity of antibody binding to the neo-epitope on LCCD present before the targeted therapeutic molecule has been introduced to the subject, or to a sample from a subject ex vivo, to a second level of quantity of antibody binding to the neo-epitope on LCCD present after the targeted therapeutic molecule has been introduced to the subject, or to a sample from a subject ex vivo, wherein a reduction in the LCCD level, or LCCD level normalized to the total λFLC or dFLC in the second level of quantification indicates target engagement.

[0019] In certain embodiments, when the first level of the LCCD fragment present before the targeted therapeutic molecule has been introduced to the subject is greater than the second level of the LCCD fragment present after the targeted therapeutic molecule has been introduced to the subject, then target engagement has occurred by the targeted therapeutic molecule.

[0020] In certain embodiments, when the first level of the LCCD fragment present before the targeted therapeutic molecule has been introduced to the subject is the same or less than the second level of the LCCD fragment present after the targeted therapeutic molecule has been introduced to the subject, then target engagement has not occurred by the targeted therapeutic molecule. In certain embodiments, the biological sample is one of plasma, urine, serum, cerebrospinal fluid (CSF), or tissue biopsy.

[0021] Another aspect of the application is a method of assessing therapeutic response of a therapeutics for treatment of light chain amyloidosis by quantifying level of a LCCD fragment in biological samples subjected to limited Proteinase K proteolysis before and after the therapeutic molecule has been introduced to the subject, or at tw o or more different time points during the treatment, wherein a reduction in the post-treatment or later timepoint levels of LCCD fragment indicates favorable response to the therapeutic molecule, irrespective to the total level of λFLC.

[0022] Another aspect of the application is a method of assessing if an AL patient would benefit from a light chain stabilizer as a therapy by quantifying level of a LCCD fragment in biological samples from a subject being considered to receive treatment by a light chain stabilizer molecule for light chain amyloidosis, wherein the ex vivo plasma sample is incubated with the said stabilizer or a solvent control, before subjecting the samples to limited Proteinase K proteolysis, and quantification of the resulting LCCD levels, wherein a reduction of LCCD biomarker level in the sample that was incubated with the stabilizer versus the solvent control sample indicates the subject may benefit from treatment by the said light chain stabilizer.

[0023] Another aspect of the application is a method of detection of presence in a subject of amyloidogenic k free light chain ( λFLC) proteins, comprising the steps of: limited proteolysis of proteins from a biological sample, wherein the limited proteolysis results in a neo-epitope exposed on an N-terminal of the LCCD fragment of the λFLC proteins; detecting the presence of the neo-epitope on the fragment of the λFLC proteins; quantitating the level of LCCD fragment of the λFLC proteins by measuring the neo-epitope by the LCCD mAbs; and comparing LCCD fragment levels from the λFLC proteins before and after the limited proteolysis, wherein when the LCCD fragment level is greater after limited proteolysis then amyloidogenic λFLC proteins are detected.

[0024] Another aspect of the application is a method of detection of presence in a subject of amyloidogenic k free light chain (λFLC) proteins, comprising the steps of: limited proteolysis by Proteinase K of proteins from a biological sample, wherein the limited proteolysis results in a neo-epitope exposed on an N-terminal of the LCCD fragment of the λFLC proteins, and wherein the neo-epitope is xQP, where x is one of G, R or S; detecting the presence of the LCCD fragment of the λFLC proteins by binding of a monoclonal antibody to the neo-epitope, wherein the monoclonal antibody binds to the neo-epitope of xQP; quantitating the LCCD fragment level by measuring the amount of antibody binding to the neo-epitope; and comparing a first level of quantity of LCCD fragment present after the limited proteolysis in a biological sample taken from a healthy subject not having light chain amyloidosis (AL), wherein the first level is a reference level, to a second level of quantify of LCCD fragment after the limited proteolysis of a biological sample from a person suspected of having AL; and wherein when the second level of LCCD fragment after the limited proteolysis of the biological sample is greater than the reference level, then amyloidogenic λFLC proteins are detected. In certain embodiments, the LCCD fragment, as a 23KDa homodimer, or a 11.5KDa monomer, is detected and quantified using mass spectrometry. In certain embodiments, when the level of LCCD fragment from λFLC is detected after limited proteolysis by Proteinase K and at an elevated level above the reference level, then light chain amyloidosis is present in the subject. In certain embodiments, where elevated LCCD level in a subject lead to a clinical decision, wherein the clinical decision is treatment and management for light chain amyloidosis (AL). In certain embodiments, the subject has one of more of 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) of AL, and Relapse of AL. In certain embodiments, the subject received plasma cell therapy and elevated LCCD levels is used to detect residual level of amyloidogenic FLC, indicating minimal residual disease (MRD). In certain embodiments, elevated LCCD levels in a subject who had stable disease indicates disease relapse. In certain embodiments, the biological sample is one of plasma, urine, serum. CSF. or tissue biopsy.

[0025] In certain embodiments, where absence of elevated LCCD level in a subject lead to a clinical decision wherein the clinical decision is ruling out of AL diagnosis and receiving treatment for other systemic amyloidosis, wherein the other systemic amyloidosis may include transthyretin amyloidosis (ATTR).

[0026] Another aspect of the application is a kit for detection of presence in a subject of amyloidogenic k free light chain (λFLC) proteins, comprising: one or more of the antibodies of LCCD-G, LCCD-R and LCCD-S; instructions for using the kit in accordance with the methods described herein.

[0027] In certain embodiments, the kit comprises a protein with an N-terminal neoepitope as the reference standard. In certain embodiments, this protein is dLCCD. In certain embodiments, the neo-epitope is xQP, where x is one of G, R or S. In certain embodiments, the kit comprises one or more of the antibodies to the neo-epitopes described herein. In certain embodiments, the antibody is LCCD-G, LCCD-R or LCCD-S. In certain embodiment, the kit comprises an instruction to generate the LCCD fragment from λFLC by limited proteolysis by Proteinase K. The fragment LCCD and dLCCD names are used interchangeably in this application unless otherwise noted. In certain embodiments, the level of dLCCD of a particular sample is a parameter that is calculated based on one or more readings of the dLCCD signal or signals of the said sample generated under limited proteolysis conditions. BRIEF DESCRIPTION OF FIGURES

[0028] Figure 1A illustrates an exemplary SDS PAGE gel of Proteinase K (PK) digestion of recombinant lambda light chains, including a random selection of 293F cell produced recombinant amyloidogenic (sequences based on AL patients) and non- amyloidogenic (sequences based on normal healthy person) λFLC proteins, comparing with and without PK treatment. PK digestion resulted in the generation of a proteolytic fragment corresponding to the disulfide linked LC constant domain (LC-C domain, or dLCCD) homodimer.

[0029] Figure IB shows LC structure highlighting the hinge region. PK cleaves in the hinge region between LC-V and LC-C, producing the dLCCD fragment. PK treatment exposes neo-epitopes highly conserved for all LC sequences. Exposure depending on the dynamic/ amyloidogenic nature of the λFLC.

[0030] Figure 1C shows lambda LC sequence around the cleavage site, indicating the highly conserved PK cleavage site exposing the xQP neo-epitope. Based on analysis of public databases, frequency of sequence occurrence at the N-term of the dLCCD fragment after PK cleavage in AL λFLC are approximately: 84% GQP, 11% SQP. and 5% RQP.

[0031] Figure 2A and 2B show LCCD-G mAb binding to LCCD vs (a) full length WIL or (b) IgG + PK in buffer limited PK proteolysis in buffer, demonstrating 1) the LCCD mAb specifically binds to the cleaved and exposed xQP epitope, not xQP in a protein sequence; 2) the specific generation of the LCCD-G epitope specifically in amyloidogenic FLC, but not in full length immunoglobulin.

[0100] Figure 2C show LCCD mAbs highly specific for proteolysis exposed novel epitopes. LCCD-G, LCCD-R, and LCCD-S mAbs (green signal around 25 KDa) specifically detect LCCD fragments recombinant FLC proteins containing GQP, RQP, and SQP at the cleavage site, respectively, post-proteolysis by Proteinase K. Anti-λFLC polyclonal antibody (red signal around 50KDa) is used to indicate all full-length and LCCD fragments from the samples.

[0032] Figure 3 A shows LCCD-G mAb detection of LC proteolysis in 10 treatment naive AL plasma in WB.

[0033] Figure 3B shows quantification of dLCCD fragment level determined in Western blot in pre-treatment AL patients (post-PK treatment). [0034] Figure 4 shows dLCCD fragment level generated under limited proteolysis determined in Western blot in pre-treatment AL patient plasma (N=32), plasma from normal (N=23) and patient diagnosed with multiple myeloma (MM) (N=19). Statistical analysis method: one-way ANOVA. In WB and post-limited PK proteolysis treatment, AL samples have dLCCD signal 10-200x of normal under the same condition (p=0.0007). AL samples are also significantly differentiated from MM samples under the limited PK proteolysis and quantified by the LCCD mAb (p=0.0008).

[0035] Figure 5 shows an MSD dLCCD immunoassay. Figure 5A: Using LCCD-X mixture as the capture antibody, dLCCD level generated under limited proteolysis determined on MesoScale Discovery (MSD) platform. LCCD-X is an equal molar mixture of the three LCCD mAbs, LCCD-G, LCCD-S. and LCCD-R. Figure 5B: dLCCD levels differentiates λAL from normal (N=40), multiple myeloma (MM) (N=19), and TTR amyloidosis (ATTR, including cardiomyopathy and polyneuropathy phenotypes) (N=35) patients. Recombinant dLCCD was used as the reference standard.

[0036] Figure 6 shows baseline levels of dLCCD correlates to λAL patient overall survival. Figure 6A: Log-rank survival test of patients with high (solid line) and low (hatched line) levels of baseline dFLC, separated by the median value of 18 mg/dL. P=0.1474. Figure 6B: Log-rank survival test of patients with high (hatched line) and low (solid line) levels of baseline dLCCD, separated by the median level of 300 nM. P=0.0479. dLCCD levels were quantified on treatment naive plasma samples from 50 confirmed λAL patients using the MSD LCCD immunoassay. dFLC level of the same samples were determined by the FreeLite assay. OS=month. Note: the median level of 300 nM is a rough method of separating the group. This may not be the final cutoff.

[0037] Figure 7 shows concordance of dFLC and dLCCD predicts overall survival. Log-rank survival test of patients with low baseline levels of both biomarkers (solid line; left- to-right sloping down background shading in lower graph) compared to those with high levels of both biomarkers (dotted line; dotted background shading in lower graph), and those with discordant dFLC and dLCCD (dotted-and-dashed line; left-to-right upward sloping up shading in background graph). High and low levels of dFLC and dLCCD are defined by the median value of 18 mg/dL for baseline dFLC and 300 nM for dLCCD. P=0.0159. Treatment naive plasma samples from 50 confirmed λAL patients MSD LCCD immunoassay. OS=month. Note: the median level of 300 nM is a rough method of separating the group. This may not be the final cutoff. [0038] Figure 8A shows Western blot of the limited proteolysis samples. Using an anti-λFLC monoclonal antibody (band at 50KDa) and the dLCCD mAb (solid band at 23 KDa). Figure 8B: Quantification of the antibody-detected bands on the LiCor system and fitted to a variable slope (four parameters) response curve using Prism. EC_50S are similar using the two antibodies). Figure 8C: Protection against limited proteolysis by PTG-1412 in 13 ex vivo λAL plasma samples as quantified by dLCCD levels post-proteolysis, with and without PTG-1412.

DETAILED DESCRIPTION

[0039] Reference will be made in detail to certain aspects and exemplary embodiments of the application, illustrating examples in the accompanying structures and figures. The aspects of the application will be described in conjunction with the exemplary embodiments, including methods, materials and examples, such description is non-limiting and the scope of the application is intended to encompass all equivalents, alternatives, and modifications, either generally known, or incorporated here. 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 belongs. One of skill in the art will recognize many techniques and materials similar or equivalent to those described here, which could be used in the practice of the aspects and embodiments of the present application. The described aspects and embodiments of the application are not limited to the methods and materials described.

[0040] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise.

[0041] Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed 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 is also understood that when a value is disclosed that "less than or equal to "the value," greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value " 10" is disclosed the "less than or equal to 10" as well as "greater than or equal to 10" is also disclosed.

[0042] It is understood that the exact concentration and units of reagents may vary based on the source of enzymatic reagents and therefore customized method optimization may be used to achieve the same limited proteolysis results.

[0043] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one having ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0044] In describing the invention, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefit and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques. Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary' fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the invention and the claims.

[0045] In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be evident, however, to one skilled in the art that the present invention may be practiced without these specific details.

[0046] Using limited proteolysis, this application describes a method of generating neo-epitopes at the N-terminal of the λFLC cleavage product and the monoclonal antibodies (mAbs) that specifically recognize such epitopes. Under optimized proteolysis conditions, the generation of these neo-epi topes directly correlates to the lack of kinetic stability of the amyloidogenic FLC. which may be corrected with a small molecule stabilizer binding to the dimeric λFLC. Levels of amyloidogenic FLC may also be reduced by other therapeutic interventions. Immunoassays using these mAbs coupled with limited proteolysis by Proteinase K are shown to detect and quantify amyloidogenic λFLC specially in AL patient plasma samples and irrespective of the patients’ λFLC sequences. Sequence-independent quantification of proteolytically unstable plasma λFLC described here should provide specific, sensitive, and low-cost diagnosis of λAL amyloidosis, as well as methods to follow minimal residual diseases and monitor therapeutics responses. In addition to monoclonal antibodies, one of ordinary skill will understand that the methods described herein can be performed with other means to target specific epitopes, such as aptamers, nanobodies, etc. The use of monoclonal antibodies herein is not limiting on the invention.

[0047] One of ordinary’ skill will understand that the neo-epitopes described herein can be recognized by other epitope-recognizing agents, such as. but not limited to, nanobodies, aptamers, affimers, etc. One of ordinary skill will also understand that the methods of detecting a neo-epitope or novel biomarker as described herein as not limiting on the invention, and may include, but are not limited to, immunoassay (e.g., ELISA, Simoa, Olink). lateral flow assays, mass spectroscopy, etc.

Definitions

[0048] As used herein, the term “antibody” refers to a polypeptide or a polypeptide complex that specifically recognizes and binds to antigen through one or more immunoglobulin variable regions. Antibody can be a whole antibody , antigen binding fragment or a single chain thereof. The term “antibody” encompasses various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as alpha, delta, epsilon, gamma, and mu, or a, 5, E, y and p) with some subclasses among them (e.g., yl-y 4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgGl, IgG2. IgG3, IgG4, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly , are within the scope of the instant disclosure. All immunoglobulin classes are within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules.

[0049] Antibodies or antibody antagonists of the present application may include, but are not limited to polyclonal, monoclonal, multispecific, bispecific, trispecific, human, humanized, primatized, chimeric and, single chain antibodies. Antibodies disclosed herein may be from any animal origin, including birds and mammals. In one embodiment, the antibodies are human, murine, rat, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In some embodiments, the variable region may be condricthoid in origin (e.g., from sharks). [0050] The terms “antibody fragment” or “antigen-binding fragment” are used with reference to a portion of antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library and anti-idiotypic (anti- id) antibodies. Regardless of structure, antibody fragment binds with the same antigen that is recognized by the intact antibody. The term “antibody fragment” includes DARTs and diabodies. The term “antibody fragment” also includes any synthetic or genetically engineered proteins comprising immunoglobulin variable regions that act like antibody by binding to a specific antigen to form a complex. A “single-chain fragment variable” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins. In some aspects, the regions are connected with a short linker peptide of ten to about 25 amino acids. The linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. With regard to IgGs. a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are typically joined by disulfide bonds in a “Y” configuration where the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.

[0051] Both the light and heavy chains are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CHI, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention, the numbering of the constant region domains in conventional antibodies increases as they become more distal from the antigenbinding site or amino-terminus of the antibody. In conventional antibodies, the N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy -terminus of the heavy and light chain, respectively.

[0052] As used herein, a “variant” of antibody, antibody fragment or antibody domain refers to antibody, antibody fragment or antibody domain that (1) shares a sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with the original antibody, antibody fragment or antibody domain, and (2) binds specifically to the same target that the original antibody, antibody fragment or antibody domain binds specifically. It should be understood that where a measure of sequence identity is presented in the form of the phrase “at least x % identical” or “at least x % identity ”, such an embodiment includes any and all whole number percentages equal to or above the lower limit.

[0053] Further it should be understood that where an ammo acid sequence is presented in the present application, it should be construed as additionally disclosing or embracing “variant” amino acid sequences having a sequence identity of 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% identity to that amino acid sequence.

[0054] It should be understood that where a sequence homology range is presented herein, as in e.g., the phrase “about 80% to about 100%”, such an embodiment includes any and all subranges within, wherein the lower number can be any whole number between 80 and 100.

[0055] As used herein, the phrase “humanized antibody” refers to 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 which exhibits diminished immunogenicity as compared to the parental antibody when administered to humans.

[0056] As used herein, the phrase “chimeric antibody,” refers to antibody where the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial or modified in accordance with the instant disclosure) is obtained from a second species. In certain embodiments the target binding region or site will be from a non-human source (e.g., mouse or primate) and the constant region is human.

[0057] Included within the scope of the antibodies referred to herein are various compositions and methodologies, including asymmetric IgG-like antibodies (e.g., triomab/quadroma. Trion Pharma/Fresemus Biotech); knobs-into-holes antibodies (Genentech); Cross MAbs (Roche); electrostatically matched antibodies (AMGEN); LUZ-Y (Genentech); strand exchange engineered domain (SEED) body (EMD Serono; BioIonic, Merus); Fab-exchanged antibodies (Genmab), symmetric IgG-like antibodies (e.g. dual targeting (DT)-Ig (GSK/Domantis); two-in-one antibody (Genentech); crosslinked MAbs (Karmanos Cancer Center), mAb2 (F-star); Cov X-body (Cov X/Pfizer); dual variable domain (DVD)-Ig fusions (Abbott); IgG-like bispecific antibodies (Eli Lilly); Ts2Ab (Medimmune/AZ): BsAb (ZymoGenetics); HERCULES (Biogen Idec,TvAb, Roche); scFv/Fc fusions; SCORPION (Emergent BioSolutions/Trubion, ZymoGenetics/BMS); dual affinity retargeting technology (Fc-DART), MacroGenics; dual (scFv)2-Fabs (National Research Center for Antibody Medicine); F(ab)2 fusions (Medarex/ AMGEN); dual-action or Bis-Fab (Genentech); Dock-and-Lock (DNL, ImmunoMedics); Fab-Fv (UCB-Celltech); scFv- and diabody-based antibodies (e.g., bispecific T cell engagers (BiTEs, Micromet); tandem diabodies (Tandab, Affimed); DARTs (MacroGenics); single-chain diabodies; TCR- like antibodies (AIT, Receptor Logics); human serum albumin scFv fusion (Merrimack); COMBODIES (Epigen Biotech); and IgG/non-IgG fusions (e.g., immunocytokines (EMDSerono, Philogen, ImmunGene, ImmunoMedics).

[0058] By ‘'specifically binds” or ‘'has specificity to”, it is generally meant that antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. According to this definition, antibody is said to '‘specifically bind” to an epitope when it binds to that epitope via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term ‘'specificity” is used herein to qualify the relative affinity' by which a certain antibody binds to a certain epitope. For example, antibody “A” may be deemed to have a higher specificity for a given epitope than antibody “B,” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope “D.” In some embodiments, antibody or antibody fragment “has specificity to” antigen if the antibody or antibody fragment forms a complex with the antigen with a dissociation constant (Kd) of 10' ⁶M or less, 10'⁷M or less, 10^-8M or less, 10'⁹M or less, or 10'¹⁰M or less.

A Method of Generating A Monoclonal Antibody For Detection of a Neo-epitope Formed From Amyloidogenic λ free light chain (XFLC) proteins

[0059] An aspect of the application is a method of generating a monoclonal antibody for detection of a neo-epitope formed from amyloidogenic λ free light chain (λFLC) proteins, comprising the steps of: (a) proteolyzing a sample comprising X free light chain (λFLC) proteins with proteinase K, wherein the proteolyzing results in a neo-epitope exposed on the N-terminal of a ~23KDa fragment comprising at least part of the constant domain of the λFLC, or when λFLC is a monomer, a neo-epitope exposed on the N-terminal of a ~11.5 KDa fragment comprising at least part of the constant domain of the λFLC proteins; and wherein the neo-epitope is xQP, where x is one of G, R or S (G=Glycine, R= Arginine, S=Serine); and (b) generating a monoclonal antibody to the neo-epitope on the fragment of the λFLC, wherein the generated monoclonal antibody (LCCD) specifically binds to the neo-epitope of xQP, and wherein proteolyzing conditions are optimized so that an LCCD signal obtained after proteolyzing from the ~23KDa fragment, or the ~11 .5 KDa fragment, is at least two times stronger from an amyloidogenic λFLC compared to a non-amyloidogenic λFLC. One of ordinary skill in the art will understand that the method of determining the LCCD signal is not limiting.

[0060] In certain embodiments, the neo-epitope is GQP. In certain embodiments, the neo-epitope is RQP. In certain embodiments, the neo-epitope is SQP.

[0061] In certain embodiments, the generated antibody (LCCD or dLCCD-G) binds to the neo-epitope of GQP. In certain embodiments, the generated antibody (LCCD-R or dLCCD-R) binds to the neo-epitope of RQP. In certain embodiments, the generated antibody (LCCD-S or dLCCD-S) binds to the neo-epitope of SQP.

[0062] In certain embodiments, the fragment of the λFLC is a homodimeric fragment, which is a ~23kDa fragment under non-denaturing condition. In certain embodiments, the fragment of the λFLC is a monomeric fragment, which is a ~11.5kDa fragment under denaturing condition.

[0063] In certain embodiments, the limited proteolysis by proteinase K comprises the steps of: a liquid sample treated with an optimized proteolysis condition to produce the LCCD domain. In certain embodiments, the optimized proteolysis condition is 0.02-20. OpM Proteinase K at 37°C for 30 seconds to 20 hrs depending on the specific activity of the protease. One of ordinary skill will understand that the specific activity of the protease may be independently determined and is not limiting.

[0064] An aspect of the application is an antibody for detection of a neo-epitope formed from amyloidogenic λ free light chain (λFLC) proteins, wherein the antibody comprises heavy chain encoded by a nucleotide sequence of LCCD-G SEQ.ID. No.: 4 (HC3) and light chain encoded by a nucleotide sequence of LCCD-G SEQ. ID. No.:5 (LC7).

[0065] An aspect of the application is an antibody for detection of a neo-epitope formed from amyloidogenic λ free light chain (λFLC ) proteins, wherein the antibody comprises heavy chain encoded by a nucleotide sequence of LCCD-R SEQ.ID. No.: 6 (HC2) and light chain encoded by a nucleotide sequence of LCCD-R SEQ ID No.: 7 (LC9).

[0066] An aspect of the application is an antibody for detection of a neo-epitope formed from amyloidogenic λ free light chain (λFLC) proteins, wherein the antibody comprises heavy chain needed by a nucleotide sequence of LCCD-S SEQ.ID. No.: 8 (HC2) and light chain encoded by a nucleotide sequence of LCCD-S SEQ ID No: 9 (LC5).

[0067] An aspect of the application is a neo-epitope on λFLC prepared by limited proteolysis of amyloidogenic k free light chain (λFLC) proteins by proteinase K. In certain embodiments, the neo-epitope is xQP, where x is one of G, R or S. In certain embodiments, an antibody is generated to the neo-epitope described herein. In certain embodiments, the antibody is LCCD-G, LCCD-R or LCCD-S.

[0068] An aspect of the application is a method of optimizing limited proteolysis condition using Proteinase K, comprising the steps of optimizing one or more of the following enumerated factors: (1) source of the Proteinase K; (2) composition of the testing sample; (3) concentration of Proteinase K: (4) time of proteolysis reaction; (5) temperature of proteolysis reaction; wherein a proteolysis product of the LCCD fragment is differentially obtained from amyloidogenic λFLC vs. non-amyloidogenic λFLC.

[0069] The key characteristics achieved by the immunoassays described herein include: (1) biomarker correlates with root cause of AL amyloidosis (i.e. unstable variant LC protein links to amyloidogenicity); (2) applicable to majonty if not all AL variants ( λFLC AL as the current focus); (3) easy to access immunoassay that uses biofluids (serum, plasma, and urine); (4) sensitive, specific, and quantitative assay; (5) enables early diagnosis or screening based on amyloidogenicity of each λFLC variant irrespective of the FLC primary sequence or exact λFLC level (personalized medicine); (6) assesses treatment therapeutic response; (7) follows MRD and predicts relapse.

[0070] To develop a diagnostic that can accomplish the desirable characteristics listed above, in comparing amyloidogenic versus non-amyloidogenic FLC, the key principle is that amyloidogenic FLC proteins are thermodynamically and/or kinetically less stable, causing them to be more prone to protein misfolding, proteolysis, and aggregation. With added stress, amyloidogenic FLC readily generates the LCCD fragment and reveals novel cryptic epitopes that can be detected, including with antibodies. To this end, this application discloses the specific stress (i.e. limited proteolysis by Proteinase K) to reveal such neo-epitopes on the LCCD fragment in AL patient plasma samples and mAbs specific for such novel epitopes.

[0071] Considering that different sources of Proteinase K may have varied enzymatic activity , one skilled in the art may conduct optimization of the limited proteolysis by varying the concentration, specific activity, matrix containing the test subject, concentration of test subject, reaction temperature, and reaction time to arrive at a condition where LCCD domain is generated predominantly in AL samples vs. normal or MM samples. [0072] In addition to AL diagnosis and thanks to its sensitivity, this assay is also expected to detect residual amount of amyloidogenic FLC post treatment and at the beginning of a relapse.

A Method of Detection of Presence in a Subject of Amyloidogenic Free Light Chain

(λFLC) Proteins

[0073] An aspect of the application is a method of detection of presence in a subject of amyloidogenic λ free light chain (λFLC) proteins, comprising the steps of: limited proteolysis of proteins from a biological sample, wherein the limited proteolysis results in a neo-epitope exposed on a LCCD fragment of the λFLC proteins; detecting the presence of the neo-epitope on the N-termini of the LCCD fragment of the λFLC proteins; quantitating the presence of the LCCD fragment from the λFLC proteins based on measuring the level of neoepitope on the fragment using the LCCD mAbs; and comparing levels of the LCCD fragment before and after the limited proteolysis, wherein when the level of the LCCD fragment from λFLC proteins is greater after limited proteolysis, then amyloidogenic λFLC proteins are detected. One of ordinary skill will understand that the method determining the level (e.g. quantity) of LCCD fragment is not limiting.

[0074] In certain embodiments, the method further comprises the steps of: measuring a first level of the LCCD fragment of the λFLC proteins present after the limited proteolysis in a biological sample taken from a healthy subject not having light chain amyloidosis (AL), wherein the first level is a reference level; measuring a second level of the fragment of the λFLC proteins present after the limited proteolysis of a biological sample from a person suspected of having AL; and wherein when the second level of the LCCD fragment of the λFLC proteins after the limited proteolysis of the biological sample is greater than the reference level, then amyloidogenic λFLC proteins are detected.

[0075] In certain embodiments, when the level of LCCD fragment from λFLC is detected after limited proteolysis by Proteinase K and at an elevated level above the reference level, then light chain amyloidosis is present in the subject.

[0076] In certain embodiments, an elevated LCCD fragment level in a subject leads to a clinical decision, wherein the clinical decision is treatment and management for light chain amyloidosis (AL).

[0077] In certain embodiments, where absence of an elevated LCCD fragment level in a subject leads to a clinical decision, wherein the clinical decision is ruling out of AL diagnosis and receiving treatment for other systemic amyloidosis, wherein the other systemic amyloidosis may include transthyretin amyloidosis (ATTR). [0078] In certain embodiments, the LCCD fragment is a 23KDa homodimer and is detected and quantified using mass spectrometry.

[0079] In certain embodiments, the LCCD fragment is a 11.5KDa monomer and is detected and quantified using mass spectrometry.

[0080] In certain embodiments, the subject has one of more of 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.

[0081] In certain embodiments, the biological sample is one of plasma, urine, serum, CSF, or tissue biopsy.

[0082] An aspect of the application is a kit for detection of presence in a subject of amyloidogenic λ free light chain (λFLC) proteins, comprising: the antibodies of LCCD-G, LCCD-R and LCCD-S; instructions for using the kit in accordance with the methods described herein.

[0083] In certain embodiments, the method of detection of presence in a subject of amyloidogenic λ free light chain (λFLC) proteins as described herein, further comprises: obtaining one or more readouts from dLCCD signals obtained by measuring the level of neoepitope on the fragment using the LCCD mAbs.

[0084] In certain embodiments, method of detection of presence in a subject of amyloidogenic λ free light chain (λFLC) proteins as described herein, further comprising: obtaining the readout of the dLCCD signal by use of an algorithm where the dLCCD level is a component of.

[0085] Methods of detecting the LCCD fragment resulting from limited PK proteolysis may include mass spectrometry, which will need to account for the limited number of sequence variation in the constant domain of the λFLC.

[0086] Other methods may include immunoassays using antibodies that can recognize the LCCD fragment. For example, gel electrophoresis coupled with Western blot using an anti-λFLC antibody. All commercial anti-λFLC antibodies this study has tested (>20) can recognize not only λFLC, but also λ light chains associated with immunoglobulins. As reported herein, to solve this problem, the study generated rabbit mAbs that specifically recognize one of the three unique xQP neo-epitopes at the N-termini of the LCCD fragment exposed through limited PK proteolysis. This application demonstrates that immunoassays based on the LCCD-G mAh (in other embodiments, LCCD-S and LCCD-R mAbs or a combination of the LCCD mAbs may be used) can sensitively and specifically detect and quantify the LCCD domain post proteolysis and serve as an indicator of the kinetic instability of the λFLC protein.

[0087] LCCD rabbit mAbs described here may be used alone or together in a mixture of two or three mAbs for the assays. In one embodiment, an equal-molar mixture of the three mAbs are prepared and used and is referred to LCCD-X.

[0088] The ~23KDa LCCD or dLCCD fragment is a disulfide-linked homodimer. Under reducing conditions, LCCD presents as a monomer at about 11.5KDa (SEQ ID NO: 2), yet still containing the N-terminal xQP epitope. Such a monomer can also be utilized for the uses intended by the current application.

[0089] One of ordinary skill will understand that variants of the constant domain of the λFLC proteins may include the neo-epitope; the present application also encompasses antibody or antibody fragments that recognize the neo-epitope when it is a part of variants of the constant domain of the λFLC proteins.

[0090] Solution based LCCD immunoassays such as ELISA, MSD, Simoa and Olink can afford improved sensitivity over Western blot method. Such improved sensitivity is especially valuable when applied to early diagnosis and following minimal residual diseases of AL, where the total λFLC levels are much lower.

[0091] Through a combination of limited PK proteolysis and specific detection and quantification of LCCD fragment, this application drastically simplified the detection of amyloidogenic λFLC. In addition, the ease of the LCCD immunoassays using biofluids such as plasma, serum, and urine samples may enable point-of-care usage of such assays, further expanding its utility.

[0092] The LCCD immunoassay can be used to demonstrate target engagement for treatment targeting stabilization of amyloidogenic λFLC (as described in, but not limited to, WO2022/226166). This method may be used as companion diagnostics to select AL patients who are most likely to respond to an LC stabilizer in the clinic, enabling personalized medicine.

[0093] LCCD signals can also be used in combination with other clinical biomarkers and assessments, such as the λFLC level, dFLC, Troponin, and NT-proBNP to guide clinical practice. Levels of the LCCD biomarker measured using ex vivo samples from the patient can also be used to assess AL disease prognosis. Due to the pathogenic nature of the LCCD biomarker, higher level of LCCD level may indicate worse clinical outcomes. [0094] The method of combining limited proteolysis on λFLC and detection of the resulting LCCD fragment can also be used in the course of drug discovery in identifying molecules that can influence λFLC stability, and for the purpose of finding potent drug candidates.

[0095] Other biological samples containing λFLC, such as urine and bone marrow biopsy may also be used in the limited Proteinase K proteolysis coupled with the LCCD immunoassay or mass spectrometry methods for the diagnosis and treatment response assessment of AL.

[0096] Other reagents that specifically bind to the N-terminal exposed xQP neoepitope, such as aptamer, nanobody, etc. may also be generated and used for the detection and quantification of the LCCD biomarker.

A Method of Assessing Target Engagement of a FLC Stabilizer or Treatment Response for Treatment of Light Chain Amyloidosis

[0097] An aspect of the application is assessing target engagement of a FLC stabilizer for treatment of light chain amyloidosis, comprising the steps of: limited proteolysis of proteins from a biological sample, wherein the biological sample is a recombinant protein or from a subject receiving treatment by a targeted therapeutic molecule for light chain amyloidosis, and wherein the limited proteolysis results in a neo-epitope exposed on a LCCD fragment of the λFLC proteins; detecting the presence of the neo-epitope on an N-termini of the LCCD fragment of the λFLC proteins; and quantitating the level of the fragment of the λFLC proteins resulting from the limited proteolysis based on the neo-epitope presence detected, wherein target engagement is assessed by comparing a first level of quantity of the LCCD fragment present before the targeted therapeutic molecule has been introduced to the subject, or to a sample from a subject ex vivo, to a second level of quantity of the LCCD fragment present after the targeted therapeutic molecule has been introduced to the subject, or to a sample from a subject ex vivo, wherein a reduction in the LCCD level, or the LCCD level normalized to the total λFLC in the second level of quantification indicates target engagement.

[0098] An aspect of the application is a method of assessing therapeutic response of a therapeutics for treatment of light chain amyloidosis by quantifying a LCCD fragment in plasma samples before and after the therapeutic molecule has been introduced to the subject, or at two different time points during the treatment, wherein a reduction in the post-treatment or second timepoint level of LCCD indicates favorable response to the therapeutic molecule, irrespective to the total level of λFLC. Said therapeutics include but are not limited to those that are Light Chain stabilizers, plasma cell-targeted therapies, gene and cell therapies, stem cell transplant, and chemotherapeutic agents. Assessment based on the dLCCD biomarker may be done one time or multiple times following the said therapeutic treatment.

[0099] An aspect of the application is a method of assessing if an AL patient would benefit from a light chain stabilizer as a therapy by quantifying a LCCD fragment in plasma samples from a subject being considered to receive treatment by a light chain stabilizer molecule for light chain amyloidosis, wherein the ex vivo plasma sample is incubated with the said stabilizer or a solvent control, before subjecting the samples to limited proteolysis, and quantification of the resulting LCCD levels, wherein a reduction of LCCD level in the sample that was incubated with the stabilizer versus the solvent control sample indicates the subject may benefit from treatment by the said light chain stabilizer.

[0100] In certain embodiments, when the first level of the LCCD fragment present before the targeted therapeutic molecule has been introduced to the subject is greater than the second level of the LCCD fragment present after the targeted therapeutic molecule has been introduced to the subject, then target engagement or treatment response has occurred by the targeted therapeutic molecule.

[0101] In certain embodiments, when the first level of the LCCD fragment present before the targeted therapeutic molecule has been introduced to the subject is the same or less than the second level of the LCCD fragment present after the targeted therapeutic molecule has been introduced to the subject, then target engagement or treatment response has not occurred by the targeted therapeutic molecule. In certain embodiments, the biological sample is one of plasma, urine, serum, cerebrospinal fluid (CSF), or tissue biopsy.

[0102] In addition to its important utilities in AL diagnosis and target engagement monitoring, xQP mAbs may have other general utilities in biotechnology. xQP may be the smallest non-charged N-terminal protein tag known. N-terminal or C-terminal tags are widely used in recombinant proteins and cellular systems for the purpose of target protein detection and/or purification. Antibodies binding to these tags are important tools in studying target proteins and protein complexes. Commonly used tags include His tag, Flag tag, HA tag, and myc tag, all containing 6-10 charged amino acids. The three new mAbs (LCCD-G, LCCD-S, and LCCD-R) discussed in the application specifically bind to small N-terminal tags of 1-5 amino acids, with GQP and SQP non-charged. In certain aspects, such small tags (epitopes) can be placed at the N-terminal of a protein of interest through genetic engineering to facilitate such utility. Clinical utilities of Limited proteolysis in combination with the LCCD mAbs

[0103] In certain embodiments, the methods and compositions described herein can be used for diagnosis of Light Chain Amyloidosis (AL) amongst plasma cell disorder patients suspected of Multiple myeloma (MM), Light Chain Amyloidosis (AL), Smoldering Multiple Myeloma (SMM), Monoclonal Gammopathy of Undetermined Significance (MGUS), and IgM-AL, also including those who have no clinical symptoms.

[0104] In certain embodiments, the methods and compositions described herein can be used to demonstrate target engagement by measuring the stabilization effect of a light chain stabilizer clinical candidate (or drug) for its protection against proteolysis in individual AL patient plasma sample post treatment.In certain embodiments, the methods and compositions described herein can be used to monitor response to light chain stabilizer or plasma cell-targeting therapeutic treatment in AL amyloidosis patients.

[0105] In certain embodiments, the methods and compositions described herein can be used for prognostic biomarkers, such as the level of LCCD biomarker can be used to indicate disease severity and clinical outcomes.

[0106] In certain embodiments, the methods and compositions described herein can be used for detection of Minimal Residual Disease (MRD) and Relapse: Detection of Light Chain Amyloidosis (AL) MRD and relapse by measuring the amount of LCCD signal in patient plasma samples.

[0107] In certain embodiments, the methods and compositions described herein can be used for companion diagnostics, such as to test the ability of a LC stabilizer clinical candidate (or drug) for its protection against proteolysis in individual pre-treatment AL patient plasma sample ex vivo and use the outcome to determine if the patient would benefit from treatment with the LC stabilizer.

[0108] In certain embodiments, the methods and compositions described herein can be used for surrogate biomarkers or therapeutic response markers, such as to demonstrate the proteolysis protection effect of a LC stabilizer clinical candidate (or drug) and use that as an indication of clinical efficacy.

[0109] For the purpose of the above uses, other biological samples, such as urine, serum, CSF, tissue biopsy, etc. may also be used.

[0110] The present application is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures and Tables, are incorporated herein by reference. EXAMPLES

ELISA assay using LCCD mAb

[0111] LCCD mAbs were tested for their binding to WIL and IgG with and without treatment of Proteinase K (PK) in a Sandwich ELISA binding assay. Briefly, goat anti-IgG (H+L) antibody from Bethyl on a high-binding ELISA plate at 1 pg/mL concentration in a pH 9.6 carbonate buffer and incubated at 4 °C for overnight. After blocking with Superblock, serial dilutions of WIL LC and purified human IgG with and without PK (0.2 pM) digestion were added to the plate and incubated at room temperature for 1 hour. After washing away the unbound protein. LCCD mAb at 1 pg/mL 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, the plate was washed thoroughly, then the enzyme substrate tetramethylbenzidine (TMS) was added to each well. The reaction was stopped by adding 0.5 M of sulfuric acid and absorbance at 450 nm was read on a plate reader.

Limited Proteolysis Method for Generation of LCCD from λFLC

[0112] In one embodiment, plasma samples were incubated with IpM of Proteinase K at 37°C for 2 hrs before the reaction was stopped by adding phenylmethylsulfonyl fluoride protease inhibitor.

Detection of dLCCD in plasma using Western Blot

[0113] Plasma samples treated with Proteinase K using the limited proteolysis condition were run on non-reducing SDS-PAGE gels, followed by standard Western blot procedure. In one embodiment, the gel was transferred onto a nitrocellulose membrane. The membrane was then blocked with blocking reagent and incubated with 0.5 pg/mL LCCD-G mAb at 4 °C overnight. Anti-rabbit conjugated with 800 dye was used and the washed blot was imaged on an LiCor Odyssey system. The fluorescence intensity of the dLCCD band at around 23 KDa was recorded and the fluorescence signal quantified.

Example 1: Identification of a neo-epitope after limited proteolysis of

[0114] Amyloidogenic /.FLC tends to be kinetically less stable compared to the non- amyloidogenic counterparts, rendering it susceptible to denaturing conditions. Limited proteolysis has been widely used to probe protein structural dynamicity. Herein, kinetic stability of λFLC is assessed using limited proteolysis where the FLC protein is incubated with a protease. Quantification of the intact full-length FLC protein remaining or the proteolysis product(s) is used as an indication of the FLC protein stability. Proteinase K (PK) has previously been used to study the stability of λFLC. although it was assumed λFLC would yield small peptides after PK treatment, and only the full-length intact λFLC was followed and analyzed. PK is considered a broad-spectrum protease. The predominant site of PK cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups. Proteins can be completely digested by PK if the incubation time is long and PK concentration high enough. 5pM of the LC recombinant protein was treated with 200nM Proteinase K at 37°C for 2 hrs. The gel was run under nonreducing condition, stained and visualized with Coomassie dye. Under currently optimized limited PK treatment, an about 23KDa molecular weight band was consistently generated (nonreducing condition), from recombinant amyloidogenic λFLC protein, such as WIL (Figure 1 A), regardless of the primary sequence of the amyloidogenic lambda LC constructs. This fragment was not readily generated under the same condition using non-amyloidogenic lambda LC proteins such as JTO. Edman degradation sequencing identified GQPKA at the N-term of this 23KDa band, abbreviated herein as the dLCCD, LCCD, dLCCD fragment, dLCCD biomarker, or dLCCD domain, corresponds to the disulfide bond-linked homodimeric constant domain of the protein, (Figure IB). Under reducing condition, a fragment of about 11.5KDa representing the monomeric LCCD will be generated. The cleavage site constituted by the GQPKA sequence is a neo-epitope exposed only through limited proteolysis by PK.

[0115] The consistent generation of this fragment on λFLC is unexpected given the ambiguous digestion nature of Proteinase K known in the art.

[0116] The PK recognition site, residing at the immediate N-terminal of this neoepitope, is >99% conserved in λFLC and was confirmed by analyzing the public databases ABYSIS and AL Bases containing >6,000 different λFLC sequences (Figure 1C). Sequence analysis of the λFLC sequence databases indicates that the Gly, Ser, or Arg at the beginning of the neo-epitope accounts for around 84%, 11% and 5% of all λFLC sequences, respectively. The neo-epitopes are therefore referred to as xQP, where x = G, S, or R in the sequence.

EXAMPLE 2: Generation and characterization of LCCD monoclonal antibodies

[0117] Monoclonal antibodies (mAb) were generated through immunizing rabbits using peptide-KLH conjugates as the antigens. Peptide antigen contains the sequence of xQPKA, where x = G, S, or R in the sequence. Anti-serum titer was monitored using ELISA against the antigen. Upon detection of high titer, antigen-specific B cells were isolated for specific recognition of the antigen. Cells were expanded and antibody genes amplified and cloned. The amplified antibody heavy and light chains were expressed in HEK293 cell, and the supernatant assayed by ELISA to confirm the antibody specificity. mAbs from the immunizations were selected, sequenced, and the mAbs were named LCCD-G, LCCD-S, and LCCD-R, respectively, based on the identity of x in the xQP neo-epitopes.

[0118] Purified recombinant LC proteins were first used to test the specificity of the LCCD mAb in a Sandwich ELISA assay, where LCCD mAb was used as the capture antibody, and a goat polyclonal anti-LC antibody was used as the detection antibody. Recombinant WIL, an amyloidogenic λFLC protein, before and after limited PK digestion were subjected to the ELISA assay. The data indicated that LCCD-G mAb binds strongly to only the PK digested WIL LC and has minimum or no binding to full-length undigested LC (Figure 2A). In human proteome, the same light chain protein can also heterodimerize with heavy chain proteins and form full-length immunoglobulin. The study investigated if the light chain in immunoglobulin is stable under the limited proteolysis condition. To test that, purified human IgG was treated with PK and the resulting samples subjected to the LCCD ELISA assay. As shown in Figure 2B. no signal was detected after PK treatment of IgG. EXAMPLE 3; Quantification of LCCD in AL plasma after limited proteolysis

[0119] LCCD mAb was used to detect and quantify the proteolysis product in AL patient plasma samples using standard Western blot (WB) and imaged on a LiCor system (Figure 3A). LCCD mAb is highly specific to the LCCD or dLCCD fragment generated after the limited proteolysis and does not detect any other signal in the AL plasma samples (Figure 3A). Of the 43 plasma samples from treatment naive AL patients, 37 samples (86.0%) have detectable LCCD-G signal under the limited proteolysis condition.

[0120] A moderate correlation between dLCCD fluorescence signal and λFLC measured by the FreeLite clinical test based on turbidimetry was observed in 20 of the treatment naive AL plasma samples (Figure 3B). No such correlation can be draw n for normal or MM plasma samples under the current limited proteolysis condition.

[0121] Under optimized stress condition, the mAb detects signal in 32 randomly selected λAL plasma samples in this application employing standard Western blot analysis using a few microliters of samples. The signal is much lower if not undetectable in normal (N=23) and 18 out of the 19 MM plasma samples under the same stress condition. In fact, the mean signal in AL samples is 10x-200x above that of normal or most MM plasma samples and the result is statistically significant. It may be that the single MM patient with high signal had co-existing AL. This result establishes the assay’s utility as AL diagnostics. EXAMPLE 4; LCCD immunoassay differentiates AL from normal plasma

[0122] Under the limited proteolysis condition, LCCD mAb differentiates AL plasma samples from normal or MM plasma samples (Figure 4, p=0007 and 0.0008, respectively) using Western Blot. This method is therefore used to specifically diagnose AL patients or follow MRD and relapse based on the dLCCD signal in the patient samples.

[0123] The ability to specifically detect amyloidogenic FLC through the detection and quantification of LCCD signal with the aid of limited proteolysis in biological samples forms the foundation for the diagnostics utility of LCCD-based assays.

EXAMPLE 5; LCCD immunoassay for quantification of dLCCD

[0124] Figure 5A shows the steps of an ELISA-based dLCCD immunoassay that differentiates AL from MM, Normal, and ATTR post limited proteolysis using ex vivo blood samples. The Meso Scale Discovery (MSD) platform is used in the example. The steps of the plasma/serum assay led to a combination of a tagged detection antibody (anti-LC pAb), which binds to the analy te (dLCCD), which is in turn bound to a capture antibody (dLCCD mAbs). which is supported on a working electrode. The MSD electrochemiluminescence has high sensitivity, and a large dynamic range (>400x); typically, a 96- or 384-well format is used with as little as approx. 2pl biofluid per assay. A readout is obtained in a few hours. Figure 5B shows the outcome of a dLCCD assay with plasma sample post limited proteolysis. dLCCD levels differentiate λAL from normal (N=40), multiple myeloma (MM) (N=19), and TTR amyloidosis (ATTR, including cardiomyopathy and polyneuropathy phenotypes) (N=35) patients. Recombinant dLCCD (homodimer of SEQ ID NO: 2 linked by disulfide bond) was used as the reference standard.

EXAMPLE 6; High baseline dLCCD correlates to poor overall survival

[0125] Figure 6 shows baseline levels of dLCCD correlates to λAL patient overall survival. dLCCD in the experiment was detected at the 2 hour time point. Figure 6A shows log-rank survival test of patients with high (hatched line) (N=25) and low (solid line) (N=25) levels of baseline dFLC. dFLC level of the same samples were determined by the FreeLite assay. Figure 6B shows log-rank survival test of patients with high (solid line) (N=25) and low (hatched line) (N=25) levels of baseline dLCCD. The dLCCD levels were quantified on treatment naive plasma samples from 50 confirmed λAL patients using the MSD dLCCD immunoassay described herein.

EXAMPLE 7; dFLC18 and dLCCD300 concordance predicts overall survival

[0126] Figure 7 shows concordance of dFLC and dLCCD predicts overall survival based on the data from Figure 6. The figures shows a log-rank survival test of patients with low baseline levels of both biomarkers (red curve) (N=17) comparing to those with high levels of both biomarkers (green curve) (N=17). and those with discordant dFLC and dLCCD (blue curve) (N=16). High and low levels of dFLC and dLCCD are defined by the median value of 18 mg/dL for baseline dFLC and 300 nM for dLCCD. The results are based on treatment naive plasma samples from 50 confirmed λAL patients using an MSD LCCD immunoassay as described herein. There are significant differences in OS; in particular, median OS: dFLC^low/dLCCD^low 162 months; dFLC^high/dLCCD^high 36 months; discordant 16 months. dLCCD level further correlates to cTnT and NT-proBNP (p=0.05). dLCCD is a pathogenic-indicating biomarker in most λAL patients. Baseline dLCCD is a better OS predictor than dFLC. Accordingly, reducing dLCCD levels by therapeutics is important to achieve clinical benefits; for example, by eradicating amyloidogenic FLC producing clonal plasma cells, or by stabilizing λFLC to minimize its amyloidogenicity, or a combination of the above two.

Example 8; dLCCD biomarker level as an indicator of LC stabilizer target engagement in biofluids or as response to therapy

[0127] Small molecules that bind directly to the dimer interface of the dimeric λFLC protein were shown to stabilize the protein and reduce its amyloidogenicity (Morgan et al. 2019, PMC6486714). Such small molecules are considered light chain (LC) stabilizers. By comparing the levels of dLCCD generated in ex vivo plasma samples before and after compound treatment and post limited proteolysis using Proteinase K, one can quantify the level of unstable or amyloidogenic λFLC in the biofluids and use that as an indicator of target engagement. In Figure 8, LC stabilizer PTG-1412 w as incubated with the ex vivo AL plasma, and the assay showed dose-dependent protection of the λFLC in the sample.

[0128] One skilled in the art may conduct the same assay on biofluid from an AL patient before and after therapeutic treatment and use the difference in the dLCCD levels as an indicator for response to therapy or the lack thereof.

[0129] While various embodiments have been described above, it should be understood that such disclosures have been presented by way of example only and are not limiting. Thus, the breadth and scope of the subject compositions and methods should not be limited by any of the above-described exemplary' embodiments but should be defined only in accordance with the following claims and their equivalents.

[0130] The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present invention, and it is not intended to detail all those obvious modifications and variations of it which will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present invention, which is defined by the following claims. The claims are intended to cover the components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates the contrary.

REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0131] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on February 21, 2024, is named “PROT 119 WO.xmT’ and is 19,125 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.

APPENDIX

Monomeric LCCD sequence (SEQ ID NO: 1):

GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSS PVKAGVETTTPSKQS

NNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS

The N-terminal G can be S or R in the xQP epitope. Monomeric LCCD can form dimeric

LCCD protein through disulfide bonding via the C-terminal Cys (underlined).

A few other human constant domain sequences exist but retain the N-terminal xQP epitope.

These are collectively referred to as LCCD, dLCCD, dLCCD biomarker, LCCD fragment,

LCCD Biomarker, or LCCD domain.

Amyloidogemc FLC protein WIL (SEQ ID No.: 2):

NFLLTQPHSVSES PGKTVTISCTRSSGS IANNYVHWYQQRPGSS PTWI FEDDHRPSGVPDR FSGSVDTSSNSASLTISGLKTEDEADYYCQSYDHNNQVFGGGTKLTVLGQPKAAPSVTLFPP SSEELQANKATLVCLISDFYPGAVTVAWKADSS PVKAGVETTTPSKQSNNKYAAS SYLSLTP EQWKSHKSYSCQVTHEGSTVEKTVAPTECS

Non-amyloidogenic FLC protein JTO (SEQ ID No.: 3):

NFMLNQPHSVSES PGKTVTISCTRSSGNIDSNYVQWYQQRPGSAPITVIYEDNQRPSGVPDR FAGS IDRSSNSASLTISGLKTEDEADYYCQSYDARNWFGGGTRLTVLGQPKAAPSVTLFPP SSEELQANKATLVCLISDFYPGAVTVAWKADSS PVKAGVETTTPSKQSNNKYAAS SYLSLTP EQWKSHKSYSCQVTHEGSTVEKTVAPTECS

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

ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAG

TCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTC ACCTGCACAGTCTCTGGATTCTCCCTCAGTAGTTATGCATTGAGCTGGGTCCGCC AGGCTCCAGGGAAGGGGCTGGAATGGGTCGGAATGATTGATACTGATGATATCA TATATTATGCGAGCTGGGCAAAAGGCCGATTCACCATCTCCAAAACCTCGACCAC

GGTGGATCTGAGAGTGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTG TGCCAGAGAGCCTGATGTTGGTAGTAGTTGGGACATCTGGGGCCCAGGCACCCT GGTCACCGTCTCCTTAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCC

TGCTGCGGGGACACACCCAGCTCCACGGTCACCCTGGGTTGTCTTGTGAAGGGATACCTCC CGGAACCCGTGACCGTGACCTGGAACTCCGGCACCCTGACCAATGGAGTGCGGACCTTCCCG AGCGTCAGGCAGTCCTCCGGGTTGTACAGCTTGTCTAGCGTGGTGTCCGTGACGTCGTCAAG CCAGCCTGTGACTTGCAATGTGGCCCATCCGGCCACCAACACCAAGGTCGACAAGACCGTGG

CGCCTTCCACCTGTTCCAAGCCCACTTGCCCGCCGCCTGAGCTCCTGGGAGGACCGTCCGTG

TTCATCTTCCCTCCAAAACCCAAGGATACCCTGATGATTAGCCGCACTCCCGAAGTCACTTG

CGTGGTCGTGGACGTGTCGCAGGACGATCCTGAGGTGCAGTTCACTTGGTACATCAACAACG

AACAAGTCCGGACAGCTAGACCACCGCTGCGCGAGCAGCAGTTCAACTCAACTATCCGGGTG GTGTCCACCCTGCCGATCGCGCATCAGGATTGGCTGCGGGGGAAGGAGTTCAAGTGCAAAGT CCACAACAAGGCCCTGCCCGCCCCCATCGAAAAGACCATCTCCAAGGCTCGGGGCCAGCCTC TGGAGCCCAAAGTGTACACCATGGGCCCGCCTCGCGAGGAGCTCTCCTCACGCTCGGTGTCG CTGACTTGCATGATTAACGGCTTCTACCCTTCCGACATCTCCGTGGAATGGGAGAAGAACGG AAAAGCCGAAGATAACTACAAGACCACGCCCGCCGTGCTGGACTCCGACGGAAGCTATTTCC TGTACTCCAAGCTCTCCGTCCCCACTTCGGAATGGCAGAGGGGGGACGTGTTCACTTGCTCC GTGATGCACGAGGCACTCCACAACCACTACACCCAAAAGAGCATTTCGCGGTCACCTGGCAA GTAA

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

ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCA CATTTGCCGCCGTGCTGACCCAGACACCATCTCCCGTGTCTGCAGCTGTGGGAGG CACAGTCAGCATCAGTTGCCACTCCAGTCAGAGTGTTTATAATAACAACTGGTTA GCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACGATGCA TCGAAACTGGCATCTGGGGTCCCATCGCGATTCAGCGGCAGTGGATCTGGGACA GAGTTCACTCTCACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACT GTGCAGGCGGTTATGTTATTGGTGGTGGTGTGCGGGCTTGCGGCGGCGGGACCG AGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTG CTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGCGTGGCTAACAAGTACTTCCCGGAC GTGACCGTGACCTGGGAAGTCGACGGAACCACTCAGACCACTGGTATCGAGAACAGCAAGAC GCCCCAGAACTCCGCCGATTGTACTTATAACCTGTCCTCCACACTGACCCTCACCTCGACCC AGTACAATTCCCACAAGGAGTACACTTGCAAAGTCACCCAGGGAACCACTTCAGTGGTGCAG AGCTTCAACCGGGGGGATTGCTGA

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

ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAG TCGGTGAAGGAGTCCGAGGGAGGTCTCTTCAAGCCAACGGACACCCTGACACTC ACCTGCACAGTCTCTGGATTCTCCCTCAGTACCTATGGAGTGAGCTGGGTCCGCC

AGGCTCCAGGGAACGGGCTGGAATACATCGGATTCATTGGTAGTGGTGGTGGCA CATACTACGCGAGCTGGGCGAAAAGCCGATCCACCATCACCAGAAACACCAACC TGAACACGGTGACTCTGAAAATGACCAGCCTGACAGCCGCGGACACGGCCACCT ATTTCTGTGCGAGAGATACCATTGTCGATAGTGATCCTTATGAATATGTCCACTA CGGCATGGACCCCTGGGGCCCAGGGACCCTCGTCACCGTCTCTTCAGGGCAACCT AAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACG GTCACCCTGGGTTGTCTTGTGAAGGGATACC T C C C G G AAC CCGTGACCGTGACCTG G AAC T C CGGCACCCTGACCAATGGAGTGCGGACCTTCCCGAGCGTCAGGCAGTCCTCCGGGTTGTACA GCTTGTCTAGCGTGGTGTCCGTGACGTCGTCAAGCCAGCCTGTGACTTGCAATGTGGCCCAT CCGGCCACCAACACCAAGGTCGACAAGACCGTGGCGCCTTCCACCTGTTCCAAGCCCACTTG CCCGCCGCCTGAGCTCCTGGGAGGACCGTCCGTGTTCATCTTCCCTCCAAAACCCAAGGATA CCCTGATGATTAGCCGCACTCCCGAAGTCACTTGCGTGGTCGTGGACGTGTCGCAGGACGAT CCTGAGGTGCAGTTCACTTGGTACATCAACAACGAACAAGTCCGGACAGCTAGACCACCGCT GCGCGAGCAGCAGTTCAACTCAACTATCCGGGTGGTGTCCACCCTGCCGATCGCGCATCAGG ATTGGCTGCGGGGGAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCCCTGCCCGCCCCCATC GAAAAGACCATCTCCAAGGCTCGGGGCCAGCCTCTGGAGCCCAAAGTGTACACCATGGGCCC GCCTCGCGAGGAGCTCTCCTCACGCTCGGTGTCGCTGACTTGCATGATTAACGGCTTCTACC CTTCCGACATCTCCGTGGAATGGGAGAAGAACGGAAAAGCCGAAGATAACTACAAGACCACG CCCGCCGTGCTGGACTCCGACGGAAGCTATTTCCTGTACTCCAAGCTCTCCGTCCCCACTTC GGAATGGCAGAGGGGGGACGTGTTCACTTGCTCCGTGATGCACGAGGCACTCCACAACCACT

ACACCCAAAAGAGCATTTCGCGGTCACCTGGCAAGTAA LCCD-R mAb LC9 (SEQ ID NO.: 7)

ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCC AGGTGC C A GATGTGCTGACATTGTGATGACCCAGACTCCAGCCTCCGTGGAGGCAGCTGTGG GAGGCACAGTCACCATCAAGTGCCAGGCCAGTGAGAACATTTATACCAATTTAG

CCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTATGATGCATC CGATCTGGCATCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGATCTGGGACAGA CTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGT CAAGGCGGTTATTATAGTAGTAGTAATACTTATACTTGTAATGCTTTCGGCGGAG GGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCA CCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTG TGCGTGGCTAACAAGTACTT CCCGGACGTGACCGTGACCTGGGAAGTCGACGGAACCACTCAGACCACTGGTATCGAGAACA GCAAGACGCCCCAGAACTCCGCCGATTGTACTTATAACCTGTCCTCCACACTGACCCTCACC TCGACCCAGTACAATTCCCACAAGGAGTACACTTGCAAAGTCACCCAGGGAACCACTTCAGT GGTGCAGAGCTTCAACCGGGGGGATTGCTGA

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

ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAG TCAGTGAAGGAGTCCGAGGGAGGTCTCTTCAAGCCAACGGATACCCTGACACTC ACCTGCACAGTCTCTGGATTCTCCCTCGGTAGGTACAACATGGCCTGGGTCCGCC AGGCTCCAGGGGAGGGGCTGGAATACATCGGATGGATTACTTATAATAATTACG TGCACTACGCGAGCTGGGCGAAAAGCCGATCCACCGTCACCAGAAACACCAACG AGAACACGGTGACTCTGAAAATGACCAGTCTGACAGCCGCGGACACGGCCACCT ATTTCTGTGCGAGAGAGGGGGATGTTGGTGAGAATGACATCTGGGGCCCAGGCA CCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGC CCCCTGCTACGGGGACACACCCAGCTCCACGGTCACCCTGGGTTGTCTTGTGAAGGGATAC CTCCCGGAACCCGTGACCGTGACCTGGAACTCCGGCACCCTGACCAATGGAGTGCGGACCTT CCCGAGCGTCAGGCAGTCCTCCGGGTTGTACAGCTTGTCTAGCGTGGTGTCCGTGACGTCGT CAAGCCAGCCTGTGACTTGCAATGTGGCCCATCCGGCCACCAACACCAAGGTCGACAAGACC GTGGCGCCTTCCACCTGTTCCAAGCCCACTTGCCCGCCGCCTGAGCTCCTGGGAGGACCGTC CGTGTTCATCTTCCCTCCAAAACCCAAGGATACCCTGATGATTAGCCGCACTCCCGAAGTCA CTTGCGTGGTCGTGGACGTGTCGCAGGACGATCCTGAGGTGCAGTTCACTTGGTACATCAAC AACGAACAAGTCCGGACAGCTAGACCACCGCTGCGCGAGCAGCAGTTCAACTCAACTATCCG GGTGGTGTCCACCCTGCCGATCGCGCATCAGGATTGGCTGCGGGGGAAGGAGTTCAAGTGCA AAGTCCACAACAAGGCCCTGCCCGCCCCCATCGAAAAGACCATCTCCAAGGCTCGGGGCCAG CCTCTGGAGCCCAAAGTGTACACCATGGGCCCGCCTCGCGAGGAGCTCTCCTCACGCTCGGT GTCGCTGACTTGCATGATTAACGGCTTCTACCCTTCCGACATCTCCGTGGAATGGGAGAAGA ACGGAAAAGCCGAAGATAACTACAAGACCACGCCCGCCGTGCTGGACTCCGACGGAAGCTAT TTCCTGTACTCCAAGCTCTCCGTCCCCACTTCGGAATGGCAGAGGGGGGACGTGTTCACTTG CTCCGTGATGCACGAGGCACTCCACAACCACTACACCCAAAAGAGCATTTCGCGGTCACCTG GCAAGTAA

LCCD-S mAb LC5 (SEQ ID NO.: 9) ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCA

CATTTGCCCAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGG CACAGTCACCATCAACTGCCAGGCCAGTCAGAGTCTTTATAATAACAAAAATTTA GCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATTATGCAT CCACTCTGGCAACTGGGGTCCCATCACGGTTCAAAGGCAGTGGATCTGGGACAC AGTTCACTCTCACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTTTTG

TCAAGGCGAATTTCCTTGTAGCAGTGCTGATTGTGGTGCTTTCGGCGGAGGGACC GGGGTGGTGGTC A A AGGTGATC C AGTTGC AC C TACTGTCCTCATCTTCCCACCAGCT GCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGT G C GT G GCT AAC AAGT ACT T C C C G GA CGTGACCGTGACCTGGGAAGTCGACGGAACCACTCAGACCACTGGTATCGAGAACAGCAAGA CGCCCCAGAACTCCGCCGATTGTACTTATAACCTGTCCTCCACACTGACCCTCACCTCGACC

CAGTACAATTCCCACAAGGAGTACACTTGCAAAGTCACCCAGGGAACCACTTCAGTGGTGCA GAGCTTCAACCGGGGGGATTGCTGA

Claims

WHAT IS CLAIMED IS:

1. A method of generating a monoclonal antibody for detection of a neo-epitope formed from amyloidogenic λ free light chain (λFLC) proteins, comprising the steps of:

(a) proteolyzing a sample comprising λ free light chain (λFLC) proteins with proteinase K, wherein the proteolyzing results in a neo-epitope exposed on the N-terminal of a ~23KDa fragment comprising at least part of the constant domain of the λFLC, or when λFLC is a monomer, a neo-epitope exposed on the N-terminal of a ~11.5 KDa fragment comprising at least part of the constant domain of the λFLC proteins; and wherein the neo-epitope is xQP, where x is one of G, R or S (G=Glycine, R= Arginine, S=Serine); and

(b) generating a monoclonal antibody to the neo-epitope on the fragment of the λFLC, wherein the generated monoclonal antibody (LCCD) specifically binds to the neo-epitope of xQP, and wherein proteolyzing conditions are optimized so that an LCCD signal obtained after proteolyzing from the ~23KDa fragment, or the ~11.5 KDa fragment, is at least two times stronger from an amyloidogenic λFLC compared to a non-amyloidogenic λFLC.

2. The method of Claim 1, wherein the neo-epitope is GQP.

3. The method of Claim 1 , wherein the neo-epitope is RQP.

4. The method of Claim 1, wherein the neo-epitope is SQP.

5. The method of Claim 2, wherein the generated antibody (LCCD-G) binds to the neoepitope of GQP.

6. The method of Claim 3, wherein the generated antibody (LCCD-R) binds to the neoepitope of RQP.

7. The method of Claim 4, wherein the generated antibody (LCCD-S) binds to the neoepitope of SQP.

8. The method of any one of Claims 1-7, wherein the fragment of the λFLC is a homodimeric fragment, which is a ~23kDa fragment under non-denaturing condition.

9. The method of any one of Claims 1-7, wherein the fragment of the λFLC is a monomeric fragment, which is a ~11.5kDa fragment under denaturing condition.

10. The method of any one of Claims 1-9. wherein the limited proteolysis by proteinase K comprises the steps of: a liquid sample treated with an optimized proteolysis condition to produce the LCCD domain.

11. The method of any one of Claims 1-10, wherein the optimized proteolysis condition is 0.02-20. OpM Proteinase K at 37°C for 30 seconds to 20 hours depending on the specific activity of the protease.

12. An antibody for detection of a neo-epitope formed from amyloidogenic λ free light chain (λFLC) proteins, wherein the antibody comprises heavy chain encoded by a nucleotide sequence of LCCD-G SEQ.ID. No.: 4 (HC3) and light chain encoded by a nucleotide sequence of LCCD-G SEQ.

ID. No.:5 (LC7).

13. An antibody for detection of a neo-epitope formed from amyloidogenic λ free light chain (λFLC) proteins, wherein the antibody comprises heavy chain encoded by a nucleotide sequence of LCCD-S SEQ.ID. No.: 8 (HC2) and light chain encoded by a nucleotide sequence of LCCD-S SEQ ID No: 9 (LC5).

14. An antibody for detection of a neo-epitope formed from amyloidogenic λ free light chain (λFLC) proteins, wherein the antibody is encoded by a nucleotide sequence encoding LCCD-S (SEQ.ID. No.: 8 (HC2) and SEQ ID No: 9 (LC5)).

15. A method of assessing target engagement of a FLC stabilizer for treatment of light chain amyloidosis, comprising the steps of: limited proteolysis of proteins from a biological sample, wherein the biological sample is a recombinant protein or from a subject receiving treatment by a targeted therapeutic molecule for light chain amyloidosis, and wherein the limited proteolysis results in a neo-epitope exposed on a LCCD fragment of the λFLC proteins; detecting the presence of the neo-epitope on an N-termini of the LCCD fragment of the λFLC proteins; and quantitating the level of the fragment of the λFLC proteins resulting from the limited proteolysis based on the neo-epitope presence detected, wherein target engagement is assessed by comparing a first level of quantity of the LCCD fragment present before the targeted therapeutic molecule has been introduced to the subject, or to a sample from a subject ex vivo, to a second level of quantity of the LCCD fragment present after the targeted therapeutic molecule has been introduced to the subject, or to a sample from a subject ex vivo, wherein a reduction in the LCCD level, or the LCCD level normalized to the total λFLC in the second level of quantification indicates target engagement.

16. A method of assessing therapeutic response of a therapeutics for treatment of light chain amyloidosis by quantifying a LCCD fragment in plasma samples before and after the therapeutic molecule has been introduced to the subject, or at two different time points during the treatment, wherein a reduction in the post-treatment or second timepoint level of LCCD indicates favorable response to the therapeutic molecule, irrespective to the total level of λFLC.

17. A method of assessing if an AL patient would benefit from a light chain stabilizer as a therapy by quantifying a LCCD fragment in plasma samples from a subject being considered to receive treatment by a light chain stabilizer molecule for light chain amyloidosis, wherein the ex vivo plasma sample is incubated with the said stabilizer or a solvent control, before subjecting the samples to limited proteolysis, and quantification of the resulting LCCD levels, wherein a reduction of LCCD level in the sample that was incubated with the stabilizer versus the solvent control sample indicates the subject may benefit from treatment by the said light chain stabilizer.

18. The method of any one of Claims 15-17, wherein when the first level of the LCCD fragment present before the targeted therapeutic molecule has been introduced to the subject is greater than the second level of the LCCD fragment present after the targeted therapeutic molecule has been introduced to the subject, then target engagement has occurred by the targeted therapeutic molecule.

19. The method of any one of Claims 15-17, wherein when the first level of the LCCD fragment present before the targeted therapeutic molecule has been introduced to the subject is the same or less than the second level of the LCCD fragment present after the targeted therapeutic molecule has been introduced to the subject, then target engagement has not occurred by the targeted therapeutic molecule.

20. The method of any one of Claim 15-19, wherein the biological sample is one of plasma, urine, serum, cerebrospinal fluid (CSF), or tissue biopsy.

21. A method of detection of presence in a subject of amyloidogenic λ free light chain ( λFLC) proteins, comprising the steps of: limited proteolysis of proteins from a biological sample, wherein the limited proteolysis results in a neo-epitope exposed on a LCCD fragment of the λFLC proteins; detecting the presence of the neo-epitope on the N-termini of the LCCD fragment of the λFLC proteins; quantitating the presence of the LCCD fragment from the λFLC proteins based on measuring the level of neo-epitope on the fragment using the LCCD mAbs; and comparing levels of the LCCD fragment before and after the limited proteolysis, wherein when the level of the LCCD fragment from λFLC proteins is greater after limited proteolysis, then amyloidogenicx yTLC proteins are detected.

22. The method of Claim 21, further comprising the steps of: measuring a first level of the LCCD fragment of the ZFI .C proteins present after the limited proteolysis in a biological sample taken from a healthy subject not having light chain amyloidosis (AL), wherein the first level is a reference level; measuring a second level of the fragment of the λFLC proteins present after the limited proteolysis of a biological sample from a person suspected of having AL; and wherein when the second level of the LCCD fragment of the λFLC proteins after the limited proteolysis of the biological sample is greater than the reference level, then amyloidogenic λFLC proteins are detected.

23. The method of any one of Claims 21-22, wherein when the level of LCCD fragment from λFLC is detected after limited proteolysis by Proteinase K and at an elevated level above the reference level, then light chain amyloidosis is present in the subject.

24. The method of Claim 23, where an elevated LCCD fragment level in a subject leads to a clinical decision, wherein the clinical decision is treatment and management for light chain amyloidosis (AL).

25. The method of Claim 23, where absence of an elevated LCCD fragment level in a subject leads to a clinical decision, wherein the clinical decision is ruling out of AL diagnosis and receiving treatment for other systemic amyloidosis, wherein the other systemic amyloidosis may include transthyretin amyloidosis (ATTR).

26. The method of any one of Claims 21-25, wherein the LCCD fragment is a 23KDa homodimer and is detected and quantified using mass spectrometry.

27. The method of any one of Claims 21 -25, wherein the LCCD fragment is a 11 5KDa monomer and is detected and quantified using mass spectrometry.

28. The method of any one of Claims 21-27, wherein the subject has one of more of 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 of any one of Claims 21-28, wherein the biological sample is one of plasma, urine, serum, CSF, or tissue biopsy.

30. A kit for detection of presence in a subject of amyloidogenic λ free light chain (λFLC) proteins, comprising: the antibodies of LCCD-G, LCCD-R and LCCD-S; instructions for using the kit in accordance with the method of Claim 21.

31. A neo-epitope on λFLC prepared by limited proteolysis of amyloidogenic λ free light chain (λFLC) proteins by proteinase K.

32. The neo-epitope of claim 31 that is xQP. where x is one of G. R or S.

33. An antibody or antibody mixture to the neo-epitope of claim 31 or 32.

34. The antibody of claim 33 that is LCCD-G. LCCD-R or LCCD-S.

35. A method of optimizing limited proteolysis condition using Proteinase K, comprising the steps of optimizing one or more of the following enumerated factors:

(1) source of the Proteinase K;

(2) composition of the testing sample;

(3) concentration of Proteinase K;

(4) time of proteolysis reaction;

(5) temperature of proteolysis reaction; wherein a proteolysis product of the LCCD fragment is differentially obtained from amyloidogenic λFLC vs. non-amyloidogenic λFLC.

36. The method of detection of presence in a subject of amyloidogenic λ free light chain (λFLC) proteins of any one of Claims 21-29, further comprising: obtaining a readout from an LCCD signal obtained by measuring the level of neo-epitope on the fragment using the LCCD mAbs.

37. The method of detection of presence in a subject of amyloidogenic λ free light chain

(λFLC) proteins of Claim 36, further comprising: obtaining the readout of the LCCD signal by use of an algorithm.