Methods and reagents for light chain amyloidosis diagnosis

Abstract

The disclosure provides a method for detection and quantification of amyloidogenic lambda free light chain (lFLC). The disclosure further provides reagents for performing the method, including monoclonal antibodies or antigen binding fragments thereof that bind to a neo-epitope on amyloidogenic λFLC exposed to limited proteolysis. The clinical utilities of the methods described herein 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, and detection of relapse.

Description

Attorney Docket No.: PRTE-021 / 01WO 345214-2102METHODS AND REAGENTS FOR LIGHT CHAIN AMYLOIDOSIS DIAGNOSISCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 686,444, filed August 23, 2024, and US. Provisional Patent Application No. 63 / 688,766, filed August 29, 2024, all of which are incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (PRTE_021_01WO_SeqList_ST26.xml; Size: 433,088 bytes; and Date of Creation: August 18, 2025) are herein incorporated by reference in its entirety.BACKGROUND

[0003] Immunoglobulin light chain and heavy chain proteins are produced by plasma cells, a type of white blood cell found in the bone marrow, in the process of making various types of immunoglobulins, such as IgG, IgA, IgM, etc. Light chain amyloidosis (“AL amyloidosis,” also referred to as “AL” herein) is caused by aberrant clonal plasma cell proliferation. In some cases, malignant clonal plasma cells proliferate and secrete excessive amounts of amyloidogenic free light chain (FLC) into the blood circulation. As a result, high levels of FLC are detected in the blood and urine, which typically results in an abnormal serum K / FLC ratio. Amyloidogenic monoclonal FLC differs from non-amyloidogenic polyclonal FLC generated as part of the adaptive immune response in terms of the amount of circulating FLC and its amyloidogenicity. Amyloidogenic FLC is prone to misfold and misassemble into nonnative species. Misfolded conformations (soluble aggregates and fibrils), as well as fragments of amyloidogenic FLC, are considered to be toxic and can eventually deposit onto various organs as amyloids. Organ toxicity can manifest as cardiomyopathy, nephrotic syndrome and / or end-stage renal failure, collectively categorized as AL amyloidosis. Both amyloidogenic X and K FLC may cause AL, although ZFLC is frequently identified as the responsible, or involved, FLC in AL. Estimated AL incidence is 10 in every million population worldwide. ZFLC accounts for -80% of the cases amongst all AL patients.

[0004] ZFLC monomer is around 215 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). ZFLC usually presents as a homodimer linked by an inter-monomer disulfide bond at the C-terminal constant1323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 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. Such sequence diversity and the lack of means to differentiate FLC amyl oidogeni city pose unique challenges for the diagnosis and targeted treatment of AL. Thus, specific quantification of amyloidogenic FLC has been hampered by the sequence diversity of immunoglobulin light chains. Delayed AL amyloidosis diagnosis is a major contributing factor to the high early mortality of this disease.

[0005] AL amyloidosis is similar to multiple myeloma in that both are caused by a proliferation of plasma cells. However, a distinction is that the pathology of AL amyloidosis is due, at least in part, to the presence of misfolded and misassembled amyloidogenic FLC, while the pathology of multiple myeloma stems from the direct effect of the aberrant plasma cell proliferation. 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.

[0006] There is a substantial unmet need for methods of detection of amyloidogenic ZFLC that are sensitive, specific, easy to access, and inexpensive. Such a test would enable early diagnosis of AL, as well as monitor response to therapeutics and MRD. Provided herein are methods and related compositions to address this need.SUMMARY

[0007] In some aspects, provided herein is a method for detecting amyloidogenic ZFLC protein, comprising the steps of: (i) providing a sample comprising one or more of an amyloidogenic FLC protein and a non-amyloidogenic FLC protein; (ii) contacting the sample with a protease of the disclosure to obtain a digested sample, wherein the contacting is performed under conditions effective for producing an amyloidogenic ZFLC protein neo-epitope; and (iii) determining an2323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 amount of the neo-epitope in the digested sample comprising contacting the digested sample with a monoclonal antibody or antigen-binding fragment thereof of the disclosure that binds to the neo-epitope, thereby detecting amyloidogenic XFLC protein in the sample.

[0008] In some embodiments of the aspects described herein, an increased amount of neoepitope in the digested sample indicates the presence of amyloidogenic XFLC protein in the sample. In some embodiments, an increased amount of the neo-epitope in the digested sample as compared to a reference indicates the presence of amyloidogenic XFLC protein in the sample. In some embodiments, the reference is an amount of neo-epitope in a control sample comprising non-amyloidogenic XFLC protein and subjected to a method for detecting amyloidogenic XFLC protein described herein.

[0009] In another aspect, provided herein is a method for identifying a subject for treatment of AL amyloidosis, comprising: (i) obtaining a sample from the subject, the sample comprising one or more of an amyloidogenic FLC protein and a non-amyloidogenic FLC protein; (ii) contacting the sample with a protease to obtain a digested sample, wherein the contacting is performed under conditions effective for producing an amyloidogenic XFLC protein neo-epitope; and (iii) determining an amount of the neo-epitope in the digested sample comprising contacting the digested sample with a monoclonal antibody or antigen-binding fragment thereof of the disclosure that binds to the neo-epitope, wherein detection of the neo-epitope in the digested sample identifies the subject for treatment of AL amyloidosis. In some embodiments, detecting amyloidogenic XFLC protein in the sample indicates (a) the subject has AL amyloidosis, minimal residual disease (MRD) of AL amyloidosis, or a relapse of AL amyloidosis; and / or (b) the subject will respond to treatment with an FLC dimer stabilizer.

[0010] In some aspects, provided herein is a method of determining if a patient has a condition, comprising: (i) contacting a sample from the patient with a protease, wherein the sample comprises amyloidogenic XFLC protein, and wherein contacting the sample with the protease produces a peptide comprising a neo-epitope of the XFLC protein; (ii) detecting the presence of the neo-antigen of the XFLC protein by contacting the proteolytically cleaved sample with a monoclonal antibody or antigen-binding fragment thereof of the disclosure; wherein the patient has the condition if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC, and wherein the condition is selected from the group consisting of amyloidosis, AL amyloidosis, monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma, smoldering multiple myeloma, IgM light chain amyloidosis, MRD of AL amyloidosis, or relapse of AL amyloidosis.3323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102

[0011] In another aspect, provided herein is a method of determining if a patient has a condition, comprising: (i) contacting a sample from the patient with a monoclonal antibody or antigenbinding fragment thereof of the disclosure, wherein the sample comprises amyloidogenic XFLC protein that has been contacted by a protease to produce a neo-epitope of the XFLC protein; and (ii) determining if the monoclonal antibody or antigen -binding fragment thereof binds to the neo-epitope of the XFLC protein; wherein the patient has the condition if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein, and wherein the condition is selected from the group consisting of amyloidosis, AL amyloidosis, MGUS, multiple myeloma, smoldering multiple myeloma, IgM light chain amyloidosis, minimal residual disease of AL amyloidosis, or relapse of AL amyloidosis.

[0012] In another aspect, provided herein is a method of determining if a patient a condition, comprising: (i) contacting a sample from the patient with a protease, wherein the sample comprises amyloidogenic XFLC protein, and wherein contacting with the protease produces a neo-epitope of the XFLC protein; (ii) contacting the proteolytically cleaved sample with a monoclonal antibody or antigen-binding fragment thereof of the disclosure; and (ii) determining if the monoclonal antibody or antigen-binding fragment thereof binds to the XFLC; wherein the patient has the condition if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein, and wherein the condition is selected from the group consisting of AL amyloidosis, minimal residual disease of AL amyloidosis, or relapse of AL amyloidosis.

[0013] In another aspect, provided herein is a method of determining if a patient has a condition, comprising: (i) contacting a sample from the patient with a monoclonal antibody or antigenbinding fragment thereof of the disclosure, wherein the sample comprises amyloidogenic XFLC protein that has been contacted by a protease and wherein contacting with the protease produces a neo-epitope of the XFLC protein; and (ii) determining if the monoclonal antibody or antigenbinding fragment thereof binds to the neo-epitope of the XFLC protein; wherein the patient has the condition if the monoclonal antibody or antigen-binding fragment thereof binds to the neoepitope of the XFLC protein, and wherein the condition is selected from the group consisting of AL amyloidosis, minimal residual disease of AL amyloidosis, or relapse of AL amyloidosis.

[0014] In some embodiments of the aspects described herein, the method further comprises administering a treatment to the subject.

[0015] In yet another aspect, provided herein is a method of treating a patient with a condition, comprising: (i) determining if the patient has the condition by: (a) contacting a sample from the4323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 patient with a protease, wherein the sample comprises amyloidogenic XFLC protein and wherein contacting with the protease produces a neo-epitope of the XFLC protein; b) contacting the proteolytically cleaved sample with a monoclonal antibody or antigen-binding fragment thereof of the disclosure; and (c) determining if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein; wherein the patient has the condition if the monoclonal antibody or antigen -binding fragment thereof binds to the neoepitope of the XFLC protein; and (ii) administering a treatment for the condition, wherein the condition is selected from the group consisting of amyloidosis, AL amyloidosis, MGUS, multiple myeloma, smoldering multiple myeloma, IgM light chain amyloidosis, MRD of AL amyloidosis, or relapse of AL amyloidosis.

[0016] In another aspect, provided herein is a method of treating a patient with a condition, comprising: (i) determining if the patient has the condition by: (a) contacting a sample from the patient with a monoclonal antibody or antigen-binding fragment thereof of the disclosure, wherein the sample comprises amyloidogenic XFLC protein that has been contacted by a protease and wherein contacting with the protease produces a neo-epitope of the XFLC protein; and (b) determining if the monoclonal antibody or antigen-binding fragment thereof binds to the XFLC; wherein the patient has the condition if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein; and (ii) administering a treatment for the condition, wherein the condition is selected from the group consisting of amyloidosis, AL amyloidosis, MGUS, multiple myeloma, smoldering multiple myeloma, IgM light chain amyloidosis, MRD of AL amyloidosis, or relapse of AL amyloidosis.

[0017] In another aspect, provided herein is a method of treating a patient with a condition, comprising: (i) determining if the patient has the condition by: (a) contacting a sample from the patient with a protease, wherein the sample comprises amyloidogenic XFLC protein and wherein contacting with the protease produces a neo-epitope of the XFLC protein; (b) contacting the proteolytically cleaved sample with a monoclonal antibody or antigen-binding fragment thereof of the disclosure; and (c) determining if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein; wherein the patient has the condition if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein; and (ii) administering a treatment for the condition, wherein the condition is selected from the group consisting of amyloidosis, AL amyloidosis, MGUS, minimal residual disease of AL amyloidosis, or relapse of AL amyloidosis.5323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102

[0018] In another aspect, provided herein is a method of treating a patient with a condition, comprising: (i) determining if the patient has the condition by: (a) contacting a sample from the patient with a monoclonal antibody or antigen-binding fragment thereof of the disclosure, wherein the sample comprises amyloidogenic XFLC protein that has been contacted by a protease, and wherein contacting with the protease produces a neo-epitope of the XFLC protein; and (b) determining if the monoclonal antibody or antigen -binding fragment thereof binds to the XFLC; wherein the patient has the condition if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein; and (ii) administering a treatment for the condition, wherein the condition is selected from the group consisting of AL amyloidosis, minimal residual disease of AL amyloidosis, or relapse of AL amyloidosis.

[0019] In some embodiments of the aspects described herein, the treatment is a chemotherapeutic, autologous stem cells, carfilzomib, daratumumab, or a combination thereof. In some embodiments, the treatment comprises an FLC dimer stabilizer.

[0020] In some embodiments of the aspects described herein, the sample is obtained from a subject. In some embodiments, the subject is suspected of having a plasma cell disorder. In some embodiments, the subject is suspected of having an abnormal FLC level. In some embodiments, the subject has received or is receiving a treatment for a plasma cell disorder, optionally wherein the treatment comprises a plasma cell elimination chemotherapy. In some embodiments, the sample is serum, plasma, urine, cerebrospinal fluid, or tissue.

[0021] In some embodiments, the subject is receiving or has received an FLC dimer stabilizer. In some embodiments, target engagement of the FLC dimer stabilizer is determined using a method for detecting amyloidogenic XFLC protein described herein. In certain embodiments, a decreased amount of the neo-epitope in the digested sample as compared to a reference indicates target engagement of the FLC dimer stabilizer in the subject, wherein the reference is an amount of neo-epitope in a control sample obtained from the subject prior to receiving the FLC dimer stabilizer and subjected to a method for detecting amyloidogenic XFLC protein described herein. In certain embodiments, a substantially similar amount of the neo-epitope in the digested sample as compared to a reference indicates target engagement of the FLC dimer stabilizer in the subject, wherein the reference is an amount of neo-epitope in a control sample comprising non- amyloidogenic XFLC and subjected to a method for detecting amyloidogenic XFLC protein described herein.

[0022] In some embodiments of the aspects described herein, the sample is contacted with an FLC dimer stabilizer occurs prior to the method for detecting amyloidogenic XFLC protein. In6323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 some embodiments, the sample comprises recombinant amyloidogenic XFLC. In certain embodiments, the recombinant amyloidogenic XFLC is selected from the group consisting of WIL, H3, H9, and a combination thereof. In some embodiments, the sample is obtained from a subject suspected of having an AL amyloidosis. In some embodiments, a decreased amount of the neo-epitope in the digested sample as compared to a reference indicates target engagement of the FLC dimer stabilizer, wherein the reference is an amount of neo-epitope in a control sample lacking the FLC dimer stabilizer and subjected to the method for detecting amyloidogenic XFLC protein.

[0023] In some embodiments of the aspects described herein, the subject is suspected of having a plasma cell disorder. In some embodiments, the subject previously received a diagnosis of AL amyloidosis, and detection of the neo-epitope in the digested sample identifies the subject as having minimal residual disease and / or relapse of AL amyloidosis. In some embodiments, the subject did not previously receive a diagnosis of AL amyloidosis.

[0024] In some embodiments of the aspects described herein, the sample is whole blood, plasma, urine, serum, saliva, cerebrospinal fluid (CSF), bone marrow aspirate, or tissue biopsy.

[0025] In some embodiments of the aspects described herein, the protease is a recombinant protein. In some embodiments, the protease is selected from the group consisting of proteinase K, thermolysin, pepsin, trypsin, chymotrypsin, cathepsin B, cathepsin L, papain, and a peptidase S8, optionally wherein the peptidase S8 is PCSK9 or subtilisin. In some embodiments, the protease is proteinase K and / or chymotrypsin. In some embodiments, the protease is proteinase K.

[0026] In some embodiments of the aspects described herein, the conditions effective for producing an amyloidogenic XFLC protein neo-epitope comprise a temperature of about 4°C to about 45°C. In some embodiments, the conditions comprise a temperature of about 35°C to about 42°C. In some embodiments, the conditions comprise a duration of time about 10 min to about 18 hours. In some embodiments, the conditions comprise a duration of about 10 minutes to 120 minutes. In some embodiments, the conditions comprise a concentration of the protease of about 0.010 pM to about 10,000 pM. In some embodiments, the conditions comprise a concentration of protease of about 0.1 pM to 10 pM. In some embodiments, the conditions comprise a concentration of protease of about 0.1 pM to 1 pM.

[0027] In some embodiments of the aspects described herein, the step of contacting the sample with a protease of the disclosure to obtain a digested sample comprises contacting the sample with at least two proteases to obtain the digested sample. In some embodiments, the step7323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 comprises contacting the sample with 2, 3, or 4 proteases. In some embodiments, the contacting is performed under conditions effective for producing the amyloidogenic XFLC protein neoepitope and at least one second amyloidogenic XFLC protein neo-epitope. In some embodiments, the step of determining an amount of the neo-epitope in the digested sample comprises determining an amount of the neo-epitope and the at least one second neo-epitope in the digested sample, comprising contacting the digested sample with the monoclonal antibody or antigen-binding fragment thereof that specifically binds to the neo-epitope, and a monoclonal antibody or antigen-binding fragment thereof that specifically binds to the at least one second neo-epitope. In some embodiments, the at least two proteases comprise proteinase K and a second protease. In some embodiments, the second protease is selected from the group consisting of thermolysin, pepsin, trypsin, chymotrypsin, cathepsin B, cathepsin L, papain, and a peptidase S8, optionally wherein the peptidase S8 is PCSK9 or subtilisin.

[0028] In another aspect, provided herein is a method of generating a monoclonal antibody that binds to a neo-epitope of an amyloidogenic XFLC protein, comprising immunizing a subject with an immunogenic composition described herein, and obtaining the monoclonal antibody from the immunized subject. In some embodiments, the subject is a rabbit, mouse, rat, rabbit, goat, sheep, horse, or chicken.

[0029] In another aspect, provided herein is an immunogenic composition comprising: (i) a neoepitope of an amyloidogenic ZFLC protein; and (ii) a pharmaceutically acceptable buffer. In some embodiments, the neo-epitope is exposed by a method of limited proteolysis described herein. In some embodiments, the limited proteolysis comprises contacting the amyloidogenic ZFLC protein to a protease under conditions suitable for generating the neo-epitope. In some embodiments, the protease is proteinase K. In some embodiments, the protease is chymotrypsin. In some embodiments, the protease is papain. In some embodiments, the neo-epitope has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide of any one of SEQ ID NOs: 1 and 34-135, 137-239, 241-343344, GQP, SQP, and RQP. In some embodiments, the neo-epitope comprises the amino acid sequence GQP, RQP, or SQP. In some embodiments, the neo-epitope comprises GQPKA (SEQ ID NO: 134), RQPKA (SEQ ID NO: 342), or SQPKA (SEQ ID NO: 238). In some embodiments, the neo-epitope comprises from the N-terminus to the C-terminus (i) GQP, RQP, or SQP; and (ii) a polypeptide with at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or at least 100 % identical to any one of SEQ ID NOS: 351- 450, KAA, and KA. In some embodiments, the immunogenic composition comprises (iii) an8323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 adjuvant, optionally wherein the adjuvant is selected from any one of Aluminum hydroxide, Aluminum phosphate, Calcium phosphate, Freund’s complete adjuvant, Freund’s incomplete adjuvant, Montanide ISA 50, Montanide ISA 206, AS03, AS04, MF59, CpG oligodeoxynucleotides, Quil A, Liposomes, Poly(I), TLR agonists, E. coli heat-labile toxin, Chitosan, Zinc oxide, Squalene, BCG (Bacillus Calmette-Guerin), Dendritic cell vaccines, Saponins, Microparticles, Nanospheres, Micelles, Emulsions, Detoxified bacterial toxins, Oil-in- water emulsions, Water-in-oil emulsions, Polylactic acid, Polyethylene glycol, Pseudomonas exotoxin, Virol ex, Imiquimod, RIBI, Polypropylene, Pluronic F68, Eudragit, Gelatin, Alginate, Silica, Cellulose, Starch, Activated carbon, Graphene oxide, Chloroform extracts, Arginine, Mannan, Lipopeptides, Peptidoglycan, and Phospholipids.

[0030] In another aspect, provided herein is a monoclonal antibody or antigen-binding fragment thereof that binds to a neo-epitope of an amyloidogenic XFLC protein, where the monoclonal antibody is generated according to a method described herein.

[0031] In some embodiments of the aspects described herein, the monoclonal antibody or antigen-binding fragment thereof comprises: (a) a variable heavy chain CDR1 (HCDR1) comprising an amino acid sequence of SEQ ID No: 10, 16, or 22; (b) a variable heavy chain CDR2 (HCDR2) comprising an amino acid sequence of SEQ ID No: 11, 17, or 23; (c) a variable heavy chain CDR3 (HCDR3) comprising an amino acid sequence of SEQ ID No: 12, 18, or 24;(d) a variable light chain CDR1 (LCDR1) comprising an amino acid sequence of SEQ ID No: 13, 19, or 25; (e) a variable light chain CDR2 (LCDR2) comprising an amino acid sequence of SEQ ID No: 14, 20, or 26; and (f) a variable light chain CDR3 (LCDR3) comprising an amino acid sequence of SEQ ID No: 15, 21, or 27.

[0032] In some embodiments of the aspects described herein, the monoclonal antibody or antigen-binding fragment thereof comprises a HCDR1 comprising an amino acid sequence of SEQ ID No: 10; a HCDR2 comprising an amino acid sequence of SEQ ID No: 11, a HCDR3 comprising an amino acid sequence of SEQ ID No: 12, a LCDR1 comprising an amino acid sequence of SEQ ID No: 13, a LCDR2 comprising an amino acid sequence of SEQ ID No: 14, and a LCDR3 comprising an amino acid sequence of SEQ ID No: 15.

[0033] In some embodiments of the aspects described herein, the monoclonal antibody or antigen-binding fragment thereof comprises a HCDR1 comprising an amino acid sequence of SEQ ID No: 16, a HCDR2 comprising an amino acid sequence of SEQ ID No: 17, a HCDR3 comprising an amino acid sequence of SEQ ID No: 18, a LCDR1 comprising an amino acid9323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 sequence of SEQ ID No: 19, a LCDR2 comprising an amino acid sequence of SEQ ID No: 20, and a LCDR3 comprising an amino acid sequence of SEQ ID No: 21.

[0034] In some embodiments of the aspects described herein, the monoclonal antibody or antigen-binding fragment thereof comprises a HCDR1 comprising an amino acid sequence of SEQ ID No: 22, a HCDR2 comprising an amino acid sequence of SEQ ID No: 23, a HCDR3 comprising an amino acid sequence of SEQ ID No: 24, a LCDR1 comprising an amino acid sequence of SEQ ID No: 25, a LCDR2 comprising an amino acid sequence of SEQ ID No: 26, and a LCDR3 comprising an amino acid sequence of SEQ ID No: 27.

[0035] In some embodiments of the aspects described herein, the monoclonal antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable domain (VH) comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 345, and a light chain variable domain (VL) comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 346; (ii) a VH comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 347, and a VL comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 348; or (iii) a VH comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 349, and a VL comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 350.

[0036] In some embodiments of the aspects described herein, the monoclonal antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID No: 345, and a VL comprising an amino acid sequence of SEQ ID No: 346.

[0037] In some embodiments of the aspects described herein, the monoclonal antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID No: 347, and a VL comprising an amino acid sequence of SEQ ID No: 348.

[0038] In some embodiments of the aspects described herein, the monoclonal antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID No: 349, and a VL comprising an amino acid sequence of SEQ ID No: 350.

[0039] In some embodiments of the aspects described herein, the neo-epitope has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide of any10323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 one of SEQ ID NOS: 1 and 34-135, 137-239, 241-343344, GQP, SQP, and RQP. In some embodiments, the neo-epitope comprises the amino acid sequence GQP (SEQ ID NO: 136), RQP (SEQ ID NO: 344), SQP (SEQ ID NO: 240), GQPKA (SEQ ID NO: 134), RQPKA (SEQ ID NO: 342), or SQPKA (SEQ ID NO: 238). In some embodiments, the neo-epitope comprises from the N-terminus to the C-terminus (i) GQP, RQP, or SQP; and (ii) a polypeptide with at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or at least 100 % identical to any one of SEQ ID NOS: 351-450, KAA, and KA.

[0040] In yet another aspect, provided herein is a kit comprising: a monoclonal antibody or antigen-binding fragment thereof of the disclosure. In some embodiments, the kit further comprises a neo-epitope of an amyloidogenic ZFLC protein described herein. In some embodiments, the kit further comprises a protease described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1A illustrates an exemplary SDS PAGE gel of Proteinase K (PK) digested of recombinant lambda light chains, including a random selection of 293F cell produced recombinant amyloidogenic (sequences based on AL subjects) and non-amyloidogenic (sequences based on normal healthy subjects) ZFLC proteins, comparing with and without PK treatment. PK digested resulted in the generation of a proteolytic fragment corresponding to the disulfide linked LC constant domain (LC-C domain, or dLCCD) homodimer.

[0042] FIG. IB shows an LC structure highlighting the hinge region. PK cleaves in the hinge region between the VL domain and the CL domain, producing the dLCCD fragment. PK treatment exposes neo-epitopes highly conserved for all LC sequences. Exposure depending on the dynamic / amyloidogenic nature of the ZFLC.

[0043] FIG. 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, the frequency of sequence occurrence at the N-terminus of the dLCCD fragment e in AL amyloidosis ZFLC is approximately: 84% GQP, 11% SQP, and 5% RQP.

[0044] FIGs. 2A-2B show LCCD-G mAb binding to LCCD vs (a) full length WIL-46L (amyloidogenic ZFLC) (FIG. 2A) or (b) WIL-46L or human IgG with or without proteinase K (FIG. 2B).

[0045] FIG. 2C shows LCCD mAbs highly specific for proteolysis exposed novel epitopes. LCCD-G, LCCD-R, and LCCD-S mAbs (signal around 25 KDa) specifically detect LCCD11323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 fragments in recombinant FLC proteins containing GQP, RQP, and SQP at the cleavage site, respectively, post-proteolysis by Proteinase K. Anti-kFLC polyclonal antibody (around 50KDa) is used to indicate all full-length and LCCD fragments from the samples.

[0046] FIG. 3A shows LCCD-G mAb detection of amyloidogenic XFLC proteolysis in 10 treatment naive AL amyloidosis plasma samples.

[0047] FIG. 3B shows quantification of dLCCD fragment level determined in the Western blot of FIG. 3 A in pre-treatment AL amyloidosis patients (post-PK treatment).

[0048] FIG. 4 shows dLCCD fragment level generated under limited proteolysis determined in Western blot in pre-treatment AL amyloidosis patient plasma (N=32), plasma from normal (N=23) and multiple myeloma (MM) patient plasma (N=19). Statistical analysis method: oneway ANOVA. In WB and post-limited PK proteolysis treatment, AL amyloidosis samples have dLCCD signal 10-200x of normal under the same condition (p=0.0007). AL amyloidosis samples are also significantly differentiated from MM samples under the limited PK proteolysis and quantified by the LCCD mAb (p=0.0008).

[0049] FIGS. 5A-5B shows an MesoScale Discovery (MSD)dLCCD immunoassay. FIG. 5A: Using LCCD-X mixture as the capture antibody, dLCCD level generated under limited proteolysis determined on MSD platform. LCCD-X is an equal molar mixture of the three LCCD mAbs, LCCD-G, LCCD-S, and LCCD-R. FIG. 5B: dLCCD levels differentiates XFLC in AL amyloidosis samples FLC from normal (N=40), multiple myeloma ) (N=19), and TTR amyloidosis (ATTR, including cardiomyopathy and polyneuropathy phenotypes) (N=35) samples. Recombinant dLCCD was used as the reference standard.

[0050] FIGS. 6A-6B show baseline levels of dLCCD correlates to XAL amyloidosis patient overall survival. FIG. 6 A: 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. FIG. 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 UAL amyloidosis patients using the MSD LCCD immunoassay. dFLC level of the same samples were determined by the FreeLite assay. OS=month.

[0051] FIGS. 7A-7B shows concordance of dFLC and dLCCD predicts overall survival. Logrank 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 discordant12323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 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 XAL patients MSD LCCD immunoassay. OS=month.

[0052] FIG. 8A shows a Western blot analysis of samples after limited proteolysis, detected with an anti -kF LC monoclonal antibody (band at 50KDa) and the dLCCD mAb (band at 23 KDa).

[0053] FIG. 8B: Quantification of the antibody-detected bands on the LiCor system and fitted to a variable slope (four parameters) response curve using Prism.

[0054] FIG. 8C: Protection against limited proteolysis by an FLC dimer stabilizer (PTG-1412) in 13 ex vivo XAL plasma samples as quantified by dLCCD levels post-proteolysis, with and without PTG-1412.

[0055] FIGS. 9A-9C depict an example of unique fragments and neo-epitopes generated on amyloidogenic ZFLC using other proteases. Gel image stained with Coomassie blue showing different proteolytic fragments generated with increasing concentrations of thermolysin (FIG. 9A), pepsin (FIG. 9B), and trypsin (FIG. 9C).

[0056] FIG. 9A depicts a gel image stained with Coomassie blue showing different proteolytic fragments generated with increasing concentrations of thermolysin. Top panel, Coomassie gels; bottom panel, Western blot using dLCCD mAb.

[0057] FIG. 9B depicts a gel image stained with Coomassie blue showing different proteolytic fragments generated with increasing concentrations of pepsin. Top panel, Coomassie gels; bottom panel, Western blot using dLCCD mAb.

[0058] FIG. 9C depicts a gel image stained with Coomassie blue showing different proteolytic fragments generated with increasing concentrations of trypsin. Top panel, Coomassie gels; bottom panel, Western blot using dLCCD mAb.

[0059] FIG. 9D depicts a gel image of the result of chymotrypsin limited proteolysis of rhFLC comparing an amyloidogenic ZFLC sequence (WH-T46L) vs. a non-amyloidogenic normal ZFLC in buffer. Untreated (0 min), and chymotrypsin treated samples at 30, 60, 90, and 120 minutes on a SDS PAGE gel. The top panel is stained with Coomassie dye, the bottom panel is stained with the anti-LCCD-X mAb mixture.

[0060] FIG. 9E depicts the quantification of dLCCD fragment generated from FIG. 9D, where amyloidogenic rhFLC data points are presented with open symbols, and the normal rhFLC data points are presented with closed symbols.13323555102Atorney Docket No.: PRTE-021 / 01WO 345214-2102

[0061] FIG. 9F depicts the quantification of the XFLC dimer bands at around 50 KDa in the top panel of FIG. 9G, where amyloidogenic rhFLC is in open symbols, and the normal rhFLC is in closed symbols. Papain cleaved AL rhFLC (open symbol) at a much higher rate than that of normal rhFLC (closed symbol).

[0062] FIG. 9G depicts unique neo-epitopes generated on amyloidogenic XFLC using various proteases. Papain limited proteolysis of rhFLC comparing an amyloidogenic XFLC sequence (WIL) vs. a non-amyloidogenic normal XFLC in buffer. Untreated, and papain treated samples at 10, 60, and 90 minutes on a SDS PAGE gel. The top panel was stained with an anti-XFLC pAb, the middle panel was stained with the anti-dLCCD mAb, and the bottom panel is the overlay of the top two images.

[0063] FIG. 10A depicts limited proteolysis by Proteinase K with varying of an AL plasma sample incubated at 37°C over time. The resulting dLCCD fragment was quantified using anti- dLCCD mAb.

[0064] FIG. 10B depicts limited proteolysis by Proteinase K of an AL plasma sample wat 37°C (open symbols) and 30°C (closed symbols) over time.

[0065] FIGS. 11A-11C show detection of the dLCCD biomarker in serum, urine, and saliva samples from an AL amyloidosis subject post limited proteolysis by Proteinase K. FIG. 11A shows the kinetics of limited proteolysis in matching plasma (open symbols) and serum (closed symbols) samples from an AL patient. FIG. 11B shows urine and FIG. 11C shows saliva samples from two XAL patients that were subjected to limited proteolysis in the presence of Proteinase K. Western blots were probed using the dLCCD antibody, showing the fragment specifically detected in the AL samples after proteolysis.

[0066] FIG. HD shows dot blot urine and saliva LCCD detection following limited Proteinase K proteolysis.

[0067] FIG. HE shows the results of immobilized protease digestion of amyloidogenic recombinant ZFLC. Coomassie stained SDS gel. Lanes 1, 2, and 3 are molecular weight markers, undigested recombinant ZFLC, and recombinant ZFLC incubated with Proteinase K agarose beads at 37°C for 30 minutes.

[0068] FIG. HF shows the specific activity of Proteinase K from 12 repeats. 1 unit of the enzyme was used to generate 85.8 ± 13.2 nM dLCCD from 1 pM WIL rhFLC in 30 minutes at 37°C in PBS buffer (pH=7.4). 1 unit of Proteinase K is equivalent to 0.1 pM.

[0069] FIGS. 12A-12C indicate that a dLCCD fragment from XFLC can be detected dose- dependently on solid phase matrix and detected using the dLCCD mAbs. FIG. 12A depicts a dot14323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 blot with a recombinant LCCD-G standard curve after spotting onto a membrane with immunodetection. FIG. 12B depicts a dot blot with Wil-T46L digested sample at increasing time points. Top: dot blot image. Bottom: quantification of the image. FIG. 12C depicts an image of lateral flow assay using biotinylated dLCCD-G mAb, FITC labeled anti-human lambda LC, and recombinant dLCCD-G protein.

[0070] FIG. 13A and FIG. 13B depict a competition assay using dLCCD mAb to detect amyloidogenic XFLC. FIG. 13A provides a schematic of the operation of a competition assay described herein. FIG. 13B depicts the results of the competition assay of FIG. 13 A, showing dLCCD recombinant protein dose-dependently competed off the GQPK-mer-Biotin probe (solid circle). Non-biotinylated GQPK-mer was used as the negative control (filled triangle).

[0071] FIG. 14A shows that commercial XFLC assays have epitopes residing on the constant domain, which therefore detect both the full-length rhFLC and the resulting dLCCD fragment post-treatment with Proteinase K. rhFLC WIL with and without protease treatment was detected using anti-dLCCD mAb (left) and goat anti-human FLC pAb (right) using Western Blot. FIG. 14B shows Sebia XFLC ELISA assay detects both XFLC (rhFLC WIL; closed symbols) and dLCCD (open symbols) dose-dependently.

[0072] FIG. 15 provides a graph showing the levels of dLCCD detected in WIL (amyloidogenic XFLC), JTO (non-amyloidogenic XFLC), and normal serum samples after limited proteolysis.DETAILED DESCRIPTIONOverview

[0073] Amyloidogenic FLC differs from non-amyloidogenic FLC in several ways, including in the amount of circulating FLC and its amyl oidogeni city. Amyloidogenic FLC are more kinetically unstable than non-pathogenic FLC, leading to protein misfolding and the formation of pathogenic conformations including soluble aggregates and amyloids. Variations in FLC sequences are the result of the germline genes and the incorporation of somatic hypermutations. Typically, each AL amyloidosis patient possesses a single clonal plasma cell population that produces amyloidogenic FLC, and therefore one monoclonal amyloidogenic FLC sequence, amidst a polyclonal non-amyloidogenic FLC background. Such sequence diversity and the lack of means to differentiate FLC amyloidogenicity pose unique challenges for the diagnosis and targeted treatment of AL amyloidosis.

[0074] The present disclosure is based, at least in part, on the discovery and development of methods for assessing neo-epitopes present on ZFLC cleavage products as a marker of15323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 amyloidogenicity, thereby providing a means to differentiate amyloidogenic from non- amyloidogenic XFLC. Described herein are method of generating neo-epitopes by performing limited proteolysis of XFLC, methods of generating monoclonal antibodies, or antigen binding fragments thereof, that specifically bind to the neo-epitopes, and compositions comprising the neo-epitopes or neo-epitope binding agents for use in the methods described herein. Immunoassays using the monoclonal antibodies and antigen binding fragment thereof, as described herein, in combination with limited proteolysis (e.g., with limited proteolysis using Proteinase K) are shown to detect and quantify amyloidogenic XFLC, including in AL patient plasma samples. The measurements are performed in patient samples comprising different XFLC sequences, demonstrating sequence-independent quantification of amyloidogenic XFLC. Such methods are beneficial for providing specific, sensitive, and low-cost diagnosis of AL amyloidosis, minimal residual diseases and for monitoring responses to therapeutics interventions for such diseases.

[0075] The disclosure thus provides methods for detection of amyloidogenic XFLC in a sample, comprising a step of limited proteolysis. The limited proteolysis is performed under a set of conditions (e.g., comprising a particular temperature, duration of time, concentration of protease, and / or concentration of sample) that results in exposure of an amyloidogenic XFLC neo-epitope. The method further comprises detecting the amyloidogenic XFLC neo-epitope with a neoepitope binding agent (e.g., a monoclonal antibody or antigen binding fragment thereof that binds to the amyloidogenic XFLC neo-epitope described herein).

[0076] The disclosure further provides neo-epitope binding agentsfor use in the methods described herein, and methods for generating the agents.

[0077] The disclosure provides applications of the disclosed methods. The applications include, but are not limited to, clinical applications (e.g., diagnosis of AL, determining treatment responsiveness, and detecting minimal residual disease or disease relapse in subjects having AL amyloidosis) and preclinical applications (e.g., screening of compounds for stabilization of amyloidogenic XFLC).

[0078] The disclosure further provides kits for performing the disclosed methods.Definitions

[0079] 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 in16323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 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.

[0080] 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.

[0081] The term “about”, as used herein, in reference to a number or range of numbers, is understood to mean the stated number and numbers + / - 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

[0082] The term “between”, as used in a phrase as such “between A and B” or “between A-B” refers to a range including both A and B.

[0083] The terms “or” and “and / or”, as used herein, include any, and all, combinations of one or more of the associated listed items.

[0084] The terms “including”, “includes”, “included”, and other forms, as used herein, are not limiting.

[0085] The terms “comprise” and its grammatical equivalents, as used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0086] The term “antibody,” “immunoglobulin,” or “Ig” is used interchangeably herein, and is used in the broadest sense and specifically covers, for example, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, and multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity). A conventional antibody is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxy -terminal portion of each chain includes a constant region. See, e.g., Antibody Engineering (Borrebaeck, ed., 2d ed. 1995); and Kuby, Immunology (3d ed. 1997). An antibody can be human, humanized, chimeric and / or affinity matured, as well as an antibody from other species, for example, mouse and rabbit, etc. Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies or their humanized variants, and intrabodies. An antibody can be selected from any class of17323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGl, IgG2, IgG3 and IgG4 (e.g., variants of IgG4 and IgG4 nullbody). An antibody can comprise kappa or lambda light chain constant sequences.

[0087] The term “binds” or “binding”, as used herein, refers to a covalent or non-covalent interaction between molecules (e.g., forming a complex by interactions). Exemplary non- covalent interactions include hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. As used herein, the term “specifically binds” refers to binding of an antibody or an antigen binding fragment thereof to an antigen with a dissociation constant (KD) <107M. The term “KD” is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction. The ratio of dissociation rate (koff) to association rate (kon) of an antibody to a monovalent antigen (koff / kon) is the dissociation constant KD, which is inversely related to affinity. The lower the KD value, the higher the affinity of the antibody. The value of KD varies for different complexes of antibody and antigen and depends on both kon and koff. The dissociation constant KD for an antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity.

[0088] The term “binding affinity”, as used herein, refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a binding molecule X for its binding partner Y can generally be represented by the dissociation constant (KD). Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure.

[0089] The term “constant region” or “constant domain”, as used herein, refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor. This portion has a conserved amino acid sequence relative to the variable region. The18323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 constant region may contain the CHI, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0090] As used herein, the term “free light chain,” used interchangeably herein with the terms “FLC,” “free light chain protein,” and “FLC protein,” refers to immunoglobulin light chains that are not bound to heavy chains. The term encompass monomeric and dimeric forms, as well as fragments of the immunoglobulin light chains, unless otherwise indicated. The term further encompasses amyloidogenic and non-amyloidogenic FLC. The term is inclusive of lambda and kappa FLC.

[0091] As used herein, the term “pathogenic FLC,” used interchangeably herein with the term “amyloidogenic FLC,” refers to kinetically unstable FLC. Methods to measure protein kinetic stability are known in the art, and include methods to measure FLC unfolding temperature (e.g., wherein a decreased FLC unfolding temperature compared to a non-pathogenic FLC control indicates the FLC is kinetically unstable). Exemplary methods to measure FLC unfolding temperature include nuclear magnetic resonance, circular dichroism or 8-anilino-l- napthalenesulfonic acid (ANS) binding and fluorescence signal. In some embodiments, an FLC that is “kinetically unstable” is characterized by a rate of proteolysis under a set of conditions (e.g., conditions described herein for selective proteolytic digested) that is comparable to an exemplary pathogenic FLC described herein. In some embodiments, the exemplary pathogenic FLC is recombinant H9, WIL, or H3.

[0092] As used herein, the term “ZFLC” or “lambda FLC” or “ free light chain” or “lambda free light chain” each refer to FLC (e.g., FLC monomer or FLC dimer) with lambda light chains.

[0093] As used herein, the term “non-pathogenic FLC,” used interchangeably with “non- amyloidogenic FLC,” refers to kinetically stable FLC. In some embodiments, an FLC that is “kinetically stable” is characterized by a rate of proteolysis under a set of conditions (e.g., conditions described herein for selective proteolytic digested) that is comparable to an exemplary non-pathogenic FLC described herein.

[0094] The term “epitope,” as used herein, refers to a localized region of an antigen to which an antibody can bind. A “neo-epitope” is an epitope on a protein that is exposed after a modification of the protein, for example after proteolytic cleavage. For example, the polypeptides having amino acid sequences of SEQ ID NOS: 1 and 34-135, 137-239, 241- 343344, GQP, SQP, and RQP are neo-epitopes of an amyloidogenic ZFLC protein. In the case of a polypeptide antigen, for example, an epitope can be contiguous amino acids of the polypeptide (a “linear” epitope) or an epitope can comprise amino acids from two or more non-contiguous19323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 regions of the polypeptide (a “conformational,” “non-linear” or “discontinuous” epitope). It will be appreciated by one of skill in the art that, in general, a linear epitope may or may not be dependent on secondary, tertiary, or quaternary structure. In some embodiments, an antibody binds to a group of amino acids regardless of whether they are folded in a natural three dimensional protein structure. In some embodiments, an antibody requires amino acid residues making up the epitope to exhibit a particular conformation (e.g., bend, twist, turn or fold) in order to recognize and bind the epitope.

[0095] The term “Fab” or “Fab region”, as used herein, refers to an antibody region that binds to antigens. A conventional IgG usually comprises two Fab regions, each residing on one of the two arms of the Y-shaped IgG structure. Each Fab region is typically composed of one variable region and one constant region of each of the heavy and the light chain. More specifically, the variable region and the constant region of the heavy chain in a Fab region are VH and CHI regions, and the variable region and the constant region of the light chain in a Fab region are VL and CL regions. The VH, CHI, VL, and CL in a Fab region can be arranged in various ways to confer an antigen binding capability. For example, VH and CHI regions can be on one polypeptide, and VL and CL regions can be on a separate polypeptide, similarly to a Fab region of a conventional IgG. Alternatively, VH, CHI, VL and CL regions can all be on the same polypeptide and oriented in different orders.

[0096] The term “Fc region”, as used herein, refers to a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. A “functional Fc region” possesses an “effector function” of a native sequence Fc region. Exemplary “effector functions” include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor), etc. Such effector functions generally require the Fc region to be combined with20323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various assays known to those skilled in the art.

[0097] The term “fragment”, as used herein, refers to a portion of a polypeptide or polynucleotide molecule containing less than the entire polypeptide or polynucleotide sequence. In some embodiments, a fragment of a polypeptide or polynucleotide comprises at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the entire length of the reference polypeptide or polynucleotide. In some embodiments, a fragment of a polypeptide or polynucleotide comprises about 10%-99%, about 20%-99%, about 30%-99%, about 40%-99%, about 50%-99%, about 60%-99%, about 70%-99%, about 80%-99%, about 90%-99%, about 95%-99%, about 96%-99%, about 97%-99%, or about 98%-99%, of the entire length of the reference polypeptide or polynucleotide. In some embodiments, a polypeptide or polynucleotide fragment may contain about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, or more nucleotides or amino acids.

[0098] The term “antigen-binding fragment” refers to a fragment of an antibody that retains the ability to bind to the target antigen. A fragment may include one or more of the complementarity determining regions of the monoclonal antibody. Non-limiting examples of antigen-binding fragments include F(ab')2, F(ab)2, Fab', Fab, Fv, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), and fragments comprising either a VL or VH domain, diabodies.

[0099] The term “heavy chain”, when used in reference to an antibody, refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and a carboxy -terminal portion includes a constant region. The constant region can be one of five distinct types, (e.g, isotypes) referred to as alpha, delta, epsilon, gamma, and mu, based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains differ in size: alpha, delta, and gamma contain approximately 450 amino acids, while epsilon and mu contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to five well known classes (e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG, and IgM, respectively, including four subclasses of IgG, namely IgGl, IgG2, IgG3, and IgG4.21323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102

[0100] The term “light chain”, when used in reference to an antibody, refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and a carboxy -terminal portion includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two distinct types, referred to as kappa or lambda based on the amino acid sequence of the constant domains.

[0101] The term “variable region”, “variable domain”, “V region”, or “V domain”, as used herein, refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen. The variable region of the heavy chain may be referred to as “VH ” The variable region of the light chain may be referred to as “VL ” The term “variable” refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable regions. Instead, the V regions consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called “hypervariable regions” or “complementarity determining regions” that are each about 9-12 amino acids long. The variable regions of heavy and light chains each comprise four FRs, largely adopting a P sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases form part of, the P sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al, Sequences of Proteins of Immunological Interest (5th ed. 1991)).

[0102] The complementarity determining regions (CDRs) have been defined by well-known numbering systems. For example, the Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (see, e.g., Kabat, et al., supra). Chothia refers instead to the location of the structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol., 1987, 196:901-17). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and22323555102Attorney Docket No.: PRTE-021 / 01WO 345214-210235B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Rontermann and Diibel, eds., 2d ed. 2010)). The “contact” CDRs are based on an analysis of the available complex crystal structures. Another universal numbering system that has been developed and widely adopted is ImMunoGeneTics (IMGT) Information System® (Lafranc, et al, Dev. Comp. Immunol., 2003, 27(l):55-77). IMGT is an integrated information system specializing in immunoglobulins (IG), T-cell receptors (TCR), and major histocompatibility complex (MHC) of human and other vertebrates. An additional numbering system (AHon) has been developed by Honegger and Pluckthun, J. Mol. Biol., 2001, 309: 657-70. Correspondence between the numbering system, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to one skilled in the art (see, e.g., Kabat, supra, Chothia and Lesk, supra; Martin, supra, Lefranc, et al., supra). The boundaries of a given CDR may vary depending on the scheme used for identification. Thus, unless otherwise specified, the CDRs of a given antibody or region thereof, such as a variable region, should be understood to encompass the complementary determining region as defined by any of the known schemes described herein. In some instances, the scheme for identification of a particular CDR or CDRs is specified, such as the CDR as defined by the Kabat, Chothia, or Contact method. In other cases, the particular amino acid sequence of a CDR is given. As the “location” of the CDRs within the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features, CDR and framework residues are readily identified. This information can be used in grafting and replacement of CDR residues from immunoglobulins of one species into an acceptor framework from, typically, a human antibody.

[0103] The term “monoclonal antibody,” as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts, and each monoclonal antibody will typically recognize a single epitope on the antigen.

[0104] The term “pharmaceutically acceptable excipient, carrier or diluent”, as used herein, refers to any substance formulated alongside the active ingredient of a pharmaceutical composition that allows the active ingredient to retain biological activity and is non-reactive with the subject’s immune system. Such a substance can be included for the purpose of long-23323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 term stabilization, bulking up solid formulations that contain potent active ingredients in small amounts, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating absorption, reducing viscosity, or enhancing solubility. The selection of appropriate substance can depend upon the route of administration and the dosage form, as well as the active ingredient and other factors. Compositions having such substances can be formulated by well-known conventional methods (see, e.g., Remington, The Science and Practice of Pharmacy, 23rd edition, A. Adejare, ed., Academic Press, 2020).

[0105] The term “subject”, as used herein, refers to an “animal” and in particular a “mammal” such as a non-primate (e.g., mice, rats, bovines, horses, household cats, tigers and other large cats, dogs, pigs, rabbits, goats, deer, sheep, ferrets, gerbils, guinea pigs, hamsters, bats, and birds (e.g., chickens, turkeys, and ducks)) or a primate (e.g., monkeys, baboons, chimpanzees, and human). The term may be used interchangeably with the term “patient” or “individual”. In some embodiments, the subject is a mammal, e.g., a human, diagnosed with a disease or disorder provided herein. In some embodiments, the subject is a mammal, e.g., a human, at risk of developing a disease or disorder provided herein.

[0106] The term “percent identity” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared. Unless otherwise indicated, percent identity is determined using the National Center for Biotechnology Information (NCBI)’s Basic Local Alignment Search Tool (BLAST®), available at blast.ncbi.nlm.nih.gov / Blast.cgi, version BLAST+ 2.13.0. In some embodiments, the percent identity is calculated over the entire length of the compared sequences. In some embodiments, the sequence identity is calculated over a fragment of each compared sequence of about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 35 amino acids, about 40 amino acids, about 45 amino acids, about 50 amino acids, about 55 amino acids, about 60 amino acids, about 65 amino acids, about 70 amino acids, about 75 amino acids, about 80 amino acids, about 85 amino acids, about 90 amino acids, about 95 amino acids, about 100 amino acids, about 105 amino acids, about 110 amino acids, about 115 amino acids, about 120 amino acids, about 125 amino acids, about 130 amino acids, about 135 amino acids, about 140 amino acids, about 145 amino acids, about 150 amino acids, about 155 amino acids, about 160 amino acids, about 165 amino acids, about 170 amino acids, about 175 amino acids, about 180 amino acids, about 185 amino acids, about 190 amino acids, about 195 amino acids, or about 200 amino acids.24323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102

[0107] As used herein, the term “sample,” refers to a mixture comprising an immunoglobulin (e.g., an FLC). In some embodiments, the sample is obtained from a subject, such as a human patient. In some embodiments, the sample comprises a fresh, frozen, and / or preserved organ, biopsy, and / or aspirate obtained from the subject. In some embodiments, the sample comprises blood or any blood constituent (e.g., serum, plasma) obtained from the subject. In some embodiments, the sample comprises a bodily fluid (e.g., cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid) obtained from the subject. In some embodiments, the sample comprises non-natural compounds (e.g., preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics). In some embodiments, the sample comprises recombinant FLC protein (e.g., recombinant FLC protein in a buffer).

[0108] As used herein, the term “reference” refers to a sample, standard, or level used to assess the presence and / or amount of amyloidogenic XFLC in a sample.

[0109] As used herein, “FLC dimer stabilizer” refers to a small molecule that enhances the kinetic and / or thermodynamic stability of FLC dimers. Without being held to theory or mechanism, in some embodiments, a FLC dimer stabilizer binds to the interface in a FLC dimer and stabilizes the dimeric structure of a FLC dimer, thereby preventing degradation, aggregation, and / or misfolding

[0110] As used herein, the term “target engagement” refers to the binding of a FLC dimer stabilizer to a pathogenic FLC dimer.[OHl] As used herein, the term “treating” or “treat” describes the management and care of a patient for the purpose of combating a disease, condition, or disorder, to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. The term “treat” can also include treatment of a cell in vitro or an animal model.

[0112] As used herein, the term “preventing,” “prevent,” or “protecting against” describes reducing or eliminating the onset of the symptoms or complications of such disease, condition or disorder.Detecting Amyloidogenic LFLC Proteins

[0113] The present disclosure provides methods for detecting amyloidogenic ZFLC proteins (e.g., amyloidogenic ZFLC proteins). In some embodiments, the method comprises the steps of: (i) contacting a sample with a protease to obtain a digested sample, wherein the contacting is performed under conditions effective for producing an amyloidogenic ZFLC protein neoepitope (e.g., an amyloidogenic ZFLC protein neo-epitope described herein), and wherein the25323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 sample comprises one or more of an amyloidogenic FLC protein and a non-amyloidogenic FLC protein; and (ii) determining an amount of the neo-epitope in the digested sample. In some embodiments, the step of (ii) comprises contacting the digested sample with a monoclonal antibody or antigen-binding fragment thereof described herein that binds to the neo-epitope, thereby detecting amyloidogenic XFLC protein in the sample.

[0114] In some embodiments, the step of (ii) provides an amount of the neo-epitope in the digested sample as an absolute or relative value. In some embodiments, the amount of the neoepitope in the digested sample is determined as an absolute value. In some embodiments, the amount of the neo-epitope in the digested sample is determined relative to an amount of total FLC in the sample. In some embodiments, the amount of the neo-epitope in the digested sample is determined relative to an amount of FLC dimer in the sample. In some embodiments, the amount of the neo-epitope in the digested sample is determined relative to an amount of FLC monomer in the sample.

[0115] In some embodiments, the amount of the neo-epitope in the digested sample is determined relative to a reference. In some embodiments, the reference is an amount of the neoepitope in a control sample. In some embodiments, the control sample is the sample not subjected to step (i). In some embodiments, the reference is an amount of neo-epitope in a control sample comprising non-amyloidogenic XFLC protein and subjected to the steps of (i)- (ii). In some embodiments, the control sample comprises a recombinant non-amyloidogenic XFLC. In some embodiments, the control sample is obtained from a healthy subject. In some embodiments, an increase in the amount of the neo-epitope in the digested sample as compared to the amount of the neo-epitope in the control sample indicates the sample comprises amyloidogenic XFLC.

[0116] In some embodiments, a percent increase in the amount of the neo-epitope in the digested sample as compared to the control sample corresponds to the proportion of FLC in the sample that is amyloidogenic XFLC. In some embodiments, a percent increase of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% indicates the sample comprises amyloidogenic XFLC. In some embodiments, the percent increase corresponds to the proportion of XFLC in the sample that is amyloidogenic XFLC. In some embodiments, a fold decrease of at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold indicates the sample comprises amyloidogenic XFLC. In some embodiments, the fold decrease corresponds to the proportion of XFLC in the sample that is amyloidogenic XFLC. In some embodiments, a substantial similarity in the26323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 amount of the neo-epitope in the digested sample as compared to the amount of the neo-epitope in the control sample indicates the sample comprises non-amyloidogenic XFLC.

[0117] In some embodiments, the sample comprises amyloidogenic XFLC protein. In some embodiments, the method comprises contacting the sample with an FLC dimer stabilizer prior to the steps of (i)-(ii). In some embodiments, the sample is obtained by contacting a sample comprising amyloidogenic XFLC with the FLC dimer stabilizer ex vivo. In some embodiments, the sample comprising amyloidogenic XFLC is obtained from a subject suspected of having AL amyloidosis, and is further contacted with the FLC dimer stabilizer ex vivo prior to the steps of (i)-(ii). In some embodiments, the sample comprises a recombinant amyloidogenic XFLC and is further contacted with the FLC dimer stabilizer ex vivo prior to the steps of (i)-(ii). In some embodiments, the sample is obtained from a subject who has received the FLC dimer stabilizer, wherein the subject is suspected of having AL amyloidosis. In some embodiments, the sample is obtained from the subject immediately after receiving the FLC dimer stabilizer (e.g., within minutes to hours of receiving the FLC dimer stabilizer). In some embodiments, the sample is obtained from the subject following a period of time after receiving the FLC dimer stabilizer, wherein the period of time is about 1 to about 30 days. In some embodiments, the sample is a sample obtained from the subject that has been stored in an appropriate manner, e.g. frozen at - 80C. In some embodiments, the method comprises subjecting the sample comprising the amyloidogenic XFLC and the FLC dimer stabilizer to steps (i)-(ii).

[0118] In some embodiments, a decreased amount of the neo-epitope in the digested sample as compared to a reference indicates target engagement of the FLC dimer stabilizer. In some embodiments, the reference is an amount of neo-epitope in a control sample not contacted with an FLC dimer stabilizer. In some embodiments, a decreased amount of neo-epitope in the digested sample compared to an amount of neo-epitope in the control sample indicates target engagement of the FLC dimer stabilizer. In some embodiments, a decrease of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% indicates target engagement of the FLC dimer stabilizer. In some embodiments, a decrease of at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold indicates target engagement of the FLC dimer stabilizer.Limited Proteolysis

[0119] The methods of the disclosure for detecting amyloidogenic XFLC proteins comprise a step of limited proteolysis, wherein a sample is contacted with a protease under conditions27323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 effective for producing an amyloidogenic XFLC neo-epitope (e.g., an amyloidogenic XFLC neoepitope described herein). In some embodiments, the conditions effective for producing a neoepitope are characterized by one or more factors, wherein the one or more factors comprise: (1) a source of the protease; (2) a composition of a sample (e.g., a concentration of protein in the composition); (3) a concentration of the protease; (4) a duration of time for the proteolysis reaction; (5) a temperature for the proteolysis reaction; or (6) a pH for the proteolysis reaction. In some embodiments, the conditions comprise a temperature, a duration of time, a concentration of the protease, and / or a concentration of protein in the sample.

[0120] In some embodiments, contacting a sample with a protease under conditions effective for producing the neo-epitope results in at least partial cleavage of amyloidogenic XFLC in a sample to generate the neo-epitope. In some embodiments, contacting a sample with a protease under conditions effective for producing the neo-epitope results in complete cleavage of amyloidogenic XFLC to generate the neo-epitope. In some embodiments, the conditions do not result in substantial cleavage of non-amyloidogenic FLC.Samples

[0121] Methods for detecting amyloidogenic XFLC in a sample are provided herein. In some embodiments, the sample is obtained from a subject. In some embodiments, the subject is a healthy subject. In some embodiments, the subject is suspected of having a plasma cell disorder or a related condition. In some embodiments, the plasma cell disorder is a plasma cell proliferative disorder. Examples of plasma cell proliferative disorders include, but are not limited to, MGUS, monoclonal immunoglobulin deposition diseases (amyloidosis), plasmacytoma, and multiple myeloma. In some embodiments, the subject is suspected of having MGUS. In some embodiments, the subject is suspected of having plasmacytoma. In some embodiments, the subject is suspected of having multiple myeloma. In some embodiments, the subject is suspected of having amyloidosis. In some embodiments, the subject is suspected of having AL amyloidosis. In some embodiments, the subject is suspected of having an abnormal FLC level.

[0122] In some embodiments, the subject has been diagnosed with MGUS, amyloidosis, plasmacytoma, or multiple myeloma. In some embodiments, the subject has been diagnosed with MGUS. In some embodiments, the subject has been diagnosed with plasmacytoma. In some embodiments, the subject has been diagnosed with multiple myeloma. In some28323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 embodiments, the subject has been diagnosed with amyloidosis. In some embodiments, the subject has been diagnosed with AL amyloidosis.

[0123] In some embodiments, the subject that has been diagnosed is treatment naive, i.e., the subject has not received a treatment for a specific disease or condition with which they have been diagnosed. In some embodiments, the subject has received treatment for MGUS, amyloidosis, plasmacytoma, and / or multiple myeloma. In some embodiments, the subject has received treatment for MGUS. In some embodiments, the subject has received treatment for amyloidosis. In some embodiments, the subject has received treatment for plasmacytoma. In some embodiments, the subject has received treatment for multiple myeloma. In some embodiments, the subject has received treatment for AL amyloidosis. Treatments for AL amyloidosis may include, but are not limited to, a steroid, a chemotherapeutic agent (e.g. for plasma cell elimination chemotherapy), a stem cell transplantation, and anti-CD38 immunotherapy such as anti-CD38 monoclonal antibodies.

[0124] In some embodiments, the sample obtained from the subject is a liquid sample. In some embodiments, the liquid sample is whole blood, plasma, serum, ascites, urine, saliva, buccal sample, or cavity or organ rinse. In some embodiments, the sample is whole blood. In some embodiments, the sample is plasma. In some embodiments, the sample is serum. In some embodiments, the sample is urine. In some embodiments, the sample is a solid sample such as a tissue biopsy.

[0125] In some embodiments, the sample comprises FLC. In some embodiments, the FLC comprises amyloidogenic XFLC protein. In some embodiments, the FLC comprises non- amyloidogenic XFLC. In some embodiments, the FLC comprises amyloidogenic XFLC and nonamyl oidogenic XFLC.

[0126] In some embodiments, the amyloidogenic XFLC protein is a recombinant protein. In some embodiments, the amyloidogenic XFLC is a recombinant protein comprising a sequence of an amyloidogenic XFLC obtained from a subject having AL amyloidosis. In some embodiments, the recombinant amyloidogenic XFLC is selected from the group consisting of: H9, WIL, H3, and a combination thereof. In some embodiments, the recombinant amyloidogenic XFLC is WIL. In some embodiments, the recombinant amyloidogenic XFLC is H3. In some embodiments, the recombinant amyloidogenic XFLC is H9. In some embodiments, the sample is used in the methods of the disclosure as a control sample.

[0127] In some embodiments, the non-amyloidogenic XFLC is a recombinant protein. In some embodiments, the sample is used in the methods of the disclosure as a control sample.29323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102

[0128] In some embodiments, the sample comprises a recombinant amyloidogenic XFLC and an FLC dimer stabilizer. In some embodiments, the sample is used in the methods of the disclosure to measure target engagement of the FLC dimer stabilizer.

[0129] In some embodiments, the amyloidogenic XFLC is obtained from a subject suspected of having AL amyloidosis. In some embodiments, the amyloidogenic XFLC is a recombinant protein comprising the sequence of an amyloidogenic XFLC obtained from a subject suspected of having AL amyloidosis. Methods for obtaining recombinant amyloidogenic XFLC are known in the art, see, e.g., Rognoni et al (2013) PLoS One 8(9): e76022.

[0130] In some embodiments, the amyloidogenic ZFLC is one described in Oberti, et al (2017) Sci Rep 7: 16809. In some embodiments, the amyloidogenic ZFLC is one described in the Amyloid Light Chain Database (AL-Base) from Boston University (see https: / / wwwapp.bumc.bu.edu / BEDAC_ALBase). AL-Base is a curated database of antibody light chain sequences and provides information for light chain sequences known to be associated with AL amyloidosis. Both nucleotide and amino acid sequences of exemplary amyloidogenic light chain sequences can be obtained from AL-Base. AL-Base, including its uses, is described in detail in Bodi, et al (2009) Amyloid 16(1): 1-8 and Morgan, et al (2025) Amyloid 32(2): 129- 138. Sequence information for exemplary amyloidogenic ZFLCs is also accessible via public databases such as National Center for Biotechnology Information (NCBI) at https: / / www.ncbi.nlm.nih.gov / . In some embodiments, the amyloidogenic ZFLC is selected from the group consisting of: H9, WIL, H3, and a combination thereof. Sequence information for H9, WIL, and H3 is accessible via public databases, such as NCBI, with the identifier number provided in Table 1.Table 1: Sequence information for exemplary amyloidogenic LFLC

[0131] In some embodiments, the non-amyloidogenic ZFLC is obtained from a healthy subject (e.g., a subject not suspected of having a plasma disorder or AL amyloidosis). In some embodiments, the non-amyloidogenic ZFLC is a recombinant protein comprising the sequence of an non-amyloidogenic ZFLC obtained from a healthy subject. In some embodiments, the non-30323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 amyloidogenic XFLC is JTO. Sequence information of JTO, with accession 1CD0 A, is available from NCBI.Proteases

[0132] Described herein are exemplary proteases for use in limited proteolysis of proteins in a sample. In some embodiments, the sample is contacted with a single protease. In some embodiments, the sample is contacted with more than one protease (i.e., a combination of proteases). In some embodiments, the combination of protease comprises one, two, three, or four proteases. In some embodiments, the protease is a naturally occurring protease (i.e., a protease present in nature). In some embodiments, the protease is recombinant. In some embodiments, the protease comprises one or more modifications with respect to the naturally occurring protease.

[0133] In some embodiments, the protease is selected from the group consisting of: proteinaseK, thermolysin, pepsin, trypsin, chymotrypsin, cathepsin B, cathepsin L, papain, and a peptidase S8. In some embodiments, the peptidase S8 is PCSK9 or subtilisin.

[0134] In some embodiments, the sample is contacted with proteinase K and a second protease selected from the group consisting of thermolysin, pepsin, trypsin, chymotrypsin, cathepsin B, cathepsin L, papain, and a peptidase S8. In some embodiments, the protease is proteinase K. In some embodiments, the protease is trypsin. In some embodiments, the protease is chymotrypsin. In some embodiments, the protease is subtilisin. In some embodiments, the protease is papain. In some embodiments, the protease is cathepsin B. In some embodiments, the protease is cathepsinL. In some embodiments, the protease is pepsin. In some embodiments, the protease is thermolysin.

[0135] Proteinase K (EC_3.4.21.64, also called protease K, endopeptidase K, Tritirachium alkaline proteinase, Tritirachium album serine proteinase, Tritirachium album proteinase K) is a broad-spectrum serine protease. The predominant cleavage sites are the peptide bonds adjacent to the carboxyl group of hydrophobic amino acids (aliphatic, aromatic and other hydrophobic amino acids). Proteinase K may be extracted from Tritirachium album or made recombinantly. It is expected that Proteinase K from different sources will have different specific activities.

[0136] Thermolysin (EC 3.4.24.27, Bacillus thermoproteolyticus neutral proteinase, thermoase, thermoase Y10, TLN) is a thermostable neutral metalloproteinase enzyme produced by the Gram-positive bacteria Bacillus thermoproteolyticus. It requires one zinc ion for enzyme activity and four calcium ions for structural stability. Thermolysin specifically catalyzes the hydrolysis31323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 of peptide bonds containing hydrophobic amino acids. It is expected that Thermolysin from different sources will have different specific activities.

[0137] Pepsin is an endopeptidase that breaks down proteins into smaller peptides and amino acids. Pepsin is expressed as a zymogen, Pepsinogen, which is released by the gastric chief cells in the stomach wall, and upon mixing with the hydrochloric acid of the gastric juice, Pepsinogen activates to become Pepsin. Pepsin is an aspartic protease, using a catalytic aspartate in its active site. It is expected that Pepsin from different sources will have different specific activities.

[0138] Trypsin is an enzyme in the first section of the small intestine that starts the digested of protein molecules by cutting long chains of amino acids into smaller pieces. It is a serine protease from the PA clan superfamily, found in the digestive system of many vertebrates, where it hydrolyzes proteins. Trypsin is formed in the small intestine when its proenzyme form, the Trypsinogen produced by the pancreas, is activated. Trypsin cuts peptide chains mainly at the carboxyl side of the amino acids lysine or arginine. The enzymatic mechanism is similar to that of other serine proteases. These enzymes contain a catalytic triad consisting of histidine-57, aspartate- 102, and serine- 195. It is expected that Trypsin from different sources will have different specific activities.

[0139] Chymotrypsin (EC 3.4.21.1, Chymotrypsins A and B, alpha-chymar ophth, avazyme, chymar, chymotest, enzeon, quimar, quimotrase, alpha-chymar, alpha-chymotrypsin A, alphachymotrypsin) is a digestive enzyme component of pancreatic juice acting in the duodenum, where it performs proteolysis, the breakdown of proteins and polypeptides. Chymotrypsin preferentially cleaves peptide amide bonds where the side chain of the amino acid N-terminal to the scissile amide bond (the Pl position) is a large hydrophobic amino acid (tyrosine, tryptophan, and phenylalanine). These amino acids contain an aromatic ring in their side chain that fits into a hydrophobic pocket (the SI position) of the enzyme. It is expected that Chymotrypsin from different sources will have different specific activities.

[0140] Cathepsin B belongs to a family of lysosomal cysteine proteases known as the cysteine cathepsins and plays an important role in intracellular proteolysis. Cathepsin B is synthesized on the rough endoplasmic reticulum as a preproenzyme of 339 amino acids with a signal peptide of 17 amino acids. It is expected that Cathepsin B from different sources will have different specific activities.

[0141] Cathepsin L as used herein refers to Cathepsin LI and / or Cathepsin L2. Cathepsin LI is a protein that in humans is encoded by the CTSL1 gene. Cathepsin LI is a cysteine cathepsin, a lysosomal cysteine protease that plays a major role in intracellular protein catabolism. Cathepsin32323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102L2 (EC 3.4.22.43, also known as cathepsin V or cathepsin U) is a protein encoded in humans by the CTSV gene. Cathepsin L2 is a human cysteine cathepsin, a lysosomal cysteine protease with endopeptidase activity. Cathepsin L and other cysteine cathepsins tend to be secreted by macrophages and other tissue-invading immune cells when causing pathological inflammation. It is expected that Cathepsin L from different sources will have different specific activities.

[0142] Papain, also known as papaya proteinase I, is a cysteine protease (EC 3.4.22.2) enzyme present in papaya (Carica papaya) and mountain papaya (Vasconcellea cundinamarcensis). It is the namesake member of the Papain-like protease family. The mechanism by which Papain breaks peptide bonds involves the use of a catalytic dyad with a deprotonated cysteine. Asn-175 helps to orient the imidazole ring of His-159 to allow it to deprotonate the catalytic Cys-25. This cysteine then performs a nucleophilic attack on the carbonyl carbon of a peptide backbone. This forms a covalent acyl-enzyme intermediate and frees the amino terminus of the peptide. It is expected that Papain from different sources will have different specific activities.Conditions for limited proteolysis

[0143] Described herein are conditions for performing limited proteolysis of proteins in a sample.

[0144] In some embodiments, limited proteolysis results in at least partial cleavage of amyloidogenic FLC in a sample, wherein the at least partial cleavage results in exposure of an amyloidogenic ZFLC neo-epitope. In some embodiments, the limited proteolysis does not result in substantial cleavage of non-amyloidogenic ZFLC.

[0145] In some embodiments, the conditions effective for producing a neo-epitope are characterized by one or more kinetic parameters. In some embodiments, the kinetic parameter is the first-order rate constant (kcat). As appreciated by the skilled artisan, the kcat refers to the reaction rate when an enzyme is full occupied at a saturating concentration of the substrate. A higher kcat value indicates the enzyme is converting more substrate to product per unit of time. Methods for determining kcat are known in the art, and include, for example, measuring a decrease in substrate concentration over time or an increase in product formation over time and calculating the kcat under Michaelis-Mentin kinetics.

[0146] In some embodiments, the conditions are characterized by a kcat of about IxlO'2m / sec to IxlO'9m / sec for amyloidogenic ZFLC cleavage to generate the neo-epitope.

[0147] In some embodiments, the conditions are characterized by a kcat of about IxlO'2m / sec to IxlO'9m / sec for amyloidogenic ZFLC cleavage to generate the neo-epitope. In some33323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 embodiments, the conditions effective for producing a neo-epitope are characterized by a kcat of about IxlO'2m / sec to IxlO'3m / sec, about IxlO'3m / sec to IxlO'4m / sec, about IxlO'4m / sec to IxlO'5m / sec, about IxlO'5m / sec to IxlO'6m / sec, about IxlO'6m / sec to IxlO'7m / sec, about IxlO'7m / sec to IxlO'8m / sec, or about IxlO'8m / sec to IxlO'9m / sec for amyloidogenic ZFLC cleavage to generate the neo-epitope.

[0148] In some embodiments, the conditions effective for producing a neo-epitope do not result in substantial cleavage of non-amyloidogenic ZFLC cleavage. In some embodiments, the conditions effective for producing a neo-epitope are characterized by a kcat for amyloidogenic ZFLC cleavage that is higher than a kcat for non-amyloidogenic ZFLC cleavage. In some embodiments, the kcat for amyloidogenic ZFLC cleavage is at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the kcat for non-amyloidogenic ZFLC cleavage.

[0149] In some embodiments, limited proteolysis is performed at a temperature of about 2°C- 50°C. In some embodiments, the temperature is about 2°C-8°C, about 4°C-20°C, about 18°C- 32°C, about 32°C-42°C, about 35°C-42°C, about 37°C-40°C, or about 40°C-50°C. In some embodiments, the temperature is about 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, or 42°C. In some embodiments, the temperature is about 37°C. In some embodiments, the temperature is about 40°C. In some embodiments, the temperature is about 42°C.

[0150] In some embodiments, limited proteolysis is performed for a duration of time of about 1 minute to about 18 hours. In some embodiments, the duration of time is about 1-5 minutes, about 5-10 minutes, about 10-20 minutes, about 20-30 minutes, about 30-60 minutes, about 60- 90 minutes, about 90-120 minutes, about 120-150 minutes, about 150-180 minutes, about 3-6 hours, about 6-9 hours, about 9-12 hours, about 12-15 hours, or about 15-18 hours. In some embodiments, the duration of time is about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 150 minutes, about 180 minutes, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, or about 18 hours. In some embodiments, the duration of time is about 2 hours. In some embodiments, the duration of time is about 90 minutes. In some embodiments, the duration of time is about 60-120 minutes. In some embodiments, the duration of time is about 60-90 minutes.34323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102

[0151] In some embodiments, limited proteolysis is performed by contacting a sample with a protease at a protease concentration of about 0.01 pM to about 10,000 pM. In some embodiments, the concentration of the protease is about 0.01 pM to about 1 pM, about 0.05 pM to about 1 pM, about 0.1 pM to about 1 pM, about 0.5 pM to about 2 pM, about 0.5 pM to about 5 pM, about 1 pM to about 3 pM, about 1 pM to about 5 pM, or about 5 pM to about 10 pM. In some embodiments, the concentration of the protease is about 3pM. In some embodiments, the concentration of the protease is about 0.1 pM to about 10 pM, about 0.5 pM to about 10 pM, about 1 pM to about 10 pM, about 5 pM to about 20 pM, about 5 pM to about 50 pM, about 10 pM to about 30 pM, about 10 pM to about 50 pM, or about 50 pM to about 100 pM. In some embodiments, the concentration of the protease is about 100 pM to about 500 pM, about 500 pM to about 1000 pM, about 1000 pM to about 2000 pM, about 2000 pM to about 3000 pM, about 3000 pM to about 4000 pM, about 4000 pM to about 5000 pM, about 5000 pM to about 6000 pM, about 6000 pM to about 7000 pM, about 7000 pM to about 8000 pM, about 8000 pM to about 9000 pM, or about 9000 pM to about 10000 pM. In some embodiments, the concentration of the protease is about 0.1 pM to 10 pM. In some embodiments, the concentration of the protease is about 0.1 pM to 1 pM.

[0152] In some embodiments, limited proteolysis is performed at a pH of about 2 to about 12. In some embodiments, the pH is about 2 to about 6, about 2 to about 4, about 3 to about 5, about 4 to about 6, about 4 to about 7, about 5 to about 8, about 6 to about 8, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 8 to about 10, about 8 to about 12, about 9 to about 12, about 9 to about 11, or about 10 to about 12. In some embodiments, limited proteolysis is performed at a pH of about 2, 3, or 4. In certain embodiments where limited proteolysis is performed at a pH of less than or equal to about 4, the method further comprises adjusting the pH to about 6 to about 8 following limited proteolysis of proteins in a sample. In specific embodiments, the method further comprises adjusting the pH to about 6, about 7, or about 8 following limited proteolysis.

[0153] In some embodiments, limited proteolysis of proteins in a sample is terminated. In some embodiments, the termination comprises contacting the sample with a protease inhibitor. In some embodiments, the termination comprises contacting the sample with a combination of protease inhibitors. In some embodiments, the protease inhibitor comprises 4-(2- Aminoethyljbenzenesulfonyl fluoride hydrochloride (AEBSF), aprotinin, bestatin, E-64, leupeptin, pepstatin A, or a combination thereof.35323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102Neo-Epitopes

[0154] The present disclosure provides neo-epitopes resulting from limited proteolysis of amyloidogenic XFLC protein for use in the methods of the disclosure (e.g., to generate antibodies that bind the neo-epitope and / or to detect amyloidogenic XFLC protein in a sample).

[0155] In some embodiments, the neo-epitope of an amyloidogenic XFLC protein is present on a lambda XFLC exposed to limited proteolysis. In some embodiments, a neo-epitope is generated through limited proteolysis of the amyloidogenic XFLC protein using a protease as described herein.

[0156] In some embodiments, the neo-epitope of an amyloidogenic XFLC protein is not present on non-amyloidogenic XFLC proteins after limited proteolysis. In some embodiments, the neoepitope is generated by limited proteolysis of amyloidogenic XFLC proteins by proteinase K. In some embodiments, the neo-epitope is generated by limited proteolysis of amyloidogenic XFLC proteins by thermolysin. In some embodiments, the neo-epitope is generated by limited proteolysis of amyloidogenic XFLC proteins by pepsin. In some embodiments, the neo-epitope is generated by limited proteolysis of amyloidogenic XFLC proteins by trypsin. In some embodiments, the neo-epitope is generated by limited proteolysis of amyloidogenic XFLC proteins by chymotrypsin. In some embodiments, the neo-epitope is generated by limited proteolysis of amyloidogenic XFLC proteins by cathepsin B. In some embodiments, the neoepitope is generated by limited proteolysis of amyloidogenic XFLC proteins by cathepsin L. In some embodiments, the neo-epitope is generated by limited proteolysis of amyloidogenic XFLC proteins by papain. In some embodiments, the neo-epitope is generated by limited proteolysis of amyloidogenic XFLC proteins by a protein of the peptidase S8 family. In some embodiments, the neo-epitope is generated by limited proteolysis of amyloidogenic XFLC proteins by PCSK9. In some embodiments, the neo-epitope is generated by limited proteolysis of amyloidogenic XFLC proteins by subtilisin.

[0157] An aspect of the application is a neo-epitope on XFLC prepared by limited proteolysis of amyloidogenic XFLC proteins by proteinase K. In certain embodiments, the neo-epitope is xQP, where x is one of G, R or S. In certain embodiments, the neo-epitope is GQP. In certain embodiments, the neo-epitope is RQP. In certain embodiments, the neo-epitope is SQP.

[0158] In certain embodiments, the neo-epitope comprises at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide of any one of SEQ ID NOS: 1 and 34-135, 137-239, 241-343344, GQP, SQP, and RQP. In certain embodiments, the neoepitope comprises the amino acid sequence GQP (SEQ ID NO: 136), RQP (SEQ ID NO: 344),36323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102SQP (SEQ ID NO: 240), GQPKA (SEQ ID NO: 134), RQPKA (SEQ ID NO: 342), or SQPKA (SEQ ID NO: 238). In certain embodiments, the neo-epitope comprises, from N-terminus to C- terminus, (i) GQP, RQP, or SQP; and (ii) a polypeptide with at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or 100 % sequence identity to any one of SEQ ID NOS: 351-450, KAA, and KA.

[0159] In embodiments, the neo-epitope has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide of any one of SEQ ID NOs: 1 and 34-135, 137-239, 241-343344, GQP, SQP, and RQP. In embodiments, the neo-epitope comprises the amino acid sequence GQP, RQP, or SQP. In embodiments, the neo-epitope comprises from the N-terminus to the C-terminus (i) GQP, RQP, or SQP; and (ii) a polypeptide with at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or at least 100 % identical to any one of SEQ ID NOS: 351- 450, KAA, and KA.Exemplary Limited Proteolysis

[0160] An aspect of the application is a method of selecting conditions for limited proteolysis using proteinase K, including but not limited to: (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. In some embodiments, the conditions are selected to differentially obtain a neo-epitope from amyloidogenic XFLC vs. non-amyloidogenic XFLC.

[0161] In some embodiments, limited proteolysis is performed using proteinase K under conditions effective for exposing a neo-epitope.

[0162] In certain embodiments, the limited proteolysis by proteinase K comprises the steps of: contacting a sample with proteinase K under conditions effective for producing an amyloidogenic XFLC protein neo-epitope described herein. In certain embodiments, the conditions comprise a proteinase K concentration (e.g., 0.02-20. OpM Proteinase K), a temperature (e.g., 37°C), and a reaction time (e.g., 30 seconds to 20 hrs). In some embodiments, the conditions are selected based on the specific activity of the protease.

[0163] In some embodiments, the sample is contacted with proteinase K as a single protease. In some embodiments, the sample is contacted with proteinase K in combination with at least one other protease.

[0164] In some embodiments, limited proteolysis is performed at a temperature ranging from about 4°C to 45°C. In some embodiments, the temperature is about 4°C-20°C, about 18°C-32°C,37323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 about 32°C-42°C, about 35°C-42°C, about 37°C-40°C, or about 40°C-45°C. In some embodiments, the temperature is about 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, or 42°C. In some embodiments, the temperature is about 37°C. In some embodiments, the temperature is about 40°C. In some embodiments, the temperature is about 42°C.

[0165] In some embodiments, limited proteolysis is performed for a duration of time of about 1 minute to 18 hours. In some embodiments, the duration of time is about 1-5 minutes, about 5-10 minutes, about 10-20 minutes, about 20-30 minutes, about 30-60 minutes, about 60-90 minutes, about 60-120 minutes, about 90-120 minutes, about 120-150 minutes, about 150-180 minutes, about 3-6 hours, about 6-9 hours, about 9-12 hours, about 12-15 hours, or about 15-18 hours. In some embodiments, the duration of time is about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 150 minutes, about 180 minutes, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, or about 18 hours. In some embodiments, the duration of time is about 2 hours. In some embodiments, the duration of time is about 90 minutes.

[0166] In some embodiments, limited proteolysis is performed with a concentration of Proteinase K of about 0.01 pM to about 10 pM. In some embodiments, the concentration of the protease is about 0.01 pM to about 0.05 pM, about 0.01 pM to about 1 pM, about 0.05 pM to about 1 pM, about 0.1 pM to about 1 pM, about 0.5 pM to about 2 pM, about 0.5 pM to about 5 pM, about 1 pM to about 3 pM, about 1 pM to about 5 pM, or about 5 pM to about 10 pM. In some embodiments, the concentration of the protease is about 3 pM. In some embodiments, the concentration of the protease is about 0.1 pM to 10 pM. In some embodiments, the concentration of the protease is about 0.1 pM to 1 pM.Applications

[0167] The disclosure provides applications for the methods, immunoassays, and kits described herein. The applications include, but are not limited to, diagnosis of AL amyloidosis in a subject, treatment monitoring (e.g., detection of amyloidogenic ZFLC in a subject to determine treatment efficacy and / or to detect relapse), and screening target engagement of candidate FLC dimer stabilizers.38323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102Diagnosis

[0168] The methods of the disclosure provide for diagnosis of AL amyloidosis in a subject. AL is often diagnosed late or goes undiagnosed until later stage, at least in part because the signs and symptoms of AL mimic those of other common diseases, such as other plasma cell disorders. Clinical symptoms of specific organ involvement are the most common trigger of suspicion of the disease. Confirmation of AL diagnosis typically takes 6-12 months with visits to several physicians and specialists. The delay in AL amyloidosis diagnosis leads to many subjects having advanced organ involvement upon diagnosis, which is often associated with poor prognosis. For example, the mortality rate at six months after AL diagnosis approaches 25%, with mean survival of around four years.

[0169] 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 ZFLC and KFLC) and K / 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 spectrometry -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 amyl oidogeni city.

[0170] A challenge associated with accurate diagnosis of AL amyloidosis is differentiating the disease from other conditions associated with similar symptoms, such as a plasma cell disorder (e.g., multiple myeloma, Waldenstrom Macroglobulinemia, and Monoclonal Gammopathy of Undetermined Significance (MGUS)) and other types of amyloidosis (e.g., AA (inflammatory) amyloidosis, ATTR (transthyretin) amyloidosis, or hereditary amyloidosis). Accordingly, in some embodiments, the methods of the disclosure are applicable for diagnosis of AL amyloidosis in a subject suspected of having a plasma cell disorder selected from the group consisting of Multiple myeloma (MM), AL amyloidosis, Smoldering Multiple Myeloma (SMM), Waldenstrom Macroglobulinemia, MGUS, IgM-AL, ATTR amyloidosis, and hereditary amyloidosis. In some embodiments, the methods of the disclosure are applicable for diagnosis of AL amyloidosis in a subject who presents without clinical symptoms associated with a plasma cell disorder. In certain embodiments, the methods and compositions described herein can be used for diagnosis of AL amyloidosis amongst plasma cell disorder patients suspected of39323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 multiple myeloma (MM), AL amyloidosis, smoldering multiple myeloma (SMM), monoclonal gammopathy of undetermined significance (MGUS), and IgM-AL, also including those who have no clinical symptoms.

[0171] In addition to AL amyloidosis diagnosis, and due at least in part to its sensitivity, the diagnostic applications of the disclosure include detection of residual amyloidogenic FLC post treatment and at the beginning of a relapse.

[0172] In some embodiments, the methods for diagnosis of AL amyloidosis described herein are used in combination with an assessment of the following clinical parameters: free light chain levels, organ function biomarkers (e.g., N-terminal pro-brain natriuretic peptide (NT-proBNP), Troponin T, eGFR), and tests for organ function, including the Kansas City Cardiomyopathy Questionnaire (KCCQ), which is described in the following reference which is incorporated by reference herein in its entirety: Spertus et al. Interpreting the Kansas City Cardiomyopathy Questionnaire in Clinical Trials and Clinical Care: JACC State-of-the-Art Review, Journal of the American College of Cardiology, Volume 76, Issue 20, 2020, Pages 2379-2390; the 6-minute walk, which is used to assess aerobic capacity and endurance, and is described in the following reference which is incorporated by reference herein in its entirety: Casano et al. “Six -Minute Walk Test,” Matos Casano HA, Anjum F. Six-Minute Walk Test. [Updated 2023 Aug 14], In: StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2024; and a cardiopulmonary exercise test (CPET).

[0173] 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.

[0174] In certain embodiments, the methods and compositions described herein can be used for companion diagnostics, such as to test the ability of an FLC dimer stabilizer for its protection against proteolysis in individual pre-treatment AL amyloidosis patient plasma sample ex vivo and use the outcome to determine if the patient would benefit from treatment with the FLC dimer stabilizer.

[0175] 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 FLC dimer stabilizer, wherein the effect is an indication of clinical efficacy.

[0176] For the purpose of the above uses, other biological samples, such as urine, serum, CSF, tissue biopsy, etc. may also be used.40323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102

[0177] In some embodiments, the methods of the disclosure are applicable for generating and detecting a neo-epitope described herein in a sample obtained from subject suspected of having a plasma cell disorder, wherein a determination that the sample comprises an amyloidogenic XFLC neo-epitope is an indicator that the subject has AL amyloidosis. An advantage of the methods described herein is sensitive detection of amyloidogenic XFLC (through detection of the presence and / or quantification of the amount of a neo-epitope of an XFLC protein), thereby allowing for diagnosis of AL amyloidosis at an early stage (e.g., prior to or at the time of clinical symptom onset).

[0178] In some embodiments, the method for diagnosis of AL amyloidosis in a subject suspected of having a plasma cell disorder comprises the steps of: (i) exposing a biological sample obtained from the subject to limited proteolysis as described herein to obtain a digested sample, and (ii) determining an amount of amyloidogenic XFLC neo-epitope in the digested sample using a method of detection described herein (e.g., a method comprising contacting the digested sample with a monoclonal antibody or antigen-binding fragment thereof that binds to the neo-epitope), thereby providing a diagnostic indicator for AL amyloidosis. In some embodiments, an increased amount of the neo-epitope in the digested sample indicates the presence of amyloidogenic XFLC protein in the sample. In some embodiments, an increased amount of the neo-epitope in the digested sample as compared to a reference indicates the presence of amyloidogenic XFLC protein in the sample.

[0179] In some embodiments, the subject has received or is receiving a treatment for a plasma cell disorder. In some embodiments, the subject has not received a treatment for a plasma cell disorder. In some embodiments, the subject is experiencing symptoms associated with a plasma cell disorder.

[0180] In some embodiments, the sample comprises a blood or tissue sample obtained from the subject.

[0181] In some embodiments, the reference is an amount of amyloidogenic XFLC neo-epitope detected in a control sample subjected to limited proteolysis. In some embodiments, the control sample comprises non-amyloidogenic XFLC protein and is subjected to the limited proteolysis. In some embodiments, the reference is an amount of amyloidogenic XFLC neo-epitope detected in a control sample not subjected to limited proteolysis. In some embodiments, the control sample is obtained from the same source as the sample. In some embodiments, the control sample is processed by the method in parallel with the sample. In some embodiments, an increase in an amount of amyloidogenic XFLC neo-epitope in the digested sample as compared41323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 to the control sample is a diagnostic for AL amyloidosis. In some embodiments, an increase of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% is a diagnostic for AL amyloidosis. In some embodiments, an increase of at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold is a diagnostic for AL amyloidosis.

[0182] In some embodiments, the reference is an amount of neo-epitope in a control sample comprising amyloidogenic XFLC. In some embodiments, the control sample is obtained from the same source as the sample, wherein the control sample is modified by the addition of recombinant amyloidogenic XFLC. In some embodiments, the control sample is not obtained from the subject. In some embodiments, the control comprises recombinant amyloidogenic XFLC in a buffer. In some embodiments, the control sample is subjected to the limited proteolysis. In some embodiments, the control sample is processed by the method in parallel with the sample. In some embodiments, a substantially similar amount of amyloidogenic XFLC neo-epitope in the digested sample as compared to the control sample is a diagnostic for AL amyloidosis. In some embodiments, an amount of amyloidogenic XFLC neo-epitope in the digested sample that is not more than about ±10%, about ±20%, about ±30%, about ±40%, or about ±50% as compared to the control sample is a diagnostic for AL amyloidosis.

[0183] In some embodiments, the diagnostic for AL amyloidosis indicates the subject has AL amyloidosis, the subject will respond to treatment for a plasma cell disorder, the subject has amyloidogenic XFLC, or a combination thereof.Treatment Monitoring

[0184] The methods of the disclosure also provide for determining responsiveness of a subject suspected of having AL amyloidosis to a treatment.

[0185] In some embodiments, the treatment comprises a standard of care regimen. In some embodiments, the standard of care comprises administering an anti-CD38 antibody or antigen binding fragment thereof. Exemplary anti-CD38 antibodies include, but are not limited to, daratumumab and isatuximab. In some embodiments, the standard of care comprises a chemotherapy. In some embodiments, the chemotherapy comprises a proteasome inhibitor. Exemplary proteasome inhibitors include, but are not limited to, bortezomib, carfilzomib, and ixazomib. In some embodiments, the chemotherapy comprises an immunomodulatory drug. Exemplary immunomodulatory drugs include, but are not limited to, thalidomide, lenalidomide, and pomalidomide. In some embodiments, the chemotherapy comprises a DNA damage agent.42323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102Exemplary DNA damage agents include, but are not limited to, cyclophosphamide and melphalan. In some embodiments, the standard of care regimen comprises administering a combination of an anti-CD38 antibody or antigen binding fragment thereof and a chemotherapy. In some embodiments, the standard of care regimen comprises administering a combination of an anti-CD38 antibody or antigen binding fragment thereof and a proteasome inhibitor. In some embodiments, the standard of care regimen comprises administering a combination of an anti- CD38 antibody or antigen binding fragment thereof and an immunomodulatory drug. In some embodiments, the standard of care regimen comprises administering a combination of an anti- CD38 antibody or antigen binding fragment thereof and a DNA damage agent.

[0186] In some embodiments, the treatment comprises administration of an FLC dimer stabilizer. A FLC dimer stabilizer as described herein is a small molecule that enhances the kinetic and / or thermodynamic stability of FLCs. In some embodiments, the FLC stabilizer is a compound according to Formula Io or Ip of U.S. Patent App. Pub. No. 2024 / 0174672, herein incorporated by reference. In some embodiments, the FLC dimer stabilizer is a compound according to Formula I, as described in U.S. Patent App. Pub. No. 2024 / 0174672. In some embodiments, the FLC dimer stabilizer is any one of Compounds 1-35, as described in U.S. Patent App. Pub. No. 2024 / 0174672. In some embodiments, the FLC dimer stabilizer is a compound described in Lederber, et al (2024) J Med Chem 67:21070, herein incorporated by reference. In some embodiments, the FLC dimer stabilizer is a compound described in Morgan, et al (2019) PNAS 116:8360.

[0187] In some embodiments, the treatment comprises administering the FLC dimer stabilizer in combination with a standard of care regimen. In some embodiments, the FLC dimer stabilizer is administered to a subject who has already received the standard of care regimen. In some embodiments, the FLC dimer stabilizer is administered to a subject who is receiving the standard of care regimen.

[0188] In some embodiments, the methods of the disclosure are applicable for detecting an amyloidogenic ZFLC neo-epitope in a sample obtained from subject who has received or is receiving treatment. In some embodiments, the detection of amyloidogenic ZFLC in the sample indicates the subject is unresponsive or partially responsive to treatment. In some embodiments, the detection of substantially no amyloidogenic ZFLC in the sample indicates the subject is completely responsive to treatment.

[0189] In some embodiments, the methods of the disclosure are applicable for detecting an amyloidogenic ZFLC neo-epitope in a sample obtained from subject suspected of having AL43323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 amyloidosis who has received treatment. In some embodiments, the subject is no longer receiving treatment. In some embodiments, detection of amyloidogenic XFLC neo-epitope in the sample indicates the subject has relapsed following treatment. In some embodiments, detection of amyloidogenic XFLC neo-epitope in the sample indicates the subject has residual AL amyloidosis disease.

[0190] In some embodiments, the method for determining treatment outcome for a subject who is or who has received treatment for AL amyloidosis comprises the steps of: (i) exposing a sample obtained from the subject to limited proteolysis as described herein to obtain a digested sample, and (ii) determining an amount of an amyloidogenic XFLC neo-epitope in the digested sample using a method described herein (e.g., a method comprising contacting the digested sample with a monoclonal antibody or antigen-binding fragment thereof that binds to the neoepitope).

[0191] In some embodiments, the sample is obtained from the subject during an ongoing treatment regimen. In some embodiments, the sample is obtained from the subject after finishing a treatment regimen. In some embodiments, the sample is obtained from the subject within about 1 day to about 7 days, about 1 day to about 30 days, about 14 days to about 60 days, about 30 days to about 90 days, about 60 days to about 180 days after finishing a treatment regimen.

[0192] In some embodiments, the sample comprises a blood or tissue sample obtained from the subject.

[0193] In some embodiments, the amount of an amyloidogenic XFLC neo-epitope in the digested sample is determined as an absolute value. In some embodiments, the amount of an amyloidogenic XFLC neo-epitope is normalized to a reference. In some embodiments, the reference comprises an amount of an amyloidogenic XFLC neo-epitope in a control sample, and / or an amount of an internal standard in the digested sample.

[0194] In some embodiments, the reference is a control sample obtained from the subject prior to receiving the treatment. In some embodiments, the control sample is subjected to the limited proteolysis. In some embodiments, the control sample is processed by the method in parallel with the sample. In some embodiments, a decrease in an amount of an amyloidogenic XFLC neo-epitope in the digested sample as compared to the control sample indicates therapeutic efficacy for AL amyloidosis. In some embodiments, a decrease of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% indicates therapeutic efficacy for AL amyloidosis. In some embodiments, a decrease of at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or44323555102Attorney Docket No.: PRTE-021 / 01WO 345214-210210-fold indicates therapeutic efficacy for AL amyloidosis. In some embodiments, a substantially similar amount of FLC dimer in the digested sample as compared to the control sample indicates therapeutic efficacy for AL amyloidosis. In some embodiments, an amount of an amyloidogenic XFLC neo-epitope in the digested sample that is not more than about ±10%, about ±20%, about ±30%, about ±40%, or about ±50% as compared to the control sample indicates therapeutic efficacy for AL amyloidosis

[0195] In some embodiments, the reference is an amount of an amyloidogenic XFLC neoepitope in a control sample, wherein the control sample comprises non-amyloidogenic XFLC. In some embodiments, the control sample does not comprise amyloidogenic XFLC. In some embodiments, the control sample is obtained from a subject not suspected of having an AL amyloidosis. In some embodiments, the control sample comprises recombinant non- amyloidogenic XFLC. In some embodiments, the control sample is subjected to the limited proteolysis. In some embodiments, the control sample is processed by the method in parallel with the sample. In some embodiments, a substantially similar amount of an amyloidogenic XFLC neo-epitope in the digested sample as compared to the control sample indicates therapeutic efficacy for AL amyloidosis. In some embodiments, an amount of an amyloidogenic XFLC neo-epitope in the digested sample that is not more than about ±10%, about ±20%, about ±30%, about ±40%, or about ±50% as compared to the control sample indicates therapeutic efficacy for AL amyloidosis.

[0196] In some embodiments, the reference is an amount of an amyloidogenic XFLC neoepitope in a control sample, wherein the control sample comprises an FLC dimer stabilizer. In some embodiments, the control sample is obtained from the same source as the sample, and is further modified ex vivo by introducing the FLC dimer stabilizer. In some embodiments, the control sample is subjected to the limited proteolysis. In some embodiments, the control sample is processed by the method in parallel with the sample. In some embodiments, a substantially similar amount of an amyloidogenic XFLC neo-epitope in the digested sample as compared to the control sample indicates therapeutic efficacy for AL amyloidosis. In some embodiments, an amount of an amyloidogenic XFLC neo-epitope in the digested sample that is not more than about ±10%, about ±20%, about ±30%, about ±40%, or about ±50% as compared to the control sample indicates therapeutic efficacy for AL amyloidosis.45323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102Assessing Target Engagement

[0197] The methods of the disclosure provide for assessing target engagement of a candidate FLC dimer stabilizer. In certain embodiments, the methods and compositions described herein can be used to demonstrate target engagement by measuring the stabilization effect of an FLC dimer stabilizer for preventing proteolysis in an AL patient sample post treatment. In certain embodiments, the methods and compositions described herein can be used to monitor response to an FLC dimer stabilizer or plasma cell-targeting therapeutic treatment in AL amyloidosis patients.

[0198] In some embodiments, the candidate FLC dimer stabilizer is evaluated for stabilization of amyloidogenic XFLC in a subject suspected of having AL amyloidosis prior to receiving the FLC dimer stabilizer (e.g., as a companion diagnostic). In some embodiments, the candidate FLC dimer stabilizer is evaluated for stabilization of amyloidogenic XFLC as a measure of preclinical or clinical efficacy.

[0199] In some embodiments, the method for assessing target engagement of a candidate FLC dimer stabilizer comprises the steps of: (i)(a) obtaining a sample comprising amyloidogenic XFLC, and (b) contacting the sample with the candidate FLC dimer stabilizer, thereby providing a sample, (ii) exposing the sample to limited proteolysis as described herein to obtain an digested sample, (iii) determining an amount of an amyloidogenic XFLC neo-epitope in the digested sample using a method described herein (e.g., a method comprising contacting the digested sample with a monoclonal antibody or antigen-binding fragment thereof that binds to the neo-epitope), thereby providing a measure of target engagement for the candidate FLC dimer stabilizer.

[0200] In some embodiments, the sample is not obtained from a subject. In some embodiments, the sample comprises recombinant amyloidogenic XFLC. In some embodiments, the sample is obtained from a subject suspected of having AL amyloidosis.

[0201] In some embodiments, the amount of an amyloidogenic XFLC neo-epitope in the digested sample is determined as an absolute value. In some embodiments, the amount of an amyloidogenic XFLC neo-epitope is normalized to a reference. In some embodiments, the reference comprises an amount of an amyloidogenic XFLC neo-epitope in a control sample, and / or an amount of an internal standard in the digested sample.

[0202] In some embodiments, the reference is an amount of an amyloidogenic XFLC neoepitope in a control which does not comprise amyloidogenic XFLC. In some embodiments, the control sample is subjected to the limited proteolysis. In some embodiments, the control sample46323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 is processed by the method in parallel with the sample. In some embodiments, a substantially similar amount of an amyloidogenic XFLC neo-epitope in the control sample as compared to the digested sample indicates target engagement of the candidate FLC dimer stabilizer. In some embodiments, an amount of an amyloidogenic XFLC neo-epitope in the digested sample that is not more than about ±10%, about ±20%, about ±30%, about ±40%, or about ±50% as compared to the control sample indicates target engagement of the candidate FLC dimer stabilizer.Methods of Generating Amyloidogenic LFLC Neo-Epitope-Binding Monoclonal Antibodies of the Disclosure

[0203] The present disclosure provides methods of generating a monoclonal antibody that binds to an amyloidogenic XFLC protein neo-epitope.

[0204] In some embodiments, the method comprises providing an immunogenic composition comprising the amyloidogenic XFLC protein neo-epitope, and immunizing a subject with the immunogenic composition, thereby generating a monoclonal antibody that specifically binds to the amyloidogenic XFLC protein neo-epitope.

[0205] In some embodiments, the subject is a rabbit, mouse, rat, rabbit, goat, sheep, horse, or chicken. In some embodiments, the subject is a camel, a llama, or an alpaca.

[0206] In some embodiments, the antibody is harvested from a sample obtained from the subject. In some embodiments, the sample is a blood sample. In some embodiments, the antibody is generated from a hybridoma.

[0207] In some embodiments, the method comprises contacting the immunogenic composition with an antibody library, and selecting an antibody clone from the library that specifically binds to the amyloidogenic ±FLC protein neo-epitope. In some embodiments, the antibody library is a phage display library. In some embodiments, the antibody library is a yeast display library.Immunogenic Compositions

[0208] The present disclosure provides immunogenic compositions comprising an amyloidogenic ±FLC protein neo-epitope. In some embodiments, the immunogenic composition is derived from a sample comprising an amyloidogenic ±FLC protein. In some embodiments, obtaining the immunogenic composition comprises contacting the sample with a protease under conditions effective for producing an amyloidogenic ±FLC neo-epitope described herein. In some embodiments, the sample is obtained from a subject suspected of having AL amyloidosis. In some embodiments, the sample comprises a recombinant amyloidogenic XFLC. In some47323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 embodiments, the recombinant amyloidogenic XFLC is selected from the group consisting of: H9, WIL, H3, and a combination thereof.

[0209] In embodiments, provided herein are immunogenic compositions comprising (i) a neoepitope of an amyloidogenic XFLC protein; and (ii) a pharmaceutically acceptable buffer. In some embodiments, the immunogenic composition comprises (i) a neo-epitope of an amyloidogenic XFLC protein; and an adjuvant. In embodiments, the neo-epitope has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide of any one of SEQ ID NOs: 1 and 34-135, 137-239, 241-343344, GQP, SQP, and RQP. In embodiments, the neo-epitope comprises the amino acid sequence GQP, RQP, or SQP. In embodiments, the neo-epitope comprises from the N-terminus to the C-terminus (i) GQP, RQP, or SQP; and (ii) a polypeptide with at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or at least 100 % identical to any one of SEQ ID NOS: 351-450, KAA, and KA. In embodiments, the immunogenic compositions further comprise (iii) an adjuvant. In embodiments, the adjuvant is selected from one or more of Aluminum hydroxide, Aluminum phosphate, Calcium phosphate, Freund’s complete adjuvant, Freund’s incomplete adjuvant, Montanide ISA 50, Montanide ISA 206, AS03, AS04, MF59, CpG oligodeoxynucleotides, Quil A, Liposomes, Poly(I), TLR agonists, E. coli heat-labile toxin, Chitosan, Zinc oxide, Squalene, BCG (Bacillus Calmette- Guerin), Dendritic cell vaccines, Saponins, Microparticles, Nanospheres, Micelles, Emulsions, Detoxified bacterial toxins, Oil-in-water emulsions, Water-in-oil emulsions, Polylactic acid, Polyethylene glycol, Pseudomonas exotoxin, Virolex, Imiquimod, RIB I, Polypropylene, Pluronic F68, Eudragit, Gelatin, Alginate, Silica, Cellulose, Starch, Activated carbon, Graphene oxide, Chloroform extracts, Arginine, Mannan, Lipopeptides, Peptidoglycan, and Phospholipids.

[0210] In certain embodiments, the fragment of the XFLC is a homodimeric fragment, which is a ~23kDa fragment under non-denaturing condition. In certain embodiments, the fragment of the XFLC is a monomeric fragment, which is a ~11.5kDa fragment under denaturing condition.Monoclonal Antibodies of the Disclosure

[0211] The present disclosure provides antibodies and antigen-binding fragments thereof generated by a method described herein. The antibodies and antigen-binding fragments thereof provided herein bind to a neo-epitope of an amyloidogenic XFLC protein and can be used for the detection of a neo-epitope exposed on a fragment of an amyloidogenic XFLC protein generated48323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 through limited proteolysis. Detection of the presence and / or quantification of the levels of proteins with a neo-epitope can be used for (1) diagnosis of AL amyloidosis in a subject, (2) monitoring of AL amyloidosis treatment (e.g., detection of amyloidogenic XFLC in a subject to determine treatment efficacy and / or to detect relapse), and / or (3) screening target engagement of a candidate LC dimer stabilizer.

[0212] 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. 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.

[0213] In embodiments, the antibody or antigen-binding fragment thereof comprises a VH that comprises a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3); and a VL that comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3). In some embodiments, the HCDR1-3 each comprise a sequence as set forth in Table 2. In some embodiments, the LCDR1-3 each comprise a sequence as set forth in Table 3.

[0214] In embodiments, the HCDR1 comprises an amino acid sequence of SEQ ID No: 10. In embodiments, the HCDR1 comprises an amino acid sequence of SEQ ID No: 16. In embodiments, the HCDR1 comprises an amino acid sequence of SEQ ID No: 22. In embodiments, the HCDR1 comprises an amino acid sequence that is 100 % identical to any one of SEQ ID NOs: 10, 16, or 22, except for the addition, mutation, or deletion of up to 1 or up to 2 amino acids.

[0215] In embodiments, the HCDR2 comprises an amino acid sequence of SEQ ID No: 11. In embodiments, the HCDR2 comprises an amino acid sequence of SEQ ID No: 17. In embodiments, the HCDR2 comprises an amino acid sequence of SEQ ID No: 23. In embodiments, the HCDR2 comprises an amino acid sequence that is 100 % identical to any one of SEQ ID NOs: 11, 17, or 23, except for the addition, mutation, or deletion of up to 1 or up to 2 amino acids.

[0216] In embodiments, the HCDR3 comprises an amino acid sequence of SEQ ID No: 12. In embodiments, the HCDR3 comprises an amino acid sequence of SEQ ID No: 18. In embodiments, the HCDR3 comprises an amino acid sequence of SEQ ID No: 24. In embodiments, the HCDR3 comprises an amino acid sequence that is 100 % identical to any one49323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 of SEQ ID NOs: 12, 18, or 24, except for the addition, mutation, or deletion of up to 1 or up to 2 amino acids.

[0217] In embodiments, the LCDR1 comprises an amino acid sequence of SEQ ID No: 13. In embodiments, the LCDR1 comprises an amino acid sequence of SEQ ID No: 19. In embodiments, the LCDR1 comprises an amino acid sequence of SEQ ID No: 25. In embodiments, the LCDR1 comprises an amino acid sequence that is 100 % identical to any one of SEQ ID NOs: 13, 19, or 25, except for the addition, mutation, or deletion of up to 1 or up to 2 amino acids.

[0218] In embodiments, the LCDR2 comprises an amino acid sequence of SEQ ID No: 14. In embodiments, the LCDR2 comprises an amino acid sequence of SEQ ID No: 20. In embodiments, the LCDR2 comprises an amino acid sequence of SEQ ID No: 26. In embodiments, the LCDR2 comprises an amino acid sequence that is 100 % identical to any one of SEQ ID NOs: 14, 20, or 26, except for the addition, mutation, or deletion of up to 1 or up to 2 amino acids.

[0219] In embodiments, the LCDR3 comprises an amino acid sequence of SEQ ID No: 15. In embodiments, the LCDR3 comprises an amino acid sequence of SEQ ID No: 21. In embodiments, the LCDR3 comprises an amino acid sequence of SEQ ID No: 27. In embodiments, the LCDR3 comprises an amino acid sequence that is 100 % identical to any one of SEQ ID NOs: 15, 21, or 27, except for the addition, mutation, or deletion of up to 1 or up to 2 amino acids.

[0220] In some embodiments, the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively. In some embodiments, the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively. In some embodiments, the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of EQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively.

[0221] In some embodiments, the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. In some embodiments, the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively. In some embodiments, the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17,50323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 and SEQ ID NO: 18, respectively; and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively.

[0222] In some embodiments, the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively. In some embodiments, the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively. In some embodiments, the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively.

[0223] In embodiments, the antibody or antigen-binding fragment thereof comprises a VH, wherein the VH comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to a sequence in Table 4. In some embodiments, the VH comprises a sequence in Table 4.

[0224] In embodiments, the antibody or antigen-binding fragment thereof comprises a VL, wherein the VL comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to a sequence in Table 5. In some embodiments, the VL comprises a sequence in Table 5.

[0225] In some embodiments, the VH comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 345. In some embodiments, the VH comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 347. In some embodiments, the VH comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 349. In some embodiments, the VH comprises a leader sequence. In embodiments, a polypeptide comprising the VH and a leader sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 28. In embodiments, a polypeptide comprising the VH and a leader sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 30. In embodiments, a polypeptide comprising the VH and a leader sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 32.

[0226] In some embodiments, the VL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 346. In some embodiments, the VL comprises an amino acid sequence that is at least 90%, 91%, 92%,51323555102Attorney Docket No.: PRTE-021 / 01WO 345214-210293%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 348. In some embodiments, the VL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 350. In some embodiments, the VL comprises a leader sequence. In embodiments, a polypeptide comprising the VL and a leader sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 29. In embodiments, a polypeptide comprising the VL and a leader sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 31. In embodiments, a polypeptide comprising the VL and a leader sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 33.

[0227] In some embodiments, the VH comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to the amino acid sequence of SEQ ID NO: 345 and the VL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 346.

[0228] In some embodiments, the VH comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to the amino acid sequence of SEQ ID NO: 347 and the VL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 348.

[0229] In some embodiments, the VH comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to the amino acid sequence of SEQ ID NO: 349 and the VL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 350.

[0230] In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 28. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 32. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 345. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 347. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 349.

[0231] In some embodiments, the VH consists of an amino acid sequence of SEQ ID NO: 28. In some embodiments, the VH consists of an amino acid sequence of SEQ ID NO: 30. In some52323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 embodiments, the VH consists of an amino acid sequence of SEQ ID NO: 32. In some embodiments, the VH consists of an amino acid sequence of SEQ ID NO: 345. In some embodiments, the VH consists of an amino acid sequence of SEQ ID NO: 347. In some embodiments, the VH consists of an amino acid sequence of SEQ ID NO: 349.

[0232] In some embodiments, the VL comprises an amino acid sequence of SEQ ID NO: 29. In some embodiments, the VL comprises an amino acid sequence of SEQ ID NO: 31. In some embodiments, the VL comprises an amino acid sequence of SEQ ID NO: 33. In some embodiments, the VL comprises an amino acid sequence of SEQ ID NO: 346. In some embodiments, the VL comprises an amino acid sequence of SEQ ID NO: 348. In some embodiments, the VL comprises an amino acid sequence of SEQ ID NO: 350.

[0233] In some embodiments, the VL consists of an amino acid sequence of SEQ ID NO: 29. In some embodiments, the VL consists of an amino acid sequence of SEQ ID NO: 31. In some embodiments, the VL consists of an amino acid sequence of SEQ ID NO: 33. In some embodiments, the VL consists of an amino acid sequence of SEQ ID NO: 346. In some embodiments, the VL consists of an amino acid sequence of SEQ ID NO: 348. In some embodiments, the VL consists of an amino acid sequence of SEQ ID NO: 350.

[0234] In some embodiments, the VH comprises an amino acid sequence selected from any one of SEQ ID NOs: 28, 30, 32, 345, 347, and 349, and the VL comprises an amino acid sequence selected from any one of SEQ ID NOs: 29, 31, 33, 346, 348, and 350.

[0235] In some embodiments, the VH consists of an amino acid sequence selected from any one of SEQ ID NOs: 28, 30, 32, 345, 347, and 349, and the VL consists of an amino acid sequence selected from any one of SEQ ID NOs: 29, 31, 33, 346, 348, and 350.

[0236] In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 28, and the VL comprises an amino acid sequence of SEQ ID NO: 29.

[0237] In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 30, and the VL comprises an amino acid sequence of SEQ ID NO: 31.

[0238] In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 32, and the VL comprises an amino acid sequence of SEQ ID NO: 33.

[0239] In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 345, and the VL comprises an amino acid sequence of SEQ ID NO: 346.

[0240] In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 347, and the VL comprises an amino acid sequence of SEQ ID NO: 348.53323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102

[0241] In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 349, and the VL comprises an amino acid sequence of SEQ ID NO: 350.

[0242] In some embodiments, the VH consists of an amino acid sequence of SEQ ID NO: 28, and the VL consists of an amino acid sequence of SEQ ID NO: 29.

[0243] In some embodiments, the VH consists of an amino acid sequence of SEQ ID NO: 30, and the VL consists of an amino acid sequence of SEQ ID NO: 31.

[0244] In some embodiments, the VH consists of an amino acid sequence of SEQ ID NO: 32, and the VL consists of an amino acid sequence of SEQ ID NO: 33.

[0245] In some embodiments, the VH consists of an amino acid sequence of SEQ ID NO: 345, and the VL consists of an amino acid sequence of SEQ ID NO: 346.

[0246] In some embodiments, the VH consists of an amino acid sequence of SEQ ID NO: 347, and the VL consists of an amino acid sequence of SEQ ID NO: 348.

[0247] In some embodiments, the VH consists of an amino acid sequence of SEQ ID NO: 349, and the VL consists of an amino acid sequence of SEQ ID NO: 350.

[0248] In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain (HC), wherein the HC comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a sequence in Table 6. In some embodiments, the HC comprises a sequence in Table 6.

[0249] In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain (HC), wherein the HC comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a sequence in Table 7. In some embodiments, the HC comprises a sequence in Table 7.

[0250] In some embodiments, the heavy chain comprises an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 454, 456, 458, 460, 462, and 464. In some embodiments, the light chain comprises an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 455, 457, 459, 461, 463, and 465. In some embodiments, the heavy chain comprises an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or54323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102100% identity to any one of SEQ ID NOs: 454, 456, 458, 460, 462, and 464, and the light chain comprises an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 455, 457, 459, 461, 463, and 465.

[0251] In embodiments, the heavy chain comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 454, and the light chain comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 455.

[0252] In embodiments, the heavy chain comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 456, and the light chain comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 457.

[0253] In embodiments, the heavy chain comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 458, and the light chain comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 459.

[0254] In embodiments, the heavy chain comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 460, and the light chain comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 461.

[0255] In embodiments, the heavy chain comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 462, and the light chain comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 463.

[0256] In embodiments, the heavy chain comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 464, and the light chain comprises at least 85%, at55323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 465.

[0257] In embodiments, the heavy chain comprises a polypeptide of SEQ ID NO: 454, and the light chain comprises a polypeptide of SEQ ID NO: 455.

[0258] In embodiments, the heavy chain comprises a polypeptide of SEQ ID NO: 456, and the light chain comprises a polypeptide of SEQ ID NO: 457.

[0259] In embodiments, the heavy chain comprises a polypeptide of SEQ ID NO: 458, and the light chain comprises a polypeptide of SEQ ID NO: 459.

[0260] In embodiments, the heavy chain comprises a polypeptide of SEQ ID NO: 460, and the light chain comprises a polypeptide of SEQ ID NO: 461.

[0261] In embodiments, the heavy chain comprises a polypeptide of SEQ ID NO: 462, and the light chain comprises a polypeptide of SEQ ID NO: 463.

[0262] In embodiments, the heavy chain comprises a polypeptide of SEQ ID NO: 464, and the light chain comprises a polypeptide of SEQ ID NO: 465.

[0263] In embodiments, the heavy chain consists of a polypeptide of SEQ ID NO: 454, and the light chain consists of a polypeptide of SEQ ID NO: 455.

[0264] In embodiments, the heavy chain consists of a polypeptide of SEQ ID NO: 456, and the light chain consists of a polypeptide of SEQ ID NO: 457.

[0265] In embodiments, the heavy chain consists of a polypeptide of SEQ ID NO: 458, and the light chain consists of a polypeptide of SEQ ID NO: 459.

[0266] In embodiments, the heavy chain consists of a polypeptide of SEQ ID NO: 460, and the light chain consists of a polypeptide of SEQ ID NO: 461.

[0267] In embodiments, the heavy chain consists of a polypeptide of SEQ ID NO: 462, and the light chain consists of a polypeptide of SEQ ID NO: 463.

[0268] In embodiments, the heavy chain consists of a polypeptide of SEQ ID NO: 464, and the light chain consists of a polypeptide of SEQ ID NO: 465.

[0269] An aspect of the application is an antibody for detection of a neo-epitope formed from amyloidogenic XFLC 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).

[0270] An aspect of the application is an antibody for detection of a neo-epitope formed from amyloidogenic XFLC proteins, wherein the antibody comprises heavy chain encoded by a56323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 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).

[0271] An aspect of the application is an antibody for detection of a neo-epitope formed from amyloidogenic XFLC proteins, wherein the antibody comprises heavy chain ncoded 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).

[0272] 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 exemplary monoclonal antibodies (e.g., LCCD-G, LCCD-S, and LCCD-R) described herein specifically bind to small N-terminal tags of 1-5 amino acids (e.g., GQP and SQP noncharged). In some embodiments, the tag (epitopes) are placed at the N-terminus of a protein of interest, and a monoclonal antibody described herein is used for detection of the protein of interest via binding to the tag.

[0273] In some embodiments, the antibody or antigen binding fragment thereof comprises a tag for detection and / or purification, n some embodiments, the antibody or antigen binding fragment thereof comprises multiple tags. In some embodiments, the tag is at the N-terminus or C- terminus of the antibody or antigen binding fragment heavy chain. In some embodiments, the tag is at the N-terminus or C-terminus of the antibody or antigen binding fragment light chain. In some embodiments, the tag or the multiple tags are operably linked to a constant region of the antibody or antigen binding fragment. In some embodiments, the tag or the multiple tags are operably linked to an Fc region of the antibody or antigen binding fragment.57323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102Table 2: Heavy Chain CDRs for AntibodiesTable 3: Light Chain CDRs for AntibodiesTable 4: VH Amino Acid Sequences for Antibodies58323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102Table 5: VL Amino Acid Sequences for AntibodiesTable 6: Heavy Chain Amino Acid Sequences59323555102Atorney Docket No.: PRTE-021 / 01WO 345214-210260323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102Table 7: Light Chain Amino Acid Sequences61323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102Kits

[0274] The present disclosure provides kits for detection of amyloidogenic ZFLC proteins in a sample. In some embodiments, the kit comprises a monoclonal antibody or antigen binding fragment thereof that binds to a neo-epitope described herein, and instructions for using the kit in accordance with the methods described herein. In some embodiments, the kit comprises a monoclonal antibody or antigen binding fragment thereof that binds to a neo-epitope described herein, a protease described herein, and instructions for using the kit in accordance with the methods described herein. In some embodiments, the kit comprises a monoclonal antibody or antigen binding fragment thereof that binds to a neo-epitope described herein, a compound62323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 comprising the neo-epitope, and instructions for using the kit in accordance with the methods described herein.

[0275] An aspect of the application is a kit for detection of presence in a subject of amyloidogenic ZFLC proteins, comprising: the antibodies of LCCD-G, LCCD-R and LCCD-S; instructions for using the kit in accordance with the methods described herein.

[0276] In some embodiments, the kit further comprises reagents or components for performing the methods described herein. In some embodiments, the kit comprises a surface comprising the monoclonal antibody or antigen binding fragment thereof. In some embodiments, the kit comprises a surface comprising the neo-epitope. In some embodiments, the surface is a membrane or a plate.Other Embodiments

[0277] The present disclosure provides the following embodiments.

[0278] Embodiment 1-1. A method of detecting the presence in a subject of amyloidogenic X free light chain (ZFLC) protein, comprising the steps of: exposing a sample obtained from a subject to a protease and permitting up to limited proteolysis of ZFLC protein, if present, in the sample, wherein the limited proteolysis results in a neo-epitope exposed on a greater than 30 amino acid fragment of the ZFLC protein; detecting the presence of the neo-epitope on the fragment of the ZFLC protein in the sample subjected to limited proteolysis and quantitating the presence of the fragment in the sample subjected to limited proteolysis by or based on measuring a level of the neo-epitope; quantitating, if present, a corresponding fragment of the ZFLC protein, if present, in the same type of biological samples from a healthy control under the same limited proteolysis condition, and determine a reference level of the neo-epitope; and comparing the level of the fragment of the ZFLC protein in the subject sample exposed to the same limited proteolysis condition with the reference level of the corresponding fragment of the ZFLC protein, if present, in the healthy control sample, wherein when the level of the fragment of the ZFLC protein in the subject sample exposed to limited proteolysis is above the reference level established in the healthy control samples exposed to the same limited proteolysis conditions, then amyloidogenic ZFLC protein is detected.

[0279] Embodiment 1-2. The method of embodiment I- 1, further comprising the steps of: measuring a first level of the fragment of the ZFLC protein present after the limited proteolysis in a biological sample taken from a subject suspected of having AL; measuring a second level of the fragment of the ZFLC protein present in a separate aliquot of the same sample without63323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 limited proteolysis; and wherein when the first level of the fragment of the XFLC protein after the limited proteolysis of the biological sample is greater than the second level of the fragment without the limited proteolysis beyond expected experimental error, then amyloidogenic XFLC protein is detected.

[0280] Embodiment 1-3. A method of detection of presence in a subject of amyloidogenic X free light chain (XFLC) proteins, comprising the steps of exposing at least a portion of a sample to two or more different proteases and permitting up to limited proteolysis of XFLC proteins, if present, in the sample, wherein the limited proteolysis results in a neo-epitope exposed on two or more greater than 30 amino acid fragments of the XFLC proteins; detecting a presence of the neo-epitope of the two or more fragments of the XFLC proteins in the sample subjected to limited proteolysis and quantitating the presence of the two or more fragments in the sample subjected to limited proteolysis by or based on measuring a level of the neo-epitope; quantitating, if present, the two or more corresponding fragments of the XFLC proteins in at least a portion of the sample not exposed to limited proteolysis by or based on measuring a level of the neo-epitope; and comparing the level of the two or more fragments in the sample exposed to limited proteolysis with the level of the corresponding two or more fragments in the sample not exposed to limited proteolysis, wherein when the level of at least one of the two or more fragments is greater in the sample exposed to limited proteolysis compared with the sample not exposed to limited proteolysis, then amyloidogenic XFLC proteins are detected.

[0281] Embodiment 1-4. The method of embodiment 1-3, further comprising the steps of measuring a first level of the two or more fragments of the XFLC 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 of each of the two or more fragments is a reference level; measuring a second level of the two or more fragments of the ZFLC proteins present after the limited proteolysis of a biological sample from a person suspected of having AL; and wherein when the second level of at least one of the two or more fragments of the ZFLC proteins after the limited proteolysis of the biological sample is greater than the reference level, then amyloidogenic ZFLC proteins are detected.

[0282] Embodiment 1-5. The method of embodiment 1-1, wherein when the level of the fragment of the ZFLC protein is 2x or more greater in the subject sample exposed to limited proteolysis compared with the reference level, then light chain amyloidosis is detected in the subject.64323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102

[0283] Embodiment 1-6. The method of embodiment 1-2, wherein when the first level of the fragment of the XFLC proteins after the limited proteolysis of the biological sample is 2x or more greater than the second level, where the sample not exposed to limited proteolysis, then light chain amyloidosis is detected in the subject.

[0284] Embodiment 1-7. The method of embodiment 1-3, wherein when the level of at least one of the two or more fragments is 2x or greater in the sample exposed to limited proteolysis compared with the sample not exposed to limited proteolysis, then light chain amyloidosis is detected in the subject.

[0285] Embodiment 1-8. The method of embodiment 1-4, wherein when the second level of at least one of the two or more fragments of the XFLC proteins after the limited proteolysis of the biological sample is 2x or greater than the reference level, then light chain amyloidosis is detected in the subject.

[0286] Embodiment 1-9. The method of any one of embodiments 1-1 to 1-8, wherein the protease is Proteinase K, Thermolysin, Pepsin, Trypsin, Chymotrypsin, Cathepsin B, Cathepsin L, Papain, the peptidase S8 family, represented by PCSK9 and Subtilisin, or another natural or synthetic protease.

[0287] Embodiment I- 10. The method of any one of embodiments 1-1 to 1-9, wherein the neoepitope is dLCCD and / or papLC.

[0288] Embodiment 1-11. The method of embodiment I- 10, wherein the detecting and quantitating is performed using an antibody specific for the neo-epitopes on dLCCD and / or papLC.

[0289] Embodiment 1-12. The method of any one of embodiments 1-1 to 1-11, wherein the biological sample is one of whole blood, plasma, urine, serum, saliva, cerebrospinal fluid (CSF), bone marrow aspiration, or tissue biopsy.

[0290] Embodiment 1-13. The method of any one of embodiments 1-1 to 1-12, wherein the fragment of the XFLC protein in the sample exposed to limited proteolysis is detected and quantified using mass spectrometry.

[0291] Embodiment 1-14. The method of any one of embodiments 1-1 to 1-13, 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.65323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102

[0292] Embodiment 1-15. The method of any one of embodiments 1-1 to 1-14, wherein in the sample exposed to limited proteolysis, an elevated fragment level in a subject prompts a clinical decision, wherein the clinical decision is treatment initiation or treatment modulation for light chain amyloidosis (AL).

[0293] Embodiment 1-16. The method of any one of embodiments 1-1 to 1-13, wherein in the sample exposed to limited proteolysis, an absence of an elevated fragment level in a subject prompts 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).

[0294] Embodiment 1-17. The method of any one of embodiments 1-1 to 1-15, further comprising administering treatment for 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.

[0295] Embodiment 1-18. The method of embodiment 1-17, wherein the treatment comprises administering therapeutically effective amount of cyclophosphamide, bortezomib, and dexamethasone, lenalidomide, pomalidomide, an antibody targeting CD38, anti-BCMA, a bispecific antibody, a cell therapy targeting plasma cells, venetoclax, an anti-fibril treatments and / or a treatment set forth U.S. Patent No. 11,945,806, U.S. Patent Application Publication No. 2024-0051960, and PCT Publication Nos. WO 2022 / 226166, WO 2024 / 092037, W02024 / 092040, and / or WO 2024 / 092043.

[0296] Embodiment 1-19. The method of embodiment 1-17, wherein the treatment comprises administering chemotherapy and / or autologous stem cell transplant.

[0297] Embodiment 1-20. The method of embodiment 1-18, wherein the treatment further comprises administering chemotherapy and / or autologous stem cell transplant.

[0298] Embodiment 1-21. The method of any one of embodiments 1-1 to 1-20, wherein the method is conducted as a routine blood screening test.

[0299] Embodiment 1-22. A method of generating a monoclonal antibody A method for the detection and quantification of amyloidogenic X free light chain (XFLC) proteins in biological fluids through the artificial generation of a neo-epitope and the specific detection of such neoepitope using monoclonal antibodies, comprising the steps of: (a) proteolyzing a sample comprising X free light chain (XFLC) proteins with one or more of Thermolysin, Pepsin, Trypsin, Chymotrypsin, Cathepsin B, Cathepsin L, Papain, the peptidase S8 family, represented66323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 by PCSK9 and Subtilisin, and / or another natural or synthetic protease, wherein the proteolyzing results in a neo-epitope exposed on a XFLC fragment comprising at least part of the constant domain of the XFLC; and (b) generating a monoclonal antibody to the neo-epitope on the fragment of the XFLC, wherein the generated monoclonal antibody specifically binds to the neo-epitope, and wherein proteolyzing conditions are optimized so that a signal obtained after proteolyzing from the fragment of the XFLC, is at least two times stronger from an amyloidogenic XFLC compared to a non-amyloidogenic XFLC.

[0300] Embodiment 1-23. A method of generating a monoclonal antibody for detection of a neo-epitope formed from amyloidogenic X free light chain (XFLC) proteins, comprising the steps of: (a) proteolyzing a sample comprising X free light chain (XFLC) proteins with Proteinase K and one or more of Thermolysin, Pepsin, Trypsin, Chymotrypsin, Cathepsin B, Cathepsin L, Papain, the peptidase S8 family, represented by PCSK9 and Subtilisin, and / or another natural or synthetic protease, wherein the Proteinase K proteolyzing results in a first neo-epitope exposed on the N-terminus of a ~23KDa fragment comprising at least part of the constant domain of the XFLC, or when XFLC is a monomer, a second neo-epitope exposed on the N-terminus of a ~11.5 KDa fragment comprising at least part of the constant domain of the XFLC proteins, and wherein the proteolyzing with Thermolysin, Pepsin, Trypsin, Chymotrypsin, Cathepsin B, Cathepsin L, Papain, the peptidase S8 family, represented by PCSK9 and Subtilisin, and / or another natural or synthetic protease results in a third or more neo-epitope exposed on the N-terminus of a XFLC fragment comprising at least part of the constant domain of the XFLC; and wherein the proteinase K produced neo-epitope is xQP, where x is one of G, R or S (G=Glycine, R= Arginine, S=Serine); and (b) generating two or more monoclonal antibodies to the first / second and third or more neo-epitopes on the fragment of the XFLC, wherein the first / second generated monoclonal antibody (LCCD) specifically binds to the neo-epitope of xQP, and wherein proteolyzing conditions are optimized so that an LCCD papLC, or another neo-epitope specific signal obtained after proteolyzing from the XFLC fragments, is at least two times stronger from an amyloidogenic XFLC compared to a non-amyloidogenic XFLC.

[0301] Embodiment 1-24. The method of embodiment 1-23, wherein the first / second neoepitope is GQP.

[0302] Embodiment 1-25. The method of embodiment 1-23, wherein the first / second neoepitope is RQP.

[0303] Embodiment 1-26. The method of embodiment 1-23, wherein the first / second neoepitope is SQP.67323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102

[0304] Embodiment 1-27. The method of embodiment 1-24, wherein the first / second generated antibody (LCCD-G) binds to the neo-epitope of GQP.

[0305] Embodiment 1-28. The method of embodiment 1-25, wherein the first / second generated antibody (LCCD-R) binds to the neo-epitope of RQP.

[0306] Embodiment 1-29. The method of embodiment 1-26, wherein the first / second generated antibody (LCCD-S) binds to the neo-epitope of SQP.

[0307] Embodiment 1-30. The method of any one of embodiments 1-22 to 1-29, wherein the first fragment of the XFLC is a homodimeric fragment, which is a ~23kDa fragment under nonreducing condition.

[0308] Embodiment 1-31. The method of any one of embodiments 1-22 to 1-29, wherein the first fragment of the XFLC is a monomeric fragment, which is a ~11.5kDa fragment under reducing condition.

[0309] Embodiment 1-32. The method of any one of embodiments 1-22 to 1-31, wherein the limited proteolysis by proteinase K, Thermolysin, Pepsin, Trypsin, Chymotrypsin, Cathepsin B, Cathepsin L, Papain, the peptidase S8 family, represented by PCSK9 and Subtilisin, and / or another natural or synthetic protease comprises the steps of: a liquid sample treated with an optimized proteolysis condition to produce the LCCD, papLC, or other neo-epitope domain.

[0310] Embodiment 1-33. The method of any one of embodiments 1-22 to 1-31, wherein the optimized proteolysis condition is 0.02-20. OpM Proteinase K, Thermolysin, Pepsin, Trypsin, Chymotrypsin, Cathepsin B, Cathepsin L, Papain, the peptidase S8 family, represented by PCSK9 and Subtilisin, and / or another natural or synthetic protease at 37°C for 30 seconds to 20 hours depending on the specific activity of the protease.

[0311] Embodiment 1-34. 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, papLC, or another fragment of the XFLC proteins; detecting the presence of the neo-epitope on an N-termini of the LCCD, papLC, or other fragment of the XFLC proteins; and quantitating the level of the fragment of the XFLC 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, papLC or other 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 of68323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 quantity of the LCCD, papLC or other 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, papLC or other fragment level, or the LCCD, papLC or other fragment level normalized to the total XFLC in the second level of quantification indicates target engagement.

[0312] Embodiment 1-35. A method of assessing therapeutic response of a therapeutics for treatment of light chain amyloidosis by quantifying a LCCD fragment, papLC fragment or other fragment comprising another neo-epitope identified by limited proteolysis of a XFLC protein 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, papLC or other fragment indicates favorable response to the therapeutic molecule, irrespective to the total level of XFLC.

[0313] Embodiment 1-36. A method of assessing if an AL patient would benefit from a light chain stabilizer as a therapy by quantifying a LCCD fragment, papLC fragment or other fragment comprising another neo-epitope identified by limited proteolysis of a XFLC protein 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, papLC or other fragment levels, wherein a reduction of LCCD, papLC or other fragment 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.

[0314] Embodiment 1-37. The method of any one of Embodiments 1-35 to 1-36, wherein when the first level of the LCCD, papLC or other fragment present before the targeted therapeutic molecule has been introduced to the subject is greater than the second level of the LCCD, papLC or other fragment present after the targeted therapeutic molecule has been introduced to the subject, then target engagement has occurred by the targeted therapeutic molecule.

[0315] Embodiment 1-38. The method of any one of Embodiments 1-34 to 1-36, wherein when the first level of the LCCD, papLC or other 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, papLC or other fragment present after the targeted therapeutic molecule has been introduced to the subject, then target engagement has not occurred by the targeted therapeutic molecule. Embodiment 1-39. The method of any one of Embodiment 1-35 to 1-38, wherein the69323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 biological sample is one of plasma, whole blood, urine, serum, saliva, cerebrospinal fluid (CSF), or tissue biopsy.

[0316] Embodiment 1-40. A kit for detection of presence in a subject of amyloidogenic X free light chain (ZFLC) proteins, comprising: an antibody specific for a LCCD, papLC or other fragment comprising another neo-epitope identified by limited proteolysis of a ZFLC protein fragment; a protease comprising Thermolysin, Pepsin, Trypsin, Chymotrypsin, Cathepsin B, Cathepsin L, Papain, the peptidase S8 family, represented by PCSK9 and Subtilisin, or another natural or synthetic protease; and instructions for using the kit in accordance with the method of any one of embodiments 1-1 to 1-21.

[0317] Embodiment 1-41. A neo-epitope on ZFLC prepared by limited proteolysis of amyloidogenic X free light chain (ZFLC) proteins by Thermolysin, Pepsin, Trypsin, Chymotrypsin, Cathepsin B, Cathepsin L, Papain, the peptidase S8 family, represented by PCSK9 and Subtilisin, or another natural or synthetic protease.

[0318] Embodiment 1-42. An antibody or antibody mixture to the neo-epitope of embodiment I- 41.

[0319] Embodiment 1-43. The antibody of embodiment 1-42 that specifically binds an LCCD, papLC or other fragment comprising another neo-epitope identified by limited proteolysis of a ZFLC protein.

[0320] Embodiment 1-44. A method of optimizing limited proteolysis condition using Thermolysin, Pepsin, Trypsin, Chymotrypsin, Cathepsin B, Cathepsin L, Papain, the peptidase S8 family, represented by PCSK9 and Subtilisin and / or another natural or synthetic protease, comprising the steps of optimizing one or more of the following enumerated factors: (1) source of the Thermolysin, Pepsin, Trypsin, Chymotrypsin, Cathepsin B, Cathepsin L, Papain, the peptidase S8 family, represented by PCSK9 and Subtilisin, and / or another natural or synthetic protease; (2) composition of the testing sample; (3) concentration of Thermolysin, Pepsin, Trypsin, Chymotrypsin, Cathepsin B, Cathepsin L, Papain, the peptidase S8 family, represented by PCSK9 and Subtilisin, and / or another natural or synthetic protease; (4) time of proteolysis reaction; (5) temperature of proteolysis reaction; wherein a proteolysis product of the LCCD, papLC or other fragment comprising another neo-epitope identified by limited proteolysis of a kFLC protein fragment is differentially obtained from amyloidogenic ZFLC vs. nonamyl oidogenic ZFLC.

[0321] Embodiment 1-45. The method of detection of presence in a subject of amyloidogenic X free light chain (ZFLC) proteins of embodiment 1-11, further comprising: obtaining a readout70323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 from an LCCD signal and / or papLC signal obtained by measuring the level of neo-epitope on the fragment using the antibody specific for LCCD and / or papLC.

[0322] Embodiment 1-46. The method of detection of presence in a subject of amyloidogenic X free light chain (XFLC) proteins of embodiment 1-45, further comprising: obtaining the readout of the LCCD and / or papLC signal by use of an algorithm.

[0323] Embodiment 1-47. The method of any one of embodiments 1-22 to 1-39 or 1-45 to 1-46, further comprising administering treatment for 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.

[0324] Embodiment 1-48. The method of embodiment 1-47, wherein the treatment comprises administering therapeutically effective amount of cyclophosphamide, bortezomib, and dexamethasone, lenalidomide, pomalidomide, an antibody targeting CD38, anti-BCMA, a bispecific antibody, a cell therapy targeting plasma cells, venetoclax, an anti-fibril treatments and / or a treatment set forth U.S. Patent No. 11,945,806, U.S. Patent Application Publication No. 2024-0051960, and PCT Publication Nos. WO 2022 / 226166, WO 2024 / 092037, W02024 / 092040, and / or WO 2024 / 092043.

[0325] Embodiment 1-49. The method of embodiment 1-47, wherein the treatment comprises administering chemotherapy and / or autologous stem cell transplant.

[0326] Embodiment 1-50. The method of embodiment 1-48, wherein the treatment further comprises administering chemotherapy and / or autologous stem cell transplant.

[0327] Embodiment 1-51. The method of any one of embodiments 1-22 to 1-39 or 1-45 to 1-50, wherein the method is conducted as a routine blood screening test.

[0328] Embodiment 11-1. An immunogenic composition comprising: (i) a neo-epitope of the amyloidogenic X free light chain (XFLC) protein; and (ii) a pharmaceutically acceptable buffer. Embodiment II-2. The immunogenic composition of embodiment II- 1, wherein the neoepitope has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide of any one of SEQ ID NOs: 1 and 34-344.

[0329] Embodiment 11-3. The immunogenic composition of embodiment II- 1, wherein the neoepitope comprises the amino acid sequence GQP (SEQ ID NO: 136), RQP (SEQ ID NO: 344), or SQP (SEQ ID NO: 240).71323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102

[0330] Embodiment II-4. The immunogenic composition of embodiment II- 1, wherein the neoepitope comprises GQPKA (SEQ ID NO: 134), RQPKA (SEQ ID NO: 342), or SQPKA (SEQ ID NO: 238).

[0331] Embodiment II-5. The immunogenic composition of embodiment II- 1, wherein the neoepitope comprises from the N-terminus to the C-terminus (i) GQP, RQP, or SQP; and (ii) a polypeptide with at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or at least 100 % identical to any one of SEQ ID NOS: 351-453.

[0332] Embodiment II-6. The immunogenic composition of any one of embodiments II- 1 to II- 5, comprising (iii) an adjuvant.

[0333] Embodiment II-7. The immunogenic composition of embodiment II-6, wherein the adjuvant is selected from any one of Aluminum hydroxide, Aluminum phosphate, Calcium phosphate, Freund’s complete adjuvant, Freund’s incomplete adjuvant, Montanide ISA 50, Montanide ISA 206, AS03, AS04, MF59, CpG oligodeoxynucleotides, Quil A, Liposomes, Poly(I), TLR agonists, E. coli heat-labile toxin, Chitosan, Zinc oxide, Squalene, BCG (Bacillus Calmette-Guerin), Dendritic cell vaccines, Saponins, Microparticles, Nanospheres, Micelles, Emulsions, Detoxified bacterial toxins, Oil-in-water emulsions, Water-in-oil emulsions, Polylactic acid, Polyethylene glycol, Pseudomonas exotoxin, Virolex, Imiquimod, RIBI, Polypropylene, Pluronic F68, Eudragit, Gelatin, Alginate, Silica, Cellulose, Starch, Activated carbon, Graphene oxide, Chloroform extracts, Arginine, Mannan, Lipopeptides, Peptidoglycan, and Phospholipids.

[0334] Embodiment II-8. A monoclonal antibody or antigen-binding fragment thereof that binds to a neo-epitope of the amyloidogenic X free light chain (XFLC) protein, wherein the monoclonal antibody or antigen-binding fragment thereof comprises: (a) a variable heavy chain CDR1 (HCDR1) comprising an amino acid sequence of SEQ ID No: 10, 16, or 22; (b) a variable heavy chain CDR2 (HCDR2) comprising an amino acid sequence of SEQ ID No: 11, 17, or 23; (c) a variable heavy chain CDR3 (HCDR3) comprising an amino acid sequence of SEQ ID No: 12, 18, or 24; (d)a variable light chain CDR1 (LCDR1) comprising an amino acid sequence of SEQ ID No: 13, 19, or 25; (e) a variable light chain CDR2 (LCDR2) comprising an amino acid sequence of SEQ ID No: 14, 20, or 26; and (f) a variable light chain CDR3 (LCDR3) comprising an amino acid sequence of SEQ ID No: 15, 21, or 27.

[0335] Embodiment II-9. The monoclonal antibody or antigen-binding fragment thereof of embodiment II-8, wherein the monoclonal antibody or antigen-binding fragment thereof72323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 comprises a HCDR1 comprising an amino acid sequence of SEQ ID No: 10; a HCDR2 comprising an amino acid sequence of SEQ ID No: 11, a HCDR3 comprising an amino acid sequence of SEQ ID No: 12, a LCDR1 comprising an amino acid sequence of SEQ ID No: 13, a LCDR2 comprising an amino acid sequence of SEQ ID No: 14, and a LCDR3 comprising an amino acid sequence of SEQ ID No: 15.

[0336] Embodiment II- 10. The monoclonal antibody or antigen-binding fragment thereof of embodiment 11-8, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a HCDR1 comprising an amino acid sequence of SEQ ID No: 16, a HCDR2 comprising an amino acid sequence of SEQ ID No: 17, a HCDR3 comprising an amino acid sequence of SEQ ID No: 18, a LCDR1 comprising an amino acid sequence of SEQ ID No: 19, a LCDR2 comprising an amino acid sequence of SEQ ID No: 20, and a LCDR3 comprising an amino acid sequence of SEQ ID No: 21

[0337] Embodiment II- 11. The monoclonal antibody or antigen-binding fragment thereof of embodiment 11-8, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a HCDR1 comprising an amino acid sequence of SEQ ID No: 22, a HCDR2 comprising an amino acid sequence of SEQ ID No: 23, a HCDR3 comprising an amino acid sequence of SEQ ID No: 24, a LCDR1 comprising an amino acid sequence of SEQ ID No: 25, a LCDR2 comprising an amino acid sequence of SEQ ID No: 26, and a LCDR3 comprising an amino acid sequence of SEQ ID No: 27.

[0338] Embodiment 11-12. The monoclonal antibody or antigen-binding fragment thereof of any one of embodiments II-8 to II- 11, wherein the monoclonal antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable domain (VH) comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 345, and a light chain variable domain (VL) comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 346; (ii) a VH comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 347, and a VL comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 348; or (iii) a VH comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 349, and a VL comprising an amino acid73323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 350.

[0339] Embodiment 11-13. The monoclonal antibody or antigen-binding fragment thereof of embodiment 11-8, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID No: 345, and a VL comprising an amino acid sequence of SEQ ID No: 346.

[0340] Embodiment 11-14. The monoclonal antibody or antigen-binding fragment thereof of embodiment 11-8, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID No: 347, and a VL comprising an amino acid sequence of SEQ ID No: 348.

[0341] Embodiment 11-15. The monoclonal antibody or antigen-binding fragment thereof of embodiment 11-8, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID No: 349, and a VL comprising an amino acid sequence of SEQ ID No: 350.

[0342] Embodiment 11-16. The monoclonal antibody or antigen-binding fragment thereof of any one of embodiments II-8 to 11-15, wherein the neo-epitope has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide of any one of SEQ ID NOS: 1 and 34-344.

[0343] Embodiment 11-17. The monoclonal antibody or antigen-binding fragment thereof of any one of embodiments II-8 to 11-15, wherein the neo-epitope comprises the amino acid sequence GQP (SEQ ID NO: 136), RQP (SEQ ID NO: 344), SQP (SEQ ID NO: 240), GQPKA (SEQ ID NO: 134), RQPKA (SEQ ID NO: 342), or SQPKA (SEQ ID NO: 238).

[0344] Embodiment 11-18. The monoclonal antibody or antigen-binding fragment thereof of any one of embodiments II-8 to 11-15, wherein the neo-epitope comprises from the N-terminus to the C-terminus (i) GQP (SEQ ID NO: 136), RQP (SEQ ID NO: 344), or SQP (SEQ ID NO: 240); and (ii) a polypeptide with at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or at least 100 % identical to any one of SEQ ID NOS: 351-453.

[0345] Embodiment 11-19. A method of generating a monoclonal antibody that binds to a neoepitope of the amyloidogenic X free light chain (XFLC) protein, comprising immunizing a subject with an immunogenic composition of any one of embodiments II- 1 to II- 7 and obtaining the monoclonal antibody from the immunized subject.74323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102

[0346] Embodiment 11-20. The method of embodiment 11-19, wherein the subject is a rabbit, mouse, rat, rabbit, goat, sheep, horse, or chicken.

[0347] Embodiment 11-21. A method of determining if a patient has amyloidosis, light chain amyloidosis, monoclonal gammopathy of undetermined Significance, multiple myeloma, smoldering multiple myeloma, IgM light chain amyloidosis, minimal residual disease of light chain amyloidosis, or relapse of light chain amyloidosis, comprising: (i) contacting a sample from the patient with a protease, wherein the sample comprises amyloidogenic X free light chain (ZFLC) protein, and wherein contacting the sample with the protease produces a peptide comprising a neo-epitope of the ZFLC protein; (ii) detecting the presence of the neo-antigen of the ZFLC protein by contacting the proteolytically cleaved sample with a monoclonal antibody or antigen-binding fragment thereof of any one of embodiments II-8 to 11-18; wherein the patient has amyloidosis, light chain amyloidosis, Monoclonal Gammopathy of Undetermined Significance (MGUS), Multiple myeloma (MM), Smoldering Multiple Myeloma (SMM), IgM light chain amyloidosis, Minimal Residual Disease (MRD) of light chain amyloidosis, or relapse of light chain amyloidosis if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the ZFLC.

[0348] Embodiment 11-22. A method of determining if a patient has amyloidosis, light chain amyloidosis, Monoclonal Gammopathy of Undetermined Significance (MGUS), Multiple myeloma (MM), Smoldering Multiple Myeloma (SMM), IgM light chain amyloidosis, Minimal Residual Disease (MRD) of light chain amyloidosis, or relapse of light chain amyloidosis, comprising: (i) contacting a sample from the patient with a monoclonal antibody or antigenbinding fragment thereof of any one of embodiments II-8 to 11-18, wherein the sample comprises amyloidogenic X free light chain (ZFLC) protein that has been contacted by a protease to produce a neo-epitope of the ZFLC protein ; and (ii) determining if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the ZFLC protein ; wherein the patient has amyloidosis, light chain amyloidosis, Monoclonal Gammopathy of Undetermined Significance (MGUS), Multiple myeloma (MM), Smoldering Multiple Myeloma (SMM), IgM light chain amyloidosis, Minimal Residual Disease (MRD) of light chain amyloidosis, or relapse of light chain amyloidosis if the monoclonal antibody or antigen-binding fragment thereof binds to the the neo-epitope of the ZFLC protein.

[0349] Embodiment 11-23. A method of determining if a patient has light chain amyloidosis, minimal residual disease of light chain amyloidosis, or relapse of light chain amyloidosis, comprising: (i) contacting a sample from the patient with a protease, wherein the sample75323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 comprises amyloidogenic X free light chain (XFLC) protein, and wherein contacting with the protease produces a neo-epitope of the XFLC protein; (ii) contacting the proteolytically cleaved sample with a monoclonal antibody or antigen-binding fragment thereof of any one of embodiments II-8 to 11-18; and (ii) determining if the monoclonal antibody or antigen-binding fragment thereof binds to the XFLC; wherein the patient has light chain amyloidosis, minimal residual disease of light chain amyloidosis, or relapse of light chain amyloidosis if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein.

[0350] Embodiment 11-24. A method of determining if a patient has light chain amyloidosis, minimal residual disease of light chain amyloidosis, or relapse of light chain amyloidosis, comprising: (i) contacting a sample from the patient with a monoclonal antibody or antigenbinding fragment thereof of any one of embodiments II-8 to 11-18, wherein the sample comprises amyloidogenic X free light chain (XFLC) protein that has been contacted by a protease and wherein contacting with the protease produces a neo-epitope of the XFLC protein; and (ii) determining if the monoclonal antibody or antigen-binding fragment thereof binds to the neoepitope of the XFLC protein; wherein the patient has light chain amyloidosis, minimal residual disease of light chain amyloidosis, or relapse of light chain amyloidosis if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein.

[0351] Embodiment 11-25. A method of treating a patient with amyloidosis, light chain amyloidosis, Monoclonal Gammopathy of Undetermined Significance (MGUS), Multiple myeloma (MM), Smoldering Multiple Myeloma (SMM), IgM light chain amyloidosis, Minimal Residual Disease (MRD) of light chain amyloidosis, or relapse of light chain amyloidosis, comprising: (i) determining if the patient has amyloidosis, light chain amyloidosis, Monoclonal Gammopathy of Undetermined Significance (MGUS), Multiple myeloma (MM), Smoldering Multiple Myeloma (SMM), IgM light chain amyloidosis, Minimal Residual Disease (MRD) of light chain amyloidosis, or relapse of light chain amyloidosis by: (a) contacting a sample from the patient with a protease, wherein the sample comprises amyloidogenic X free light chain (XFLC) protein and wherein contacting with the protease produces a neo-epitope of the XFLC protein; b) contacting the proteolytically cleaved sample with a monoclonal antibody or antigen-binding fragment thereof of any one of embodiments II-8 to 11-18; and (c) determining if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein; wherein the patient has amyloidosis, light chain amyloidosis, Monoclonal Gammopathy of Undetermined Significance (MGUS), Multiple myeloma (MM), Smoldering76323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102Multiple Myeloma (SMM), IgM light chain amyloidosis, Minimal Residual Disease (MRD) of light chain amyloidosis, or relapse of light chain amyloidosisif the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein; and (ii) administering a treatment for amyloidosis, light chain amyloidosis, Monoclonal Gammopathy of Undetermined Significance (MGUS), Multiple myeloma (MM), Smoldering Multiple Myeloma (SMM), IgM light chain amyloidosis, Minimal Residual Disease (MRD) of light chain amyloidosis, or relapse of light chain amyloidosis.

[0352] Embodiment 11-26. A method of treating a patient with amyloidosis, light chain amyloidosis, Monoclonal Gammopathy of Undetermined Significance (MGUS), Multiple myeloma (MM), Smoldering Multiple Myeloma (SMM), IgM light chain amyloidosis, Minimal Residual Disease (MRD) of light chain amyloidosis, or relapse of light chain amyloidosis, comprising: (i) determining if the patient has amyloidosis, light chain amyloidosis, Monoclonal Gammopathy of Undetermined Significance (MGUS), Multiple myeloma (MM), Smoldering Multiple Myeloma (SMM), IgM light chain amyloidosis, Minimal Residual Disease (MRD) of light chain amyloidosis, or relapse of light chain amyloidosis by: (a) contacting a sample from the patient with a monoclonal antibody or antigen-binding fragment thereof of any one of embodiments II-8 to 11-18, wherein the sample comprises amyloidogenic X free light chain (XFLC) protein that has been contacted by a protease and wherein contacting with the protease produces a neo-epitope of the XFLC protein; and (b) determining if the monoclonal antibody or antigen-binding fragment thereof binds to the XFLC; wherein the patient has amyloidosis, light chain amyloidosis, Monoclonal Gammopathy of Undetermined Significance (MGUS), Multiple myeloma (MM), Smoldering Multiple Myeloma (SMM), IgM light chain amyloidosis, Minimal Residual Disease (MRD) of light chain amyloidosis, or relapse of light chain amyloidosis if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein; and (ii) administering a treatment for amyloidosis, light chain amyloidosis, Monoclonal Gammopathy of Undetermined Significance (MGUS), Multiple myeloma (MM), Smoldering Multiple Myeloma (SMM), IgM light chain amyloidosis, Minimal Residual Disease (MRD) of light chain amyloidosis, or relapse of light chain amyloidosis. Embodiment 11-27. A method of treating a patient with light chain amyloidosis, minimal residual disease of light chain amyloidosis, or relapse of light chain amyloidosis, comprising: (i) determining if the patient has light chain amyloidosis, minimal residual disease of light chain amyloidosis, or relapse of light chain amyloidosis by: (a) contacting a sample from the patient with a protease, wherein the sample comprises amyloidogenic X free light chain (XFLC) protein and wherein contacting with77323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 the protease produces a neo-epitope of the XFLC protein; b) contacting the proteolytically cleaved sample with a monoclonal antibody or antigen-binding fragment thereof of any one of embodiments II-8 to 11-18; and (c) determining if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein; wherein the patient has light chain amyloidosis, minimal residual disease of light chain amyloidosis, or relapse of light chain amyloidosis if the monoclonal antibody or antigen-binding fragment thereof binds to the neoepitope of the XFLC protein; and (ii) administering a treatment for amyloidosis, light chain amyloidosis, Monoclonal Gammopathy of Undetermined light chain amyloidosis, minimal residual disease of light chain amyloidosis, or relapse of light chain amyloidosis.

[0353] Embodiment 11-28. A method of treating a patient with light chain amyloidosis, minimal residual disease of light chain amyloidosis, or relapse of light chain amyloidosis, comprising: (i) determining if the patient has light chain amyloidosis, minimal residual disease of light chain amyloidosis, or relapse of light chain amyloidosis by: (a) contacting a sample from the patient with a monoclonal antibody or antigen-binding fragment thereof of any one of embodiments II-8 to 11-18, wherein the sample comprises amyloidogenic X free light chain (XFLC) protein that has been contacted by a protease and wherein contacting with the protease produces a neo-epitope of the XFLC protein; and (b) determining if the monoclonal antibody or antigen-binding fragment thereof binds to the XFLC; wherein the patient has light chain amyloidosis, minimal residual disease of light chain amyloidosis, or relapse of light chain amyloidosis if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein; and (ii) administering a treatment for light chain amyloidosis, minimal residual disease of light chain amyloidosis, or relapse of light chain amyloidosis.

[0354] Embodiment 11-29. The method of any one of embodiments 11-21 to 11-28, wherein the protease is proteinase K or chymotrypsin.

[0355] Embodiment 11-30. The method of any one of embodiments 11-25 to 11-29, wherein the treatment is a chemotherapeutic, autologous stem cells, carfilzomib, daratumumab, or a combination thereof.

[0356] Embodiment 11-31. The method of any one of embodiments 11-21 to 11-30, wherein the sample is serum, plasma, urine, cerebrospinal fluid, or tissue.

[0357] Embodiment 11-32. A kit, comprising: (i) a protease; and (ii) a monoclonal antibody or antigen-binding fragment thereof of any one of embodiments II-8 to 11-18.78323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102

[0358] Embodiment 11-33. A kit comprising: (i) a protease; (ii) a monoclonal antibody or antigen-binding fragment thereof of any one of embodiments II-8 to 11-18; and (iii) a neoepitope of the amyloidogenic X free light chain (ZFLC) protein.

[0359] Embodiment 11-34. A kit, comprising: (i) a monoclonal antibody or antigen-binding fragment thereof of any one of embodiments 11-8 to 11-18; and (ii) a neo-epitope of the amyloidogenic X free light chain (ZFLC) protein.

[0360] Embodiment 11-35. The kit of any one of embodiments 11-32 to 11-33, wherein the protease is proteinase K or chymotrypsin.

[0361] Embodiment 11-36. The kit of any one of embodiments 11-33 to 11-34, wherein the neoepitope has at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or 100 % identity to a polypeptide of any one of SEQ ID NOS: 1 and 34-344.

[0362] Embodiment 11-37. The kit of any one of embodiments 11-33 to 11-34, wherein the neoepitope comprises the amino acid sequence GQP (SEQ ID NO: 136), RQP (SEQ ID NO: 344), SQP (SEQ ID NO: 240), GQPKA (SEQ ID NO: 134), RQPKA (SEQ ID NO: 342), or SQPKA (SEQ ID NO: 238)

[0363] Embodiment 11-38. The kit of any one of embodiments 11-33 to 11-34, wherein the neoepitope comprises from the N-terminus to the C-terminus (i) GQP, RQP, or SQP; and (ii) a polypeptide with at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or at least 100 % identical to any one of SEQ ID NOS: 351-453.

[0364] Embodiment 11-39. The kit of any one of embodiments 11-33 to 11-38, comprising a surface coated with the neo-epitope.

[0365] Embodiment 11-40. The kit of embodiment 11-39, wherein the surface is a membrane or a plate.

[0366] Embodiment 11-41. The kit of any one of embodiments 11-32 to 11-40, comprising a pharmaceutically acceptable buffer.ExamplesMethods and materialsELISA assay using LCCD mAh

[0367] 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) antibody79323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 from Bethyl is added 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 Blocking Buffer, serial dilutions of WIL LC and purified human IgG with and without PK (0.2 pM) digested 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 FLC

[0368] Plasma samples were incubated with IpM of Proteinase K at 37°C for 2 hours before the reaction was stopped by adding phenylmethyl sulfonyl fluoride protease inhibitor.Detection of dLCCD in plasma using Western Blot

[0369] 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 IRDye-800 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 FLC

[0370] Amyloidogenic XFLC 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 XFLC 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 XFLC, although it was assumed ZFLC would yield small peptides after PK treatment, and only the full-length intact ZFLC was followed and analyzed. PK80323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 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 non-reducing 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 XFLC protein, such as WIL (FIG. 1A), 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, (FIG. IB). Under reducing condition, a fragment of about 11.5KDa representing the monomeric LCCD will be generated. The cleavage site constituted by the GQPKA (SEQ ID NO: 134) sequence is a neo-epitope exposed only through limited proteolysis by PK.

[0371] The consistent generation of this fragment on XFLC is unexpected given the non-specific digested nature of Proteinase K known in the art.

[0372] The Proteinase K cleavage site, residing at the immediate N-terminus of this neo-epitope, is >99% conserved in XFLC and was confirmed by analyzing the public databases ABYSIS and AL Bases containing >6,000 different XFLC sequences (FIG. 1C). Sequence analysis of the XFLC 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 XFLC 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

[0373] 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,81323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 and the mAbs were named LCCD-G, LCCD-S, and LCCD-R, respectively, based on the identity of x in the xQP neo-epitopes.

[0374] 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 XFLC protein, before and after limited Proteinase K digested were subjected to the ELISA assay. The data indicated that LCCD-G mAb binds strongly to only the Proteinase K digested WIL LC and has minimum or no binding to full-length undigested LC (FIG. 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 Proteinase K and the resulting samples subjected to the LCCD ELISA assay. As shown in FIG. 2B, no signal was detected after Proteinase K treatment of IgG. FIG. 2C show LCCD mAbs highly specific for proteolysis exposed novel epitopes. LCCD-G, LCCD- R, and LCCD-S mAbs (purple signal around 25 kDa) specifically detect LCCD fragments recombinant XFLC proteins containing GQP, RQP, and SQP at the cleavage site, respectively, post-proteolysis by Proteinase K. Anti-XFLC polyclonal antibody (blue signal around 50KDa) is used to indicate all full-length and LCCD fragments from the samples.

[0375] These results demonstrate that the LCCD mAb specifically binds to the cleaved and exposed xQP epitope, not xQP in a protein sequence; and the specific generation of the LCCD- G epitope specifically in amyloidogenic FLC, but not in full length immunoglobulin.Example 3: Quantification of LCCD in AL plasma after limited proteolysis

[0376] 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 (FIG. 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 (FIG. 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.

[0377] A moderate correlation between dLCCD fluorescence signal and XFLC measured by the FreeLite clinical test based on turbidimetry was observed in 20 of the treatment naive AL plasma samples (FIG. 3B). No such correlation could be drawn for normal or MM plasma samples under the current limited proteolysis condition.82323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102

[0378] Under optimized stress condition, the mAb detects signal in 32 randomly selected XAL plasma samples in this application employing standard Western blot analysis using a few microliters of samples. The signal is lower if not undetectable in normal (N=23) and 18 out of the 19 MM plasma samples under the same stress condition. 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 was thought 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

[0379] Under the limited proteolysis condition, LCCD mAb differentiates AL plasma samples from normal or MM plasma samples (FIG. 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.

[0380] 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

[0381] FIG. 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 analyte (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. 2 pl biofluid per assay. A readout is obtained in a few hours. FIG. 5B shows the outcome of a dLCCD assay with plasma sample post limited proteolysis. dLCCD levels differentiate LAL 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.83323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102Example 6: High baseline dLCCD correlates to poor overall survival

[0382] FIGS. 6A-B show baseline levels of dLCCD correlates to XAL patient overall survival. dLCCD in the experiment was detected at the 2 hour time point. FIG. 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. FIG. 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 XAL patients using the MSD dLCCD immunoassay described herein.Example 7: dFLC 18 and dLCCD 300 concordance predicts overall survival

[0383] FIGS. 7A-7B shows concordance of dFLC and dLCCD levels predicts overall survival based on the data from FIG. 6. FIG. 7A shows a log-rank survival test of patients with low baseline levels of both biomarkers (N=17) comparing to those with high baseline levels of both biomarkers (N=17), and those with discordant dFLC and dLCCD levels (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, as shown in FIG. 7B. The results are based on treatment naive plasma samples from 50 confirmed XAL patients using an MSD LCCD immunoassay as described herein. There are significant differences in OS; in particular, median OS for dFLClow / dLCCDlowis 162 months; for dFLChlgh / dLCCDhlghis 36 months; and for discordant is 16 months. dLCCD level further correlates to cTnT and NT -proBNP (p=0.05). dLCCD is a pathogenic-indicating biomarker in most XAL patients. Baseline dLCCD is a better OS predictor than dFLC. Without being bound by theory, reducing dLCCD levels using a therapeutic approach provides clinical benefits; for example, by eradicating amyloidogenic FLC producing clonal plasma cells, or by stabilizing ZFLC 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

[0384] Small molecules that bind directly to the dimer interface of the dimeric XFLC protein were shown to stabilize the protein and reduce its amyloidogenicity (Morgan et al. 2019, PMC6486714). Such small molecules are considered free light chain (FLC) dimer 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 level84323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 of unstable or amyloidogenic XFLC in the biofluids and use that as an indicator of target engagement. FLC dimer stabilizer PTG-1412 was incubated with AL plasma samples and samples were treated with Proteinase K or left untreated. Western blot analysis of the samples after limited proteolysis with Proteinase K was conducted using with an anti-XFLC monoclonal antibody (band at 50KDa) and the dLCCD mAb (solid band at 23 KDa). As shown in the Western Blot of FIG. 8A, the LC stabilizer provided dose-dependent protection of XFLC in the samples. The antibody-detected bands on the Western Blot of FIG. 8A was quantified on the LiCor system and fitted to a variable slope (four parameters) response curve using Prism, as shown in FIG. 8B. EC50s were similar using the two antibodies. Protection against limited proteolysis by PTG-1412 in 13 XAL plasma samples, as quantified by dLCCD levels postproteolysis, with and without PTG-1412 was assayed. As shown in FIG. 8C, the FLC dimer stabilizer provided protection of XFLC in the samples.

[0385] The same assay is to detect amyloidogenic XFLC in a biofluid from an AL patient before and after therapeutic treatment and the difference in the dLCCD levels is an indicator for response to therapy or the lack thereof.Example 9: Limited proteolysis using various enzymes generate fragments that contain neoepitopes

[0386] Many bioinformatics tools, such as Expasy’s Peptide Cutter, can predict theoretical proteolysis sites based on target protein sequences and the enzyme used. However, predicting the precise cleavage site under limited proteolysis conditions is much more difficult, if not impossible currently. Based on database searches (MEROPS, Peptide Cutter, PROSPER, CutDB, and DeepCleave), a few proteases were predicted to cleave FLC at the GQP epitope. These proteases include Chymotrypsin, Pepsin, Cathepsin B, Cathepsin L, Thermolysin, the peptidase S8 family, represented by PCSK9 and Subtilisin.

[0387] Limited proteolysis of rhFLC WIL using Thermolysin, Pepsin, and Trypsin was performed. The rhFLC WIL protein was subjected to limited proteolysis using Thermolysin. FIG. 9A depicts a gel image stained with Coomassie blue showing different proteolytic fragments generated with increasing concentrations of Thermolysin. rhFLC WIL protein was also subjected to limited proteolysis using Pepsin. FIG. 9B depicts a gel image stained with Coomassie blue showing different proteolytic fragments generated with increasing concentrations of Pepsin. rhFLC WIL protein was also subjected to limited proteolysis using85323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102Trypsin. FIG. 9C depicts a gel image stained with Coomassie blue showing different proteolytic fragments generated with increasing concentrations of Trypsin.

[0388] As shown in FIGS. 9A-9C, the Thermolysin, Pepsin, and Trypsin can digest XFLC, as evidenced by the reduction of the dimeric WIL band on the Commassie-stained gel. However, none of the bands at >10 KDa were recognized with dLCCD mAb, indicating they do not generate the xQP epitope. On the other hand, some of these enzymes generated fragments larger than 10 KDa that may be used as a novel biomarker to monitor XFLC stability.

[0389] As indicated, different neo-epitopes may be generated by applying limited proteolysis using different proteases. Thermolysin, Pepsin and Trypsin are provided as non-limiting examples.

[0390] Other naturally occurring or engineered enzymes such as Cathepsin B, Cathepsin L, the peptidase S8 family, represented by PCSK9 and Subtilisin are used or generated to produce an amyloidogenic ZFLC neo-epitopes according to the methods described above and herein.Example 10: Limited Proteolysis with Chymotrypsin Generates the Neo-Epitope of the / . l.C Protein

[0391] Chymotrypsin (EC 3.4.21.1, Chymotrypsins A and B, alpha-chymar ophth, avazyme, chymar, chymotest, enzeon, quimar, quimotrase, alpha-chymar, alpha-chymotrypsin A, alphachymotrypsin) is a digestive enzyme component of pancreatic juice acting in the duodenum, where it performs proteolysis, the breakdown of proteins and polypeptides. Chymotrypsin preferentially cleaves peptide amide bonds where the side chain of the amino acid N-terminal to the scissile amide bond (the Pl position) is a large hydrophobic amino acid (tyrosine, tryptophan, and phenylalanine). These amino acids contain an aromatic ring in their side chain that fits into a hydrophobic pocket (the SI position) of the enzyme.

[0392] The ability of chymotrypsin to digest the amyloidogenic ZFLC protein and produce a neo-epitope of the ZFLC protein was tested. Briefly, rhFLC WIL was contacted with chymotrypsin at a concentration of 87.5 nM. FIG. 9D shows that chymotrypsin digested produces the neo-epitope, as evident by dLCCD mAb binding to the neo-epitope. FIG. 9E shows the quantification of dLCCD fragment generated from FIG. 9D, where amyloidogenic rhFLC is represented by open symbols, and the normal rhFLC is represented by closed symbols. Therefore, the GQP neo-epitope is generated through limited proteolysis using Proteinase K and Chymotrypsin.86323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102Example 11: Limited proteolysis using different enzymes can generate fragments that differentiates amyloidogenic vs. non-amyloidogenic FLC

[0393] Papain digested of amyloidogenic vs. non-amyloidogenic XFLC was also evaluated. Recombinant XFLC WIL and Nak proteins were subjected to limited proteolysis using Papain (FIG. 9G). Papain cleaved AL rhFLC (WIL-46T) at a much higher rate than that of Normal rhFLC (Nak). Thus, limited proteolysis by Papain could distinguish AL rhFLC from normal rhFLC. A 10-15KDa fragment was generated from both WIL and Nak rhFLC. However, this fragment was not recognized by the LCCD-G mAb described herein.

[0394] In addition, Papain generates the 10-15 KDa fragment from XFLC (FIG. 9F). This fragment may be used to follow amyl oidogeni city of XFLC in patient biofluids, similar to the dLCCD biomarker.Example 12: Generation and characterization of monoclonal antibodies to Papain generated FLC fragments

[0395] Monoclonal antibodies (mAb) are generated through immunizing rabbits using peptide- KLH conjugates as the antigens. Peptide antigen contains the Papain generated a 10-15 KDa fragment from XFLC. Anti-serum titer is monitored using ELISA against the antigen. Upon detection of high titer, antigen-specific B cells are isolated for specific recognition of the antigen. Cells are expanded and antibody genes amplified and cloned. The amplified antibody heavy and light chains are expressed in HEK293 cells, and the supernatant assayed by ELISA to confirm the antibody specificity. mAbs from the immunizations are selected, sequenced, and the mAbs are named papLC.

[0396] Purified recombinant ZFLC proteins are first used to test the specificity of the papLC mAb in a Sandwich ELISA assay, where papLC mAb is used as the capture antibody, and a goat polyclonal anti-LC antibody is used as the detection antibody. Recombinant WIL, an amyloidogenic XFLC protein, before and after limited Papain digested are subjected to the ELISA assay. The data is expected to indicate that papLC mAb binds strongly to only the Papain digested WIL LC and has minimum or no binding to full-length undigested LC. In human proteome, the same light chain protein can also heterodimerize with heavy chain proteins and form full-length immunoglobulin. The study investigates if the light chain in immunoglobulin is stable under the limited proteolysis condition. To test that, purified human IgG is treated with Papain and the resulting samples subjected to the papLC ELISA assay.87323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102Example 13: Protease activity

[0397] It is well known that enzyme activity is highly dependent on many factors, such as enzyme concentration, substrate type and concentration, time of incubation, reaction temperature, and the composition of the reaction mixture. FIG. 10A highlights the effect of enzyme concentration and reaction temperature. dLCCD generation efficiency is dependent on the condition of the limited proteolysis.

[0398] Proteinase K can also be immobilized and used in limited proteolysis, as shown in FIG. HE. Agarose-immobilized Proteinase K was incubated with rhFLC. The samples before and after agarose-immobilized Proteinase K treatment were then analyzed on SDS PAGE gel. dLCCD band at ~23KDa can be observed in the post-PK-treatment sample with Coomassie staining.

[0399] FIG. HF shows the specific activity of Proteinase K as mean ± standard deviation from 12 independent experiments following the above procedure, measured with the MSD immunoassay. As an example, a unit is defined as the amount of enzyme needed to generate 85.8±13.2nM of dLCCD from LOpM of WIL recombinant protein in phosphate buffer (pH 7.4) in 30 minutes at 37°C. In a specific batch, 1 unit of Proteinase K is equivalent to 0.1 pM of the enzyme.Example 14: inAb detection of dLCCD and papLC post limited proteolysis in biofluids

[0400] Amyloidogenic XFLC is secreted into the blood circulation by clonal plasma cells. Stable XFLC protein, as a sub-50 KDa protein, is catabolized through glomerulus and passes down nephron tubule to form urine. In addition, salivary glands also contain plasma cells that can produce XFLC.

[0401] Therefore, in addition to plasma, the present results, as shown in FIGS. 11A-11C and Table 8, demonstrate that dLCCD mAbs described herein are able to detect the dLCCD biomarker in serum, urine, and saliva samples after limited Proteinase K treatment. The dLCCD mAbs detected dLCCD at various levels in Proteinase K-treated serum, urine, and saliva samples from AL patients.

[0402] In addition, papLC mAbs described herein are used to detect the papLC biomarker in whole blood, plasma, urine, serum, saliva, cerebrospinal fluid (CSF), or tissue biopsy samples after limited proteolysis papain treatment. The papLC mAbs detect papLC at various levels in Papain-treated whole blood, plasma, urine, serum, saliva, cerebrospinal fluid (CSF), or tissue biopsy samples from AL patients.88323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102Table 8: Data corresponding to dot blot of FIG. 11DExample 15: Detection of dLCCD using other methodsDot Blot and Lateral Flow

[0403] An aspect of the present disclosure is a method of assays consist of a limited proteolysis step that generates the dLCCD biomarker specifically from amyloidogenic XFLC, and an immunoassay that detects and / or quantifies the biomarker. A person skilled in the art can develop other methods to detect and / or quantify the dLCCD biomarker. For example, a different immunoassay format. FIGS. 12A-12B are examples of using dot blot to assess dLCCD level semi -quantitatively on a solid membrane.

[0404] With regard to FIG. 12A, the assay was conducted by serially diluting recombinant dLCCD-G protein in PBS buffer. 2 or 3 pL of sample were blotted onto the nitrocellulose membrane, allowed to dry completely, then blocked at RT for 30 minutes. Thereafter 1 : 10,000 diluted LCCD-X antibody was added and incubated with tilting at 4°C overnight. The membrane was then rinse 3x with TBST, incubated for one hour at RT with 1 :25,000 Donkey anti -Rabbit IR 800, and rinse 3x with TBST, then once with water. Images were obtained on a89323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102LiCor instrument. The results show that between 317.42 picograms and 40.63 nanograms there is linear detection.

[0405] With regard to FIG. 12B, the assay was conducted using 1 pM PK using 3.5 M Wil at 37°C. Multiple reactions were prepared and at each of the time points, the reaction was stopped by the addition of PMSF. A 10-point LCCD-G standard curve was run side by side, starting at 1.78 pM. 2 pL of sample was blotted onto a nitrocellulose membrane, allowed to dry 1 hour at 37°C, then blocked at RT for 1 hour. 1 : 10,000 diluted LCCD-X antibody was added and incubated with tilting at 4°C overnight. Thereafter the membrane was rinsed 3x with TBST, incubated for one hour at RT with 1 :25,000 Donkey anti-Rabbit IR 800. Rinse 3x with TBST, then once with water. Images were obtained on a LiCor device.

[0406] FIG. 12C shows the transformation of the dot blot assays of FIG. 12A and FIG. 12B into to a lateral flow assay using biotinylated dLCCD-G mAb, FITC labeled anti-human lambda LC, and recombinant dLCCD-G protein, with the lateral flow based detection of cLCCD-G in a sample.

[0407] The methods described above for LCCD-X are reproduced in connection with papLC to produce dot plot and lateral flow assays for these biomarkers as indicated below.

[0408] The papLC mAb assays consist of a limited proteolysis step that generates the papLC biomarker specifically from amyloidogenic XFLC, and an immunoassay that detects and / or quantifies the biomarker. A person skilled in the art can develop other methods to detect and / or quantify the papLC biomarker. For example, a different immunoassay format, including on solid membranes and via lateral flow.

[0409] Using the same operable principles underpinning the solid membrane results, a lateral flow assay and device using well known lateral flow diagnostic assay technologies is developed to assess dLCCD and / or papLC presence and / or level semi -quantitatively. Urine, whole blood, plasma, CSF, serum, tissue biopsy, or another sample may be utilized in such an assay.Mass Spectrometry

[0410] The dLCCD and / or papLC level in biofluids are also be detected using mass spectrometry, which follows the full-length dLCCD and / or papLC or a smaller peptide fragment upon additional protease treatment.90323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102Competition Assay

[0411] A dLCCD level can also be quantified in biofluids through a competition assay. FIGS. 13A-13B exemplify such an assay and results. As shown in FIG. 13A, a dLCCD-G mAb is immobilized on a solid support, followed by the introduction of GQPK-mer-BIOTIN labeled probe, which binds the immobilized mAb. dLCCD recombinant protein is then added to the immobilized and labeled mAb followed by the introduction of a streptavidin-SULFO-Tag to bind any probe remaining on the immobilized mAb and MSD detection. It was found that the recombinant dLCCD recombinant protein competed off the probe in a dose dependent manner as shown in FIG. 13B.Example 16: Using FLC level post limited proteolysis to indicate instability of FLC

[0412] Amyloidogenic XFLC tends to be kinetically less stable than its non-amyloidogenic counterparts, rendering it susceptible to denaturing conditions such as limited proteolysis. dLCCD and / or papLC mAb assays incorporate a limited proteolysis step to quantitatively generate the dLCCD and / or papLC biomarker through protease degradation of the full-length XFLC. Thus, the reduction of XFLC post-limited proteolysis can be determined to monitor the instability or amyl oidogeni city of XFLC in the biofluids. Currently marketed XFLC assays utilize antibodies that bind to cryptic epitopes on the constant domain, avoiding interference from light chain signals from other immunoglobulins such as IgG, IgA, and IgM. Because the dLCCD and / or papLC product also contains the constant domain, current assays such as FreeLite cannot specifically detect full-length XFLC without interference from the dLCCD and / or papLC fragment. FIGS. 14A-14B demonstrate this finding in connection with dLCCD.

[0413] FIG. 14A shows that commercial XFLC assays have epitopes residing on the constant domain, which therefore detect both the full-length rhFLC and the resulting dLCCD fragment post-treatment with Proteinase K. rhFLC WIL with and without protease treatment was detected using anti-dLCCD mAb (left) and goat anti-human FLC pAb (right) using Western Blot. FIG. 14B shows the Sebia XFLC ELISA assay detects both XFLC (rhFLC Wil; closed symbols) and dLCCD (open symbols) dose-dependently.Example 17: Quantification of LCCD level using the MSD platform

[0414] An assay was developed to quantify LCCD (Light chain Neo Epitope) level using the MSD platform. The protocol of the assay is provided below. The LCCD is generated by Proteinase K (PK) limited proteolysis in a AL amyloidosis (AL) patient's plasma or serum91323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 sample. To demonstrate how the assay works, LCCD level was quantified in normal serum samples or serum samples spiked with JTO (non-amyloidogenic LC) or WIL (amyloidogenic LC) after limited proteolysis with Proteinase K. As shown in FIG. 15, dLCCD was detected at high levels in WIL (amyloidogenic LC) samples but not in normal serum and JTO samples.

[0415] This assay can be used for the diagnosis of AL and the assessment of target engagement of a light chain stabilizer in the clinical setting.Assay Protocol for MSD platformPart 1 - Prepare MSD Plate

[0416] (A) Blocking the Plate: (i) One day before running the assay, the plate was blocked by adding 250 pL of SuperBlock PBS, without T20, to each well, (ii) The plate was sealed, with a clear plate seal, and shaken at 750 rpm for 10 min at room temperature. The plate was moved to a 4 °C fridge and incubated overnight.

[0417] (B) Coating the Plate: (i) the captured, biotinylated mAbs were diluted with SuperBlock T20 PBS to the optimized final concentrations, using 0.2 pg / mL for biotinylated mAb-LCCDg, 0.4 pg / mL for biotinylated mAb-LCCDs and 0.1 pg / mL for biotinylated mAb-LCCDr and vortexed to mix well, (ii) 50 pL of the diluted capture antibody was pipetted into each well, (iii) the plates were sealed with a clear plate seal and the plate was shaken on a benchtop plate shaker (750 rpm) for about 1 hour at room temperature.Part 2 - PK-mediated proteolysis of Serum in 0.2-mL PCR Strip Tubes

[0418] (A) Serum samples were thawed at room temperature and then kept on ice.

[0419] (B) A 5-mM EDTA in PBS solution was prepared by adding 10 pL of EDTA (as a 0.5-M stock) to 990 pL of PBS.

[0420] (C) recombinant LC (rhLC) QC standards were prepared as shown in Table 2 below.Table 2: rhLC QC standards dilution92323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102

[0421] (D) Two-step dilution of PK: (i) 5 pL of PK stock (700 pM) was diluted into 45 pL of PBS, to yield 70 pM of PK. (ii) 34 pL of PK (70 pM) was further diluted to 1870 pL of PBS to achieve a total volume of 1904 pL of a 1.25-pM PK solution. The diluted PK was kept on ice.

[0422] (E) The PK reaction was setup in 0.2-mL PCR strip tube in a metal 96-well block on ice, as follows. The patient samples were prepared as shown in Table 3 below. The control samples were prepared as shown in Table 4 below. The circled number denotes the order of reagents adding into the tube. The order may be modified.

[0423] Table 3: Preparation of patient samplesTable 4: Preparation of control samples

[0424] When the entire set of samples were ready, the diluted PK solution (1.25 pM) was placed in a 10-mL reagent reservoir and a multi-channel pipette was used to add 8 pL of PK to each sample immediately. The final PK concentration was 0.5 pM, and the total reaction volume for each sample is 20 pL. The PCR strip tubes were inverted several times to mix the PK reaction thoroughly. The PCR tubes were spun to bring the reaction mixture to the bottom of the tubes. The PCR tubes were transferred to a 96-well thermocycler, preheated to 37 °C with a hot top (105 °C), and incubated for the desired time period (90 minutes). The PK reaction was stopped by adding 2 pL of Halt protease inhibitor (100X) to each PCR tube. The tube was tapped to mix the halt and PK reaction. The PCR tubes were spun to bring down the reaction mixture. Final halt concentration was 9X. The reaction tube was then kept on ice.

[0425] (F) Add samples to MSD plate, (i) 50 pL of each diluted sample was added to wells in the MSD plate (in duplicates), (ii) 50 pL of each reference standard was added to wells in the MSD plate (in duplicates), (iii) The MSD plates were covered with clear plate seals and incubated for 1 hour at room temperature while shaking at 750 rpm on a benchtop plate shaker.93323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102

[0426] (G) Incubation with SULFO-TAG Antibody, (i) a SULFO-TAG anti-Hu / NHP, lambda, light chain antibody (500 pg / mL) was incubated in SuperBlock T20 PBS to a final concentration of 0.5 pg / mL in the dark.

[0427] (H) Reading the Plate, (i) a lx read buffer was prepared by diluting 10 mL of 4x Read buffer T, with surfactant, with 30 mL pure water (GenPure Pro System, Thermo Fisher Scientific), (ii) the plate was washed 3 times, using 300 pL of PBST, in a plate washer, (iii) 150 pL of lx read buffer was added to each well with a multi-channel pipettor, (iv) the plate was read immediately after adding the read buffer.94323555102

Claims

Attorney Docket No.: PRTE-021 / 01WO 345214-2102CLAIMS1. A monoclonal antibody or antigen-binding fragment thereof that binds to a neo-epitope of an amyloidogenic lambda free light chain (XFLC) protein, wherein the monoclonal antibody or antigen-binding fragment thereof comprises:(a) a variable heavy chain CDR1 (HCDR1) comprising an amino acid sequence of SEQ ID No: 10, 16, or 22;(b) a variable heavy chain CDR2 (HCDR2) comprising an amino acid sequence of SEQ ID No: 11, 17, or 23;(c) a variable heavy chain CDR3 (HCDR3) comprising an amino acid sequence of SEQ ID No: 12, 18, or 24;(d) a variable light chain CDR1 (LCDR1) comprising an amino acid sequence of SEQ ID No: 13, 19, or 25;(e) a variable light chain CDR2 (LCDR2) comprising an amino acid sequence of SEQ ID No: 14, 20, or 26; and(f) a variable light chain CDR3 (LCDR3) comprising an amino acid sequence of SEQ ID No: 15, 21, or 27.

2. The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a HCDR1 comprising an amino acid sequence of SEQ ID No: 10; a HCDR2 comprising an amino acid sequence of SEQ ID No: 11, a HCDR3 comprising an amino acid sequence of SEQ ID No: 12, a LCDR1 comprising an amino acid sequence of SEQ ID No: 13, a LCDR2 comprising an amino acid sequence of SEQ ID No: 14, and a LCDR3 comprising an amino acid sequence of SEQ ID No: 15.

3. The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a HCDR1 comprising an amino acid sequence of SEQ ID No: 16, a HCDR2 comprising an amino acid sequence of SEQ ID No: 17, aHCDR3 comprising an amino acid sequence of SEQ ID No: 18, aLCDRl comprising an amino acid sequence of SEQ ID No: 19, a LCDR2 comprising an amino acid sequence of SEQ ID No: 20, and a LCDR3 comprising an amino acid sequence of SEQ ID No: 21.

4. The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a HCDR1 comprising an95323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 amino acid sequence of SEQ ID No: 22, a HCDR2 comprising an amino acid sequence of SEQ ID No: 23, a HCDR3 comprising an amino acid sequence of SEQ ID No: 24, a LCDR1 comprising an amino acid sequence of SEQ ID No: 25, a LCDR2 comprising an amino acid sequence of SEQ ID No: 26, and a LCDR3 comprising an amino acid sequence of SEQ ID No: 27.

5. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein the monoclonal antibody or antigen-binding fragment thereof comprises:(i) a heavy chain variable domain (VH) comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 345, and a light chain variable domain (VL) comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 346;(ii) a VH comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 347, and a VL comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 348; or(iii) a VH comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 349, and a VL comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 350.

6. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID No: 345, and a VL comprising an amino acid sequence of SEQ ID No: 346.

7. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID No: 347, and a VL comprising an amino acid sequence of SEQ ID No: 348.

8. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a VH comprising96323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 an amino acid sequence of SEQ ID No: 349, and a VL comprising an amino acid sequence of SEQ ID No: 350.

9. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-8, wherein the neo-epitope has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide of any one of SEQ ID NOS: 1 and 34-135, 137-239, 241- 343344, GQP, SQP, and RQP10. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-8, wherein the neo-epitope comprises the amino acid sequence GQP, RQP, SQP, GQPKA (SEQ ID NO: 134), RQPKA (SEQ ID NO: 342), or SQPKA (SEQ ID NO: 238).

11. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-8, wherein the neo-epitope comprises from the N-terminus to the C-terminus (i) GQP, RQP, or SQP; and (ii) a polypeptide with at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or at least 100 % identical to any one of SEQ ID NOS: 351-450, KAA, and KA.

12. An immunogenic composition, comprising:(i) a neo-epitope of an amyloidogenic XFLC protein; and(ii) a pharmaceutically acceptable buffer.

13. The immunogenic composition of claim 12, wherein the neo-epitope has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide of any one of SEQ ID NOs: 1 and 34-135, 137-239, 241-343, GQP, SQP, and RQP.

14. The immunogenic composition of claim 12, wherein the neo-epitope comprises the amino acid sequence GQP, RQP, or SQP.

15. The immunogenic composition of claim 12, wherein the neo-epitope comprises GQPKA (SEQ ID NO: 134), RQPKA (SEQ ID NO: 342), or SQPKA (SEQ ID NO: 238).97323555102Attorney Docket No.: PRTE-021 / 01WO 345214-210216. The immunogenic composition of claim 12, wherein the neo-epitope comprises from the N-terminus to the C-terminus (i) GQP, RQP, or SQP; and (ii) a polypeptide with at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or at least 100 % identical to any one of SEQ ID NOS: 351-450, KAA, and KA.

17. The immunogenic composition of any one of claims 12-16, comprising (iii) an adjuvant, optionally wherein the adjuvant is selected from any one of Aluminum hydroxide, Aluminum phosphate, Calcium phosphate, Freund’s complete adjuvant, Freund’s incomplete adjuvant, Montanide ISA 50, Montanide ISA 206, AS03, AS04, MF59, CpG oligodeoxynucleotides, Quil A, Liposomes, Poly(I), TLR agonists, E. coli heat-labile toxin, Chitosan, Zinc oxide, Squalene, BCG (Bacillus Calmette-Guerin), Dendritic cell vaccines, Saponins, Microparticles, Nanospheres, Micelles, Emulsions, Detoxified bacterial toxins, Oil-in-water emulsions, Water-in-oil emulsions, Polylactic acid, Polyethylene glycol, Pseudomonas exotoxin, Virolex, Imiquimod, RIBI, Polypropylene, Pluronic F68, Eudragit, Gelatin, Alginate, Silica, Cellulose, Starch, Activated carbon, Graphene oxide, Chloroform extracts, Arginine, Mannan, Lipopeptides, Peptidoglycan, and Phospholipids.

18. A method of generating a monoclonal antibody that binds to a neo-epitope of an amyloidogenic XFLC protein, comprising immunizing a subject with an immunogenic composition of any one of claims 12-17, and obtaining the monoclonal antibody from the immunized subject.

19. The method of claim 18, wherein the subject is a rabbit, mouse, rat, rabbit, goat, sheep, horse, or chicken.

20. A monoclonal antibody or antigen-binding fragment thereof that binds to a neo-epitope of an amyloidogenic AFLC protein, wherein the monoclonal antibody is generated according to the method of claim 18 or 19.

21. A method of determining if a patient has a condition, comprising:98323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102(i) contacting a sample from the patient with a protease, wherein the sample comprises amyloidogenic XFLC protein, and wherein contacting the sample with the protease produces a peptide comprising a neo-epitope of the XFLC protein;(ii) detecting the presence of the neo-antigen of the XFLC protein by contacting the proteolytically cleaved sample with a monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-11 and 20; wherein the patient has the condition if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC, wherein the condition is amyloidosis, AL amyloidosis, MGUS, multiple myeloma, smoldering multiple myeloma, IgM AL amyloidosis, MRD of AL amyloidosis, or relapse of AL amyloidosis.

22. A method of determining if a patient has a condition, comprising:(i) contacting a sample from the patient with a monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-11 and 20, wherein the sample comprises amyloidogenic XFLC protein that has been contacted by a protease to produce a neo-epitope of the XFLC protein ; and(ii) determining if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein ; wherein the patient has the condition if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein, wherein the condition is amyloidosis, AL amyloidosis, MGUS, multiple myeloma, smoldering multiple myeloma, IgM AL amyloidosis, MRD of AL amyloidosis, or relapse of AL amyloidosis.

23. A method of determining if a patient has a condition, comprising:(i) contacting a sample from the patient with a protease, wherein the sample comprises amyloidogenic XFLC protein, and wherein contacting with the protease produces a neo-epitope of the XFLC protein;(ii) contacting the proteolytically cleaved sample with a monoclonal antibody or antigenbinding fragment thereof of any one of claims 1-11 and 20; and(ii) determining if the monoclonal antibody or antigen-binding fragment thereof binds to the XFLC;99323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102 wherein the patient has the condition if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein, wherein the condition is AL amyloidosis, MRD of AL amyloidosis, or relapse of AL amyloidosis.

24. A method of determining if a patient has a condition, comprising:(i) contacting a sample from the patient with a monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-11 and 20, wherein the sample comprises amyloidogenic XFLC protein that has been contacted by a protease and wherein contacting with the protease produces a neo-epitope of the XFLC protein; and(ii) determining if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein; wherein the patient has the condition if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein, wherein the condition is AL amyloidosis, MRD of AL amyloidosis, or relapse of AL amyloidosis.

25. A method of treating a patient with a condition, comprising:(i) determining if the patient has the condition by:(a) contacting a sample from the patient with a protease, wherein the sample comprises amyloidogenic XFLC protein and wherein contacting with the protease produces a neo-epitope of the XFLC protein; b) contacting the proteolytically cleaved sample with a monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-11 and 20; and(c) determining if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein; wherein the patient has the condition if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein; and(ii) administering a treatment for the condition, wherein the condition is amyloidosis, AL amyloidosis, MGUS, multiple myeloma, smoldering multiple myeloma, IgM AL amyloidosis, MRD of AL amyloidosis, or relapse of AL amyloidosis.

26. A method of treating a patient with a condition, comprising:(i) determining if the patient has the condition by:100323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102(a) contacting a sample from the patient with a monoclonal antibody or antigenbinding fragment thereof of any one of claims 1-11 and 20, wherein the sample comprises amyloidogenic XFLC protein that has been contacted by a protease and wherein contacting with the protease produces a neo-epitope of the XFLC protein; and(b) determining if the monoclonal antibody or antigen-binding fragment thereof binds to the XFLC; wherein the patient has the condition if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein; and(ii) administering a treatment for the condition, wherein the condition is amyloidosis, AL amyloidosis, MGUS, multiple myeloma, smoldering multiple myeloma, IgM AL amyloidosis, MRD of AL amyloidosis, or relapse of AL amyloidosis.

27. A method of treating a patient with a condition, comprising:(i) determining if the patient has the condition by:(a) contacting a sample from the patient with a protease, wherein the sample comprises amyloidogenic XFLC protein and wherein contacting with the protease produces a neo-epitope of the XFLC protein; b) contacting the proteolytically cleaved sample with a monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-11 and 20; and(c) determining if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein; wherein the patient has the condition if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein; and(ii) administering a treatment for the condition, wherein the condition is AL amyloidosis, minimal residual disease of AL amyloidosis, or relapse of AL amyloidosisAL amyloidosisAL amyloidosis.

28. A method of treating a patient with a condition, comprising:(i) determining if the patient has the conditionby:(a) contacting a sample from the patient with a monoclonal antibody or antigenbinding fragment thereof of any one of claims 1-11 and 20, wherein the sample comprises amyloidogenic XFLC protein that has been contacted by a protease and wherein contacting with the protease produces a neo-epitope of the XFLC protein; and101323555102Attorney Docket No.: PRTE-021 / 01WO 345214-2102(b) determining if the monoclonal antibody or antigen-binding fragment thereof binds to the XFLC; wherein the patient has the condition if the monoclonal antibody or antigen-binding fragment thereof binds to the neo-epitope of the XFLC protein; and(ii) administering a treatment for the condition, wherein the condition is AL amyloidosis, MRD of AL amyloidosis, or relapse of AL amyloidosis.

29. The method of any one of claims 21-28, wherein the protease is proteinase K or chymotrypsin.

30. The method of any one of claims 25-29, wherein the treatment is a chemotherapeutic, autologous stem cells, carfilzomib, daratumumab, or a combination thereof.

31. The method of any one of claims 21-30, wherein the sample is serum, plasma, urine, cerebrospinal fluid, or tissue.

32. A kit, comprising:(i) a protease; and(ii) a monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-11 and 20.

33. A kit compri sing :(i) a protease;(ii) a monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-11 and 20; and(iii) a neo-epitope of the amyloidogenic ZFLC protein.

34. A kit, comprising:(i) a monoclonal antibody or antigen -binding fragment thereof of any one of claims 1-11 and 20; and(ii) a neo-epitope of the amyloidogenic ZFLC protein.

35. The kit of any one of claims 32-33, wherein the protease is proteinase K or chymotrypsin.102323555102Attorney Docket No.: PRTE-021 / 01WO 345214-210236. The kit of any one of claims 33-34, wherein the neo-epitope has at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or 100 % identity to a polypeptide of any one of SEQ ID NOS: 1 and 34-135, 137-239, 241-343344, GQP, SQP, and RQP37. The kit of any one of claims 33-34, wherein the neo-epitope comprises the amino acid sequence GQP (SEQ ID NO: 136), RQP (SEQ ID NO: 344), SQP (SEQ ID NO: 240), GQPKA (SEQ ID NO: 134), RQPKA (SEQ ID NO: 342), or SQPKA (SEQ ID NO: 238)38. The kit of any one of claims 33-34, wherein the neo-epitope comprises from the N- terminus to the C-terminus (i) GQP, RQP, or SQP; and (ii) a polypeptide with at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or at least 100 % identical to any one of SEQ ID NOS: 351-450, KAA, and KA.

39. The kit of any one of claims 33-38, comprising a surface coated with the monoclonal antibody or antigen-binding fragment thereof.

40. The kit of claim 39, wherein the surface is a membrane or a plate.

41. The kit of any one of claims 32-40, comprising a pharmaceutically acceptable buffer.

42. A method for detecting amyloidogenic ZFLC protein, comprising the steps of:(i) providing a sample comprising one or more of an amyloidogenic FLC protein and a non- amyloidogenic FLC protein;(ii) contacting the sample with a protease to obtain a digested sample, wherein the contacting is performed under conditions effective for producing an amyloidogenic ZFLC protein neoepitope; and(iii) determining an amount of the neo-epitope in the digested sample comprising contacting the digested sample with a monoclonal antibody or antigen-binding fragment thereof that binds to the neo-epitope, thereby detecting amyloidogenic ZFLC protein in the sample.

43. The method of claim 42, wherein the sample is obtained from a subject.103323555102Attorney Docket No.: PRTE-021 / 01WO 345214-210244. The method of claim 43, wherein the subject is suspected of having a plasma cell disorder.

45. The method of claim 43 or 44, wherein the subject is suspected of having an abnormalFLC level.

46. The method of any one of claims 43-45, wherein the subject has received or is receiving a treatment for a plasma cell disorder, optionally wherein the treatment comprises a plasma cell elimination chemotherapy.

47. The method of any one of claims 43-46, wherein an increased amount of neo-epitope in the digested sample indicates the presence of amyloidogenic XFLC protein in the sample.

48. The method of any one of claims 43-46, wherein an increased amount of the neo-epitope in the digested sample as compared to a reference indicates the presence of amyloidogenic XFLC protein in the sample.

49. The method of claim 48, wherein the reference is an amount of neo-epitope in a control sample comprising non-amyloidogenic XFLC protein and subjected to the steps of (ii)-(iii).

50. The method of any one of claims 47-49, wherein detecting amyloidogenic XFLC protein in the sample indicates(a) the subject has AL amyloidosis, MRD of AL amyloidosis, or a relapse of AL amyloidosis; and / or(b) the subject will respond to treatment with an FLC dimer stabilizer.

51. The method of claim 43, wherein the subject is receiving or has received an FLC dimer stabilizer.

52. The method of claim 51, wherein a decreased amount of the neo-epitope in the digested sample as compared to a reference indicates target engagement of the FLC dimer stabilizer in the subject, wherein the reference is an amount of neo-epitope in a control sample obtained from the subject prior to receiving the FLC dimer stabilizer and subjected to the steps of (ii)-(iii).104323555102Attorney Docket No.: PRTE-021 / 01WO 345214-210253. The method of claim 51, wherein a substantially similar amount of the neo-epitope in the digested sample as compared to a reference indicates target engagement of the FLC dimer stabilizer in the subject, wherein the reference is an amount of neo-epitope in a control sample comprising non-amyloidogenic FLC and subjected to the steps of (ii)-(iii).

54. The method of claim 42, wherein the step of (i) comprises contacting the sample with an FLC dimer stabilizer prior to the steps of (ii)-(iii).

55. The method of claim 54, wherein the sample comprises recombinant amyloidogenic XFLC.

56. The method of claim 55, wherein the recombinant amyloidogenic XFLC is selected from the group consisting of WIL, H3, H9, and a combination thereof57. The method of claim 54, wherein the sample is obtained from a subject suspected of having an AL amyloidosis58. The method of any one of claims 54-57, wherein a decreased amount of the neo-epitope in the digested sample as compared to a reference indicates target engagement of the FLC dimer stabilizer, wherein the reference is an amount of neo-epitope in a control sample lacking the FLC dimer stabilizer and subjected to the steps of (ii)-(iii).

59. A method for identifying a subject for treatment of AL amyloidosis, comprising:(i) obtaining a sample from the subject, the sample comprising one or more of an amyloidogenic FLC protein and a non-amyloidogenic FLC protein;(ii) contacting the sample with a protease to obtain a digested sample, wherein the contacting is performed under conditions effective for producing an amyloidogenic AFLC protein neo-epitope;(iii) determining an amount of the neo-epitope in the digested sample comprising contacting the digested sample with a monoclonal antibody or antigen-binding fragment thereof that specifically binds to the neo-epitope, wherein detection of the neo-epitope in the digested sample identifies the subject for treatment of AL amyloidosis.105323555102Attorney Docket No.: PRTE-021 / 01WO 345214-210260. The method of claim 59, wherein the subject is suspected of having a plasma cell disorder.

61. The method of claim 59 or 60, wherein the subject previously received a diagnosis of AL amyloidosis, and detection of the neo-epitope in the digested sample identifies the subject as having minimal residual disease and / or relapse of AL amyloidosis62. The method of claim 59 or 60, wherein the subject did not previously receive a diagnosis of AL amyloidosis.

63. The method of any one of claims 59-62, further comprising administering a treatment to the subject.

64. The method of claim 63, wherein the treatment comprises a chemotherapeutic, autologous stem cells, carfilzomib, daratumumab, or a combination thereof.

65. The method of claim 63 or 64, wherein the treatment comprises an FLC dimer stabilizer.

66. The method of any one of claims 59-65, wherein the sample is whole blood, plasma, urine, serum, saliva, cerebrospinal fluid (CSF), bone marrow aspirate, or tissue biopsy.

67. The method of any one of claims 42-66, wherein the protease is a recombinant protein.

68. The method of any one of claims 42-67, wherein the protease is selected from the group consisting of proteinase K, thermolysin, pepsin, trypsin, chymotrypsin, cathepsin B, cathepsin L, papain, and a peptidase S8, optionally wherein the peptidase S8 is PCSK9 or subtilisin.

69. The method of any one of claims 42-68, wherein the protease is proteinase K and / or chymotrypsin.

70. The method of any one of claims 42-69, wherein the conditions comprise a temperature of about 4°C to about 45°C.106323555102Attorney Docket No.: PRTE-021 / 01WO 345214-210271. The method of claim 70, wherein the conditions comprise a temperature of about 35°C to about 42°C.

72. The method of any one of claims 42-71, wherein the conditions comprise a duration of time about 10 min to about 18 hours.

73. The method of claim 72, wherein the conditions comprise a duration of about 10 minutes to 120 minutes.

74. The method of any one of claims 42-73, wherein the conditions comprise a concentration of the protease of about 0.010 pM to about 10,000 pM.

75. The method of any one of claims 42-73, wherein step (ii) comprises contacting the sample with at least two proteases.

76. The method of claim 75, wherein step (ii) comprises contacting the sample with 2, 3, or 4 proteases.

77. The method of claim 75 or 76, wherein the contacting is performed under conditions effective for producing the amyloidogenic XFLC protein neo-epitope and at least one second amyloidogenic XFLC protein neo-epitope.

78. The method of claim 77, wherein the neo-epitope and the at least one second neo-epitope are different.

79. The method of claim 77 or 78, wherein step (iii) comprises determining an amount of the neo-epitope and the at least one second neo-epitope in the digested sample, comprising contacting the digested sample with the monoclonal antibody or antigen-binding fragment thereof that specifically binds to the neo-epitope, and a monoclonal antibody or antigen-binding fragment thereof that specifically binds to the at least one second neo-epitope.

80. The method of any one of claims 75-79, wherein the at least two proteases comprise proteinase K and a second protease.107323555102Attorney Docket No.: PRTE-021 / 01WO 345214-210281. The method of claim 80, wherein the second protease is selected from the group consisting of thermolysin, pepsin, trypsin, chymotrypsin, cathepsin B, cathepsin L, papain, and a peptidase S8, optionally wherein the peptidase S8 is PCSK9 or subtilisin.

82. The method of any one of claims 42-81, wherein the monoclonal antibody or antigenbinding fragment comprises:(a) a variable heavy chain CDR1 (HCDR1) comprising an amino acid sequence of SEQ ID No: 10, 16, or 22;(b) a variable heavy chain CDR2 (HCDR2) comprising an amino acid sequence of SEQ ID No: 11, 17, or 23;(c) a variable heavy chain CDR3 (HCDR3) comprising an amino acid sequence of SEQ ID No: 12, 18, or 24;(d) a variable light chain CDR1 (LCDR1) comprising an amino acid sequence of SEQ ID No: 13, 19, or 25;(e) a variable light chain CDR2 (LCDR2) comprising an amino acid sequence of SEQ ID No: 14, 20, or 26; and(f) a variable light chain CDR3 (LCDR3) comprising an amino acid sequence of SEQ ID No:15, 21, or 27.

83. The method of any one of claims 42-82, wherein the monoclonal antibody or antigenbinding fragment thereof comprises a HCDR1 comprising an amino acid sequence of SEQ ID No: 10; a HCDR2 comprising an amino acid sequence of SEQ ID No: 11, a HCDR3 comprising an amino acid sequence of SEQ ID No: 12, a LCDR1 comprising an amino acid sequence of SEQ ID No: 13, a LCDR2 comprising an amino acid sequence of SEQ ID No: 14, and a LCDR3 comprising an amino acid sequence of SEQ ID No: 15.

84. The method of any one of claims 42-82, wherein the monoclonal antibody or antigenbinding fragment thereof comprises a HCDR1 comprising an amino acid sequence of SEQ ID No:16, a HCDR2 comprising an amino acid sequence of SEQ ID No: 17, a HCDR3 comprising an amino acid sequence of SEQ ID No: 18, a LCDR1 comprising an amino acid sequence of SEQ ID No: 19, a LCDR2 comprising an amino acid sequence of SEQ ID No: 20, and a LCDR3 comprising an amino acid sequence of SEQ ID No: 21.108323555102Attorney Docket No.: PRTE-021 / 01WO 345214-210285. The method of any one of claims 42-82, wherein the monoclonal antibody or antigenbinding fragment thereof comprises a HCDR1 comprising an amino acid sequence of SEQ ID No: 22, a HCDR2 comprising an amino acid sequence of SEQ ID No: 23, a HCDR3 comprising an amino acid sequence of SEQ ID No: 24, a LCDR1 comprising an amino acid sequence of SEQ ID No: 25, a LCDR2 comprising an amino acid sequence of SEQ ID No: 26, and a LCDR3 comprising an amino acid sequence of SEQ ID No: 27.

86. The method of any one of claims 42-85, wherein the monoclonal antibody or antigenbinding fragment thereof comprises:(i) a heavy chain variable domain (VH) comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 345, and a light chain variable domain (VL) comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 346;(ii) a VH comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 347, and a VL comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 348; or(iii) a VH comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 349, and a VL comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No: 350.

87. The method of any one of claims 42-86, wherein the monoclonal antibody or antigenbinding fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID No: 345, and a VL comprising an amino acid sequence of SEQ ID No: 346.

88. The method of any one of claims 42-86, wherein the monoclonal antibody or antigenbinding fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID No: 347, and a VL comprising an amino acid sequence of SEQ ID No: 348.109323555102Attorney Docket No.: PRTE-021 / 01WO 345214-210289. The method of any one of claims 42-86, wherein the monoclonal antibody or antigenbinding fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID No: 349, and a VL comprising an amino acid sequence of SEQ ID No: 350.

90. The method of any one of claims 42-89, wherein the neo-epitope has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide of any one of SEQ ID NOs: 1 and 34-135, 137-239, 241-343344, GQP, SQP, and RQP.

91. The method of any one of claims 42-89, wherein the neo-epitope comprises the amino acid sequence GQP (SEQ ID NO: 136), RQP (SEQ ID NO: 344), SQP (SEQ ID NO: 240), GQPKA (SEQ ID NO: 134), RQPKA (SEQ ID NO: 342), or SQPKA (SEQ ID NO: 238).

92. The method of any one of claims 42-89, wherein the neo-epitope comprises from the N- terminus to the C-terminus (i) GQP, RQP, or SQP; and (ii) a polypeptide with at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or at least 100 % identical to any one of SEQ ID NOS: 351-450, KAA, and KA.110323555102

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