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Multiple myeloma is a systemic malignancy of monoclonal plasma cells that accounts for 10% of hematologic cancers. With development of highly effective therapies for multiple myeloma, minimal residual disease (MRD) assessment has emerged as an important end point for management decisions. Currently, serologic assays lack the sensitivity for MRD assessment, and invasive bone marrow sampling with flow cytometry or molecular methods has emerged as the gold standard. We report a sensitive and robust targeted mass spectrometry proteomics method to detect MRD in serum, without the need of invasive, sequential bone marrow aspirates. The method detects Ig-derived clonotypic tryptic peptides predicted by sequencing the clonal plasma cell Ig genes. A heavy isotope-labeled Ig internal standard is added to patient serum at a known concentration, the Ig is enriched in a light chain type specific manner, and proteins are digested and analyzed by targeted mass spectrometry. Peptides from the constant regions of the λ or κ light chains, Ig heavy chains, and clonotypic peptides unique to the patient monoclonal Igs are targeted. This technique is highly sensitive and specific for the patient-specific monoclonal Igs, even in samples negative by multiparametric flow cytometry. Our method can accurately and precisely detect monoclonal protein in serum of patients treated for myeloma and has broad implications for management of hematologic patients., (Copyright © 2020 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Antibodies and Fc-fusion antibody-like proteins have become successful biologics developed for cancer treatment, passive immunity against infection, addiction, and autoimmune diseases. In general these biopharmaceuticals can be used for blocking protein:protein interactions, crosslinking host receptors to induce signaling, recruiting effector cells to targets, and fixing complement. With the vast capability of antibodies to affect infectious and genetic diseases much effort has been placed on improving and tailoring antibodies for specific functions. While antibody:antigen engagement is critical for an efficacious antibody biologic, equally as important are the hinge and constant domains of the heavy chain. It is the hinge and constant domains of the antibody that engage host receptors or complement protein to mediate a myriad of effector functions and regulate antibody circulation. Molecular and structural studies have provided insight into how the hinge and constant domains from antibodies across different species, isotypes, subclasses, and alleles are recognized by host cell receptors and complement protein C1q. The molecular details of these interactions have led to manipulation of the sequences and glycosylation of hinge and constant domains to enhance or reduce antibody effector functions and circulating half-life. This review will describe the concepts being applied to optimize the hinge and crystallizable fragment of antibodies, and it will detail how these interactions can be tuned up or down to mediate a biological function that confers a desired disease outcome.

Purpose: Accurate quantification of monoclonal IgM immunoglobulins is essential for response assessment in patients with Waldenström's macroglobulinaemia (WM). The propensity of IgM to form multimers in serum makes sample evaluation by current laboratory methods particularly challenging., Experimental Design: We assessed the precision and linearity of IgMκ and IgMλ heavy/light chain (HLC, Hevylite) assays, and established reference intervals using 120 normal donor sera. We compared the quantitative performance of HLC assays with serum protein electrophoresis (SPE) and total IgM nephelometry for 78 diagnostic samples and follow-up samples from 25 patients with WM. Comparisons were made between the three methods for diagnostic sensitivity and response assessment., Results: IgMκ and IgMλ HLC assays showed low imprecision and good linearity. There was good agreement between summated HLC (IgMκ + IgMλ) and total IgM (measured nephelometrically; R 2 = 0.90), but only moderate agreement between involved IgM HLC and SPE densitometry (R 2 = 0.49). Analysis of 120 normal donor sera produced the following normal ranges: IgMκ: 0.29-1.82 g/L; IgMλ: 0.17-0.94 g/L; IgMκ/IgMλ ratio: 0.96-2.30. Using these ranges, IgM HLC ratios were abnormal in all WM presentation sera tested, including 15 with non-quantifiable SPE. Despite discordance in quantitation, responses assigned with HLC assays showed excellent agreement to those based on international guidelines using SPE or total IgM; although abnormal HLC ratios indicated residual disease in some patients with negative electrophoresis results., Conclusions: Nephelometric assessment of IgMκ and IgMλ HLC pairs offers a quantitative alternative to traditional laboratory techniques for the measurement of monoclonal IgM and may aid in the management of WM. Clin Cancer Res; 22(20); 5152-8. ©2016 AACR., (©2016 American Association for Cancer Research.)

The development of a quantification method for monoclonal antibodies in serum has been accomplished by high-performance liquid chromatography multiple reactions monitoring mass spectrometry. A human monoclonal antibody (HmAb) was used as the model protein for method development and validation. A peptide from the CDR3-region of its heavy chain was selected and used for quantifying the entire mAb. This signature peptide served as a template for the internal standard. Prior to mass spectrometric analysis approximately 50% of the total serum protein content was removed by albumin depletion. The accuracy of the method ranged between 99 and 112% in cynomolgus monkey serum. The intra-assay coefficient of variation (CV) was lower than 4% at 4 microg/mL and 200 microg/mL HmAb (n = 3). The CV at 400 microg/mL corresponded to 9% (n = 3). In addition, the interassay variation was investigated in a male cynomolgus serum pool and in a female cynomolgus serum pool. The CV for the male cynomolgus pool at 4 microg/mL HmAb was 7% (n = 3). The CV obtained from the female pool was 8% (n = 3), at 4 microg/mL. The dynamic range of the method was 3 orders of magnitude. After albumin depletion of 25 microL of serum, a lowest limit of quantification of 2 microg/mL HmAb was reached in both human and cynomolgus monkey samples.

Background: Serum monoclonal proteins (M-proteins) are usually quantified by electrophoresis or immunochemical measurement. A third alternative involves immunochemical measurement of the monoclonal isotype by subtraction of the polyclonal part of the isotype calculated from the other polyclonal isotypes and light chain. We experimentally calculated heavy chain/light chain equivalence factors for three immunoglobulin (Ig) isotypes and compared serum M-protein concentrations obtained using the three approaches., Methods: Equivalence factors were calculated for 1427 samples from 322 patients with a clinical diagnosis of monoclonal gammopathy of undetermined significance and validated in serum samples with and without a monoclonal component. Serum light chain recovery by nephelometry was compared to estimates using factors for serum immunoglobulin concentrations. Some 3735 samples were measured by electrophoresis and nephelometry for comparison of M-protein concentrations., Results: The experimentally calculated factors were 3.3059 (IgGkappa), 3.5204 (IgGlambda), 3.4567 (IgAkappa), 3.5308 (IgAlambda), 4.9104 (IgMkappa) and 4.8020 (IgMlambda). Light chains quantified by nephelometry in 984 serum samples without M-protein gave recovery of 100.97%+/-6.89% compared to immunoglobulin concentration estimates. Light chain recovery of <80% was observed more often for IgMkappa (61.0%) and IgMlambda (42.5%). Method differences for M-protein were >25% for 37.4%-52.3% of the serum samples. Electrophoresis results were higher than nephelometry results for 5% of serum samples and 23.7% of the estimates, especially for M-protein <20 g/L. Method differences for concentrations >40 g/L were <25% for most IgG and IgA isotypes, but not IgM., Conclusions: No method can accurately measure serum M-proteins over the entire concentration range. Estimation using polyclonal immunoglobulins and light chains represents a simple alternative to electrophoresis that is applicable to any serum M-protein concentration, regardless of electrophoretic migration and particularly for M-protein serum <20 g/L.

Clinical utility of murine mAbs is limited because many elicit Abs to murine Ig constant and variable regions in patients. An Ab humanized by the current procedure of grafting all the complementarity determining regions (CDRs) of a murine Ab onto the human Ab frameworks is likely to be less immunogenic, except that its murine CDRs could still evoke an anti-variable region response. Previous studies with anticarcinoma mAb CC49 showed that light chain LCDR1 and LCDR2 of humanized CC49 could be replaced with the corresponding CDRs of a human Ab with minimal loss of Ag-binding activity. The studies reported in this paper were undertaken to dissect the CC49 Ag-binding site to identify 1) specificity determining residues (SDRs), the residues of the hypervariable region that are most critical in Ag-Ab interaction, and 2) those residues that contribute to the idiotopes that are potential targets of patients' immune responses. A panel of variants generated by genetic manipulation of the murine CC49 hypervariable regions were evaluated for their relative Ag-binding affinity and reactivity to sera from several patients who had been immunized with murine CC49. One variant, designated HuCC49V10, retained only the SDRs of CC49 and does not react with the anti-variable region Abs of the sera from the murine CC49-treated patients. These studies thus demonstrate that the genetic manipulation of Ab variable regions can be accomplished by grafting only the SDRs of a xenogeneic Ab onto human Ab frameworks. This approach may reduce the immunogenicity of Abs to a minimum.

After preparative nondenaturing isoelectric focusing in a Rotofor of 1 mL human serum which showed on two-dimensional electrophoresis a monoclonal kappa light chain pI < 5.0, the monoclonal kappa light chain was identified in the tubes with pH 7.29-pH 6.96, indicating that the chain was not free and was most likely part of an immunoglobulin molecule. The combined fractions of the Rotofor were concentrated back to 1 mL and aliquots were separated by two-dimensional electrophoresis, followed by Western blotting. Enzyme immunoassay of the blot with A' human IgG (gamma chain specific) revealed a monoclonal heavy chain at a molecular size of approximately 100 kDa. Diffuse areas at 50 kDa (the attributed size of gamma heavy chain), 100 kDa, and 150 kDa were stained also, but the strong, restricted (picket fence) bands indicating a monoclonal pattern were at 100 kDa. The monoclonal light chain appears to be part of a large or dimeric IgG molecule.

Three monoclonal IgG of different subclasses (IgG1, IgG2, and IgG4) and one IgA1 were isolated from the serum of patient Per suffering from an immunocytic sarcoma. All four monoclonal Ig shared the same N-terminal sequence of their H chains (VH3). Furthermore, their kappa-chains exhibited identical isoelectric charges and N-terminal sequences (VK2) and expressed the same private idiotope. A strong antitubulin activity was found in IgA1Per and in two of the three monoclonal IgGPer. The specificity was restricted to tubulin for IgA1Per and IgG4Per, whereas IgG1Per also displayed significant polyreactive bindings and IgG2Per failed to react with any of the Ag tested. The monoreactive IgG4Per, as well as the polyreactive IgG1Per, bound a large peptide in the central part of both alpha and beta subunits of tubulin (amino acid position 100 to 300). In contrast, the monoreactive IgA1Per bound to a rarely detected epitope close to residue 310 of these subunits. The tubulin epitope recognized by polyreactive IgG1Per was similar to that of germ-line-encoded polyreactive antibodies. It is hypothesized that IgG4Per- and IgA1Per-producing cells derive from the IgG1Per polyreactive clone after somatic events leading to the production of monoreactive antibodies.