Heavy/light Chain Pair Research Articles

Heavy-light chain pairs as an alternative method to monitor isatuximab-based treatment responses in multiple myeloma patients

Heavy-light chain pairs as an alternative method to monitor isatuximab-based treatment responses in multiple myeloma patients

A Customizable Suite of Methods to Sequence and Annotate Cattle Antibodies.

Studying the antibody response to infection or vaccination is essential for developing more effective vaccines and therapeutics. Advances in high-throughput antibody sequencing technologies and immunoinformatic tools now allow the fast and comprehensive analysis of antibody repertoires at high resolution in any species. Here, we detail a flexible and customizable suite of methods from flow cytometry, single cell sorting, heavy and light chain amplification to antibody sequencing in cattle. These methods were used successfully, including adaptation to the 10x Genomics platform, to isolate native heavy-light chain pairs. When combined with the Ig-Sequence Multi-Species Annotation Tool, this suite represents a powerful toolkit for studying the cattle antibody response with high resolution and precision. Using three workflows, we processed 84, 96, and 8313 cattle B cells from which we sequenced 24, 31, and 4756 antibody heavy-light chain pairs, respectively. Each method has strengths and limitations in terms of the throughput, timeline, specialist equipment, and cost that are each discussed. Moreover, the principles outlined here can be applied to study antibody responses in other mammalian species.

Immunoparesis defined by heavy/light chain pair suppression in smoldering multiple myeloma shows initial isotype specificity and involves other isotypes in advanced disease.

Smoldering multiple myeloma (SMM) is an asymptomatic and biologically heterogeneous plasma cell disorder, with a highly variable clinical course. Immunoparesis, defined by total immunoglobulin measurements, has been shown to be an independent risk factor for progression to symptomatic disease. The heavy/light chain (HLC) assay allows precise measurement of the polyclonal immunoglobulin of the same isotype, enabling the evaluation of isotype-matched immunoparesis (IMI). In this study, we prospectively characterized immunoparesis, as determined by HLC measurements, in 53 SMM patients. Severe IMI was present in 51% of patients, while severe IP of uninvolved isotypes (HLC IP) was present in 39%. Most of the patients with severe HLC IP presented with severe IMI, but not the other way around. Isotype specificity of immune suppression was suggested by lower relative values of isotype-matched HLC pairs, both for IgG and IgA SMM. Severe IMI was associated with other risk factors for progression while patients with severe IMI and severe HLC IP showed an even higher risk profile. Both severe IMI and severe IgM HLC IP showed a significantly shorter time to progression. Finally, gene expression analysis demonstrated differences in the bone marrow microenvironment between patients with IMI and IMI plus HLC IP, with an increased expression of genes associated with cytolytic cells. In conclusion, our data supports isotype specificity of early immunoglobulin suppression mechanisms. While suppression of both involved and uninvolved isotypes is associated with risk of progression, the later appears to develop with more advanced disease and could be mediated by different mechanisms.

HLC Pair Suppression as a Risk Factor for Bacterial Bloodstream Infections and Early Mortality in Newly Diagnosed Intact Immunoglobulin Multiple Myeloma Patients.

Despite the outstanding progresses in Multiple Myeloma treatment options in the last decades, it remains an incurable disease nowadays. Infectious events are a complication due to an impaired immune system associated with MM, sometimes a life-threatening one, particularly on the first months after the diagnosis. Both the underlying disease and treatment can contribute to the infection risk, so a biomarker that assess this risk could be highly relevant for a more tailored management of the patient. The measurement of the heavy+light chain (HLC) pairs of immunoglobulins in serum allows the quantification of both the monoclonal component and the non-monoclonal immunoglobulin of the same isotype. This approach has demonstrated high sensitivity for the detection of the clonality and prognostic value for MM. HLC pair suppression itself has prognostic power and it has been proposed to be a reflection of the immune system’ attempt to control the tumor. In this study we evaluated the impact of the HLC pair suppression on the rate of bloodstream infections (BSI) and early death in 115 newly diagnosed MM patients. Twenty-one percent of the patients suffered a BSI in the first 6 months after diagnosis, of which 58% died within this period, accounting to 67% of the early deaths in global and highlighting the major impact of infections on MM patients in a “real world” setting. Severe HLC pair suppression identified patients with a higher risk of early BSI (HR: 6,97, p=0,009), and extreme HLC pair suppression together with BSI event and age >65 were independent risk factors for early death (p<0,001). Based on these factors, a stratification model was generated to allow identify patients at a higher risk of early death and poorer OS, with an apparently better performance than the ISS on the early death context. In conclusion, HLC pair suppression associates with both a higher risk of life-threatening early infection and early death in newly diagnosed MM patients. Patients older than 65 with extreme HLC pair suppression and BSI are at a high risk of early death, and thus patients presenting with these criteria have a very adverse prognosis.

Dysregulation of type I IFN and IL-4R signaling regulates the development of BCR VH and VL repertoire encoding anti-Sm autoantibodies in SLE

Abstract The most specific predictor for systemic lupus erythematosus (SLE) and lupus nephritis (LN) is the presence of anti-Smith (Sm) autoAb. Mechanisms leading to selection bias for anti-Sm development is unclear. We found that anti-Sm levels in SLE correlated with increased endogenous IFN-β and interferon-stimulated genes (ISGs) in combination with low (IL-4R) network genes. Using flow cytometry analysis of 47 SLE patients, we found that anti-Sm+ patients exhibited an increased ratio of IFN-β+ activated naive (aNAV) B cells and a decrease in IL-4R+ resting naive (rNAV) B cells. Using a combined 10X single-cell transcriptomics and BCR usage analysis, anti-Sm+LN+ISGhiIL4Rlopatients (Sm+) were compared with anti-Sm−LN− ISGloIL4Rhi patients (Sm−). BCR heavy chain analysis showed that while B cells from Sm+ patients exhibit greater usage of IGHV1-2, IGHV4-34, and IGHV4-39 genes, Sm− SLE patients exhibit greater usage of IGHV3-30, IGHV3-33, and IGHV4-31 genes. Preferential Ig light chain usage for ISGhi B cells was IGLV1-51, IGLV3-21, IGLV6-57, IGKV1-5, IGKV3-15 and IGKV 6-21. Dominant heavy-light chain pair usages for the Sm+ patients were IGHV4/IGKV1 and IGHV4/IGKV3. IGHV4-34, which is intrinsically autoreactive and the target of the 9G4 anti-idiotype Ab, was found to be expressed by the ISGhiIL4Rlo B cells derived from the same Sm+ patients. The results indicated that increased levels of ISGs and decreased levels of IL-4R pathway genes are the two dominant signatures and the underlying mechanisms leading to selection and activation of anti-Sm+ B cells. Verification that the B-cell IFN-β and IL-4R phenotypes are associated with race (AA vs. EA), repertoire (anti-Sm+) and disease (LN+) would lay the basis for targeted rational therapies.

Heavy/light chain assay in the monitoring of multiple myeloma

Serum protein (SPE) and immunofixation electrophoresis (IFE) have been extensively validated for the routine use of identifying, characterising and quantifying monoclonal proteins. However, accurate quantitation of IgA monoclonal proteins can be difficult when they migrate in to the β fraction, due to co-migration with transferrin and complement components. The heavy/light chain (HLC) immunoassay is an additional tool for measuring intact immunoglobulin monoclonal proteins. Therefore, we aimed to examine the clinical utility of the HLC assay for the disease monitoring of IgG and IgA multiple myeloma (MM) patients. A total of 177 samples from 30 MM patients (21 IgG and 9 IgA) were analysed retrospectively with median number of six follow up samples per patient (range 3-13). Serum free light chains (sFLC) and HLC were quantified using Freelite and Hevylite immunoassays. Details of M-protein concentration, β-globulin levels, total immunoglobulin levels and disease treatment response were obtained from the laboratory and patient information system. Passing-Bablok regression analysis was performed to compare (i) M-protein quantification with involved HLC (iHLC) and (ii) total immunoglobulin with summated HLC pairs for each immunoglobulin type (e.g., IgGκ+IgGλ). For 127 IgG MM samples, IgG iHLC levels showed a good correlation with SPE quantification (iHLC y=0.96x+4.9; r=0.917) and summated HLC showed a good correlation with total IgG concentration (summated HLC y=0.94x+5.74; r=0.91). In total, 95/127 (75%) IgG MM follow-up samples had an abnormal HLC ratio and 122/127 (96%) had a positive SPE, probably due to the lower sensitivity of HLC assay in detecting clonality in patients with IgG MM. Consistent with this, one patient assigned a very good partial response by International Myeloma Working Group criteria would be assigned a complete response based on HLC measurements. For 50 IgA MM samples, 42/50 (84%) had an abnormal HLC ratio. Conversely, 50/50 (100%) of M-proteins showed β fraction migration and were difficult to accurately quantify by SPE. Therefore, M-protein concentration and iHLC did not correlate as well in IgA MM (y=1.9x-8.4; r=0.8) compared to IgG MM. However, there was good correlation between total IgA and summated IgA HLC (IgAκ+IgAλ y=1.35x-0.33; r=0.95). Of the 8/50 (16%) IgA samples with a normal HLC ratio, 6/8 (75%) were consistent with the disease status being in complete remission. Interestingly, in one IgA MM patient, SPE and IFE were negative, but the serum FLC ratio and involved FLC were highly abnormal, consistent with the presence of light chain escape. Our data suggest HLC measurements could add value to the current disease monitoring of MM patients. In IgG MM patients, the M-protein level correlated well with HLC values. The HLC assay complements the serum FLC assay and is especially useful for monitoring of IgA MM patients who display M-proteins migrating in the β region on SPE.

QIP-MS: A specific, sensitive, accurate, and quantitative alternative to electrophoresis that can identify endogenous m-proteins and distinguish them from therapeutic monoclonal antibodies in patients being treated for multiple myeloma

Serum protein (SPE) and immunofixation electrophoresis (IFE) have been extensively validated for the routine use of identifying, characterising and quantifying monoclonal proteins. However, accurate quantitation of IgA monoclonal proteins can be difficult when they migrate in to the β fraction, due to co-migration with transferrin and complement components. The heavy/light chain (HLC) immunoassay is an additional tool for measuring intact immunoglobulin monoclonal proteins. Therefore, we aimed to examine the clinical utility of the HLC assay for the disease monitoring of IgG and IgA multiple myeloma (MM) patients.A total of 177 samples from 30 MM patients (21 IgG and 9 IgA) were analysed retrospectively with median number of six follow up samples per patient (range 3–13). Serum free light chains (sFLC) and HLC were quantified using Freelite and Hevylite immunoassays. Details of M-protein concentration, β-globulin levels, total immunoglobulin levels and disease treatment response were obtained from the laboratory and patient information system. Passing–Bablok regression analysis was performed to compare (i) M-protein quantification with involved HLC (iHLC) and (ii) total immunoglobulin with summated HLC pairs for each immunoglobulin type (e.g., IgGκ+IgGλ).For 127 IgG MM samples, IgG iHLC levels showed a good correlation with SPE quantification (iHLC y=0.96x+4.9; r=0.917) and summated HLC showed a good correlation with total IgG concentration (summated HLC y=0.94x+5.74; r=0.91). In total, 95/127 (75%) IgG MM follow-up samples had an abnormal HLC ratio and 122/127 (96%) had a positive SPE, probably due to the lower sensitivity of HLC assay in detecting clonality in patients with IgG MM. Consistent with this, one patient assigned a very good partial response by International Myeloma Working Group criteria would be assigned a complete response based on HLC measurements. For 50 IgA MM samples, 42/50 (84%) had an abnormal HLC ratio. Conversely, 50/50 (100%) of M-proteins showed β fraction migration and were difficult to accurately quantify by SPE. Therefore, M-protein concentration and iHLC did not correlate as well in IgA MM (y=1.9x–8.4; r=0.8) compared to IgG MM. However, there was good correlation between total IgA and summated IgA HLC (IgAκ+IgAλ y=1.35x–0.33; r=0.95). Of the 8/50 (16%) IgA samples with a normal HLC ratio, 6/8 (75%) were consistent with the disease status being in complete remission. Interestingly, in one IgA MM patient, SPE and IFE were negative, but the serum FLC ratio and involved FLC were highly abnormal, consistent with the presence of light chain escape.Our data suggest HLC measurements could add value to the current disease monitoring of MM patients. In IgG MM patients, the M-protein level correlated well with HLC values. The HLC assay complements the serum FLC assay and is especially useful for monitoring of IgA MM patients who display M-proteins migrating in the β region on SPE.

A Novel Risk-Model to Predict Time to First Treatment (TTT) in Chronic Lymphocytic Leukemia Based on Heavy+Light Chain Immunoparesis and Serum Free Light Chain Analysis: Results from the Israeli CLL Study Group

Introduction:Chronic Lymphocytic Leukemia (CLL) is frequently accompanied by immune dysregulation. Hypogammaglobulinemia is one of the most important immune defects encountered, and all three classes of immunoglobulins (IgG, A and M) can be involved. Recently, novel heavy+light chain (HLC) immunoassays have become available that quantify the light chain types of each immunoglobulin class (e.g. IgGk and IgGl). These assays are measured in pairs and provide information on the isotype produced by a tumour (the “involved” HLC), the non-clonal (“uninvolved”) HLC, and the ratio (e.g. IgGk/IgGl) - which indicates monoclonality. HLC assays have been shown to improve monitoring of plasma cell dyscrasias, but their role in CLL is yet to be studied.MethodsThis is a multi-center study performed in collaboration with the Israeli CLL Study Group and involved 10 medical centers.The cohort included 122 patients with CLL and 26 healthy controls. Baseline was defined as the time the blood sample was taken. Serum samples were analyzed for levels of IgG subclasses (IgG1, IgG2, IgG3, IgG4), heavy+light chains (HLC) (IgGκ, IgGλ, IgAκ, IgAλ, IgMκ or IgMλ) and free light chains (sFLC).HLC-pair suppression was defined as an abnormal HLC ratio and uninvolved HLC levels below the normal reference range (i.e. in g/L: IgGκ<3.84; IgGλ<1.91; IgAκ<0.57; IgAλ<0.44; IgMκ<0.19; IgMλ<0.12).HLC immunoparesis was defined as HLC isotype levels below the normal reference range, regardless of HLC ratio (i.e. HLC immunoparesis of at least 1 isotype indicates at least one of IgGκ, IgGλ, IgAκ, IgAλ, IgMκ or IgMλ below the normal reference range).Severe HLC-pair suppression or severe HLC immunoparesis was defined as a concentration of the uninvolved HLC or any HLC isotype that is suppressed by >50% below the normal reference range. The association of variables with time to first treatment (TTT) was performed with Cox proportional hazard model.ResultsCurrent analysis was performed on 105 CLL patients who had complete data available; median age was 68 years, 64% were males and Binet stage A, B, C, were 80%, 18.1% and 1.9% respectively. Median time from diagnosis to baseline measurements was 27 months (range 0-328 months).An abnormal sFLC ratio was identified in 70% (73/105) patients and summated k and λ concentrations (SFLC) were ≥70 mg/L in 18% of cases. An abnormal HLC ratio was present in 32% (34/105) patients, and 21% had HLC pair suppression of any 1 HLC isotype. HLC immunoparesis of 1, ≥2 and ≥3 isotypes was observed in 74 (70%), 58 (55%) and 36 (34%) of patients, respectively, with severe HLC immunoparesis identified in 40 patients (38%).Patients with IgG2 suppression were more frequently hospitalized due to infection with an Odds Ratio of 3.826 (p=0.031).In multivariate analysis, SFLC ≥70 mg/L and severe HLC immunoparesis were independently associated with TTT (HR 15.3, p<0.001; HR 80, p<0.001 respectively). Using these 2 variables, a risk-stratification model was constructed that separated CLL patients into 3 risk groups (with 0, 1 or 2 risk factors) with significantly different TTT (p<0.001, Figure 1). Patients with both risk factors (SFLC ≥70 mg/L and severe HLC immunoparesis) had the shortest TTT.ConclusionsThe findings presented here demonstrate that there is considerable potential for the use of HLC and FLC immunoassays to provide prognostic information in CLL. [Display omitted] DisclosuresTadmor:PFIEZER: Consultancy; JNJ: Consultancy; ABBVIE: Consultancy; NOVARTIS: Consultancy; ROCHE: Research Funding. Aviv:ABBVIE: Consultancy; ROCHE: Research Funding. Herishanu:ROCHE: Research Funding; JNJ: Consultancy; ABBVIE: Consultancy. Shvidel:ROCHE: Consultancy, Research Funding; ABBVIE: Consultancy, Research Funding; JNJ: Consultancy. Rahimi-Levene:ABBVIE: Consultancy. Ruchlemer:ABBVIE: Consultancy; JNJ: Consultancy. Fogl:The Binding Site Group Ltd: Employment. Polliack:ROCHE: Research Funding; ABBVIE: Consultancy. Magal:The Binding Site: Employment. Townsend:The Binding Site Group Ltd: Employment.

Cysteine-SILAC Mass Spectrometry Enabling the Identification and Quantitation of Scrambled Interchain Disulfide Bonds: Preservation of Native Heavy-Light Chain Pairing in Bispecific IgGs Generated by Controlled Fab-arm Exchange.

Bispecific antibodies (bsAbs) are one of the most versatile and promising pharmaceutical innovations for countering heterogeneous and refractory disease by virtue of their ability to bind two distinct antigens. One critical quality attribute of bsAb formation requiring investigation is the potential randomization of cognate heavy (H) chain/light (L) chain pairing, which could occur to a varying extent dependent on bsAb format and the production platform. To assess the content of such HL-chain swapped reaction products with high sensitivity, we developed cysteine-stable isotope labeling using amino acids in cell culture (SILAC), a method that facilitates the detailed characterization of disulfide-bridged peptides by mass spectrometry. For this analysis, an antibody was metabolically labeled with 13C3,15N-cysteine and incorporated into a comprehensive panel of distinct bispecific molecules by controlled Fab-arm exchange (DuoBody technology). This technology is a postproduction method for the generation of bispecific therapeutic IgGs of which several have progressed into the clinic. Herein, two parental antibodies, each containing a single heavy chain domain mutation, are mixed and subjected to controlled reducing conditions during which they exchange heavy-light (HL) chain pairs to form bsAbs. Subsequently, reductant is removed and all disulfide bridges are reoxidized to reform covalent inter- and intrachain bonds. We conducted a multilevel (Top-Middle-Bottom-Up) approach focusing on the characterization of both "left-arm" and "right-arm" HL interchain disulfide peptides and observed that native HL pairing was preserved in the whole panel of bsAbs produced by controlled Fab-arm exchange.

Prognostic Impact of Serum Heavy/Light Chain Pairs in Patients With Monoclonal Gammopathy of Undetermined Significance and Smoldering Myeloma: Long-Term Results From a Single Institution

Asymptomatic monoclonal gammopathies, such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM), are clinical conditions that usually precede symptomatic multiple myeloma. Therefore, risk stratification is crucial owing to the heterogeneous progression rate among these patients. In previous years, suppression of the uninvolved chain of specific heavy/light chain (HLC) pairs in serum has been identified as a new risk factor in MGUS. The aim of the present study was to investigate the prognostic effect of involved and uninvolved HLC pairs and HLC ratios on progression in a series of patients with MGUS and SMM. All specific serum HLC pairs were measured in 114 patients diagnosed with SMM (n= 27) and MGUS (n= 87) from 1983 to 2003. Also, the HLC ratios were calculated. Progression to symptomatic multiple myeloma was observed in 13 patients (8 with SMM and 5 with MGUS). The risk of progression was 6 times greater in those with SMM (P= .001) and 4 times greater for those with the IgA isotype (P= .01). The suppression of any IgM isotypes (IgMκ or IgMλ) in patients with IgA or IgG gammopathy or any IgA isotypes (IgAκ or IgAλ) in patients with IgG or IgM gammopathy was associated with a shorter time to progression to symptomatic gammopathy (P= .001 and P= .03, respectively). On multivariate analysis, the evolving pattern and suppression of any IgM HLC pair remained significant. HLC ratios could be a valuable tool in the risk stratification of patients with SMM and MGUS, especially patients with IgG isotypes.

Heavy/Light Chain Quantification Identifies Clonal Plasma Cell Disease in Patients with AL Amyloidosis and Normal Serum Free Light Chain Ratio

Background: Serum and urine immunofixation electrophoreses (SIFE/UIFE) are routinely used for detection of clonal immunoglobulins (Ig) in AL amyloidosis. Serum free light chain (FLC) assays (Freelite®, The Binding Site Ltd., Birmingham, UK) have significantly improved the management of patients with AL amyloidosis by providing quantitative measure for the detection and monitoring of clonal plasma cell disease. However, up to 20% of patients with AL amyloidosis may have uninformative serum free light chain values.Objective: To assess the quantitative potential of serum Heavy/Light Chain (HLC) pairs (Hevylite®, The Binding Site Ltd., Birmingham, UK) assay in identification of clonal plasma cell disease in AL amyloidosis.Methods: One hundred and ninety-nine untreated patients with AL amyloidosis were included in this study. Patients with multiple myeloma or B cell lymphoproliferative diseases associated AL amyloidosis were excluded. Serum sampleswere obtained at initial evaluation and stored at -20°C. SIFE/UIFE were performed at the time of sample collection. HLC pairs were assessed by the Hevylite® assay. HLC κ/λ normal ratios (HLCR) were: 1.12-3.21 for IgG κ/λ; 0.78-1.94 for IgA κ/λ; and 1.18-2.74 for IgM κ/λ. FLCs were assessed by the Freelite® assay; FLC κ/λ normal ratio (FLCR) was 0.26-1.65. In 103 cases, FLC testing was performed at the time of sample collection; 96 cases were tested at The Binding Site. Vital status of patients was obtained from either medical records or Social Security Death Index. Follow-up ended in June 2014.Results: An abnormal HLCR was found in 74 (37.2%), an abnormal FLCR in 163 (81.9%), and SIFE/UIFE positivity in 187 (94%) of 199 patients with AL amyloidosis. Of 36 patients with a normal FLCR, 23 (63.9%) were noted with an abnormal HLCR compared to 51 (31.3%) patients in an abnormal FLCR group (P = 0.001). In total 186/199 (93.5%) patients with AL amyloidosis had abnormalities in either HLCR or FLCR, compared to 187/199 (94%) of patients who were SIFE/UIFE+ (Table 1). The combined use of both FLCR and HLCR yielded quantifiable information in 93.5% of cases; the use of both tests in combination with SIFE/UIFE identified plasma cell clonality in 100% of patients. Seventy-two cases presented with an abnormal HLCR for a single isotype and 2 in multiple Ig isotypes. In all cases, involved LC type of abnormal HLCR matched LC type identified by SIFE/UIFE. None of 12 cases that were negative on the SIFE/UIFE presented with an abnormal HLCR, however, all showed abnormalities in FLCR.Table 1Comparative efficiency of FLCR, HLCR and Serum/Urine Immunofixation in AL Amyloidosis patients.SIFE/UIFE+ (n=187)SIFE/UIFE- (n=12)HLCR+/FLCR+51 (27.2%)-HLCR+/FLCR-23 (12.3%)-HLCR-/FLCR+100 (53.5%)12 (100%)HLCR-/FLCR-13 (7%)-Overall survival was similar in patients with and without abnormal HLCR (Log rank p=0.092; Figure 1), whereas patients with an abnormal FLCR had a significantly inferior overall survival compared to those with a normal FLCR (Log rank p=0.027; Figure 2). Combined use of both HLCR and FLCR demonstrated a trend toward superior overall survival in a group of patients with an abnormal HLCR / normal FLCR (Wilcoxon p=0.037; Log rank p=0.107; Figure 3).Conclusions: The Hevylite® assay provided information in addition to other laboratory tests for clonal plasma cell disease in AL amyloidosis. The combined use of the HLCR and FLCR provided quantifiable information in 93.5% of patients. The use of both assays in combination with SIFE/UIFE detected clonal disease in all patients. HLCR has potential to quantify clonal disease in patients with uninformative FLCR results. An abnormal HLCR was not predictive of overall survival, while an abnormal FLCR was, in this series of patients. Combined use of HLCR and FLCR could be beneficial in prognostication of outcome in AL amyloidosis. [Display omitted] [Display omitted] [Display omitted] DisclosuresMcConnell:The Binding SIte: Employment. O'hara:The Binding Site: Employment.

Usefulness of Whole-Body Diffusion-Weighted MRI (WB-MRI) With Apparent Diffusion Coefficient (ADC) In The Differentiation of Monoclonal Gammopathies

e90 on the relationship between heavy/light chain (HLC) analysis, immunofixation electrophoresis (IFE), serum free light chain (sFLC) analysis, and multicolor flowcytometry (MFC) for response evaluation in patients with MM. AIM: To compare HLC immunoassays with conventional methods of disease evaluation. Patients AND Methods: Four hundred and sixty five samples from 128 patients with IgG and IgA MM after treatment were retrospectively analyzed with HLC immunoassays. Myeloma responses were assessed serially after induction therapy according to the IMWG response criteria. Results were compared to SPE, IFE, sFLC assay, and MFC analysis. MFC negativity was defined as < 10-4 MMPCs. HLC-pair suppression was defined as the uninvolved immunoglobulin of the same isotype <50% of the normal range. Overall survival (OS) and progression-free survival (PFS) were calculated by the KaplaneMeier. Results: The number of patient samples at various responses were: sCR, n 1⁄4 99; CR, n 1⁄4 31; VGPR, n 1⁄4 160; PR, n 1⁄4 88; and at diagnosis, n1⁄487. Normal HLC ratio (HLCR) was obtained at sCR, CR, and VGPR in 83 (83%), 29 (93%), and 91 (57%) cases, respectively. In the sera from patients CR, abnormal HLCR and FLC ratio were seen at rates of 9.7% and 22.3%, respectively, and no sera had abnormal value in both assays simultaneously. Thirty-three percent of MFC negative samples were HLCR abnormal and 80% of HLCR normal samples were MFC positive among the patients VGPR. Patients with a normal HLCR had fewer MM-PCs than those with an abnormal HLCR (median 1x10-3.42 vs. 1x10-3.04, respectively, P1⁄40.01). Shorter OS were shown in patients with highly abnormal HLCR (HLCR; 100 or 0.01) at diagnosis than those with normal or less abnormal (not reached vs. not reached, P 1⁄4 0.048). Longer OS were observed in patients who achieved HLCR normalization at best response than in those who did not (not reached vs. 119.7 months, P 1⁄4 0.0007) (Figure 2B). Presence of isotype-matched HLC pair suppression at best response, not at diagnosis, was also associated with poorer OS (not reached vs. not reached, P 1⁄4 0.002). Conclusion: The findings suggest the potential usefulness of HLC immunoassays for monitoring response and prognostication in patients with myeloma. Presence of an abnormal HLCR at best response and HLC pair suppression were also associated with poorer survival.

Relationship of differences in immunoglobulin heavy/light chain pairs (Hevylite), selected laboratory parameters and stratification systems in different immunochemical types of multiple myeloma.

Advances in the diagnosis and treatment of multiple myeloma (MM), place increasing demands on accurate stratification of patients as the starting point for optimal individualized therapy. The present study focused on assessing the association between HLC levels and the HLC-r to parameters of MM activity, prognosis and tumor mass volume.The objective was to assess the correlation of immunoglobulin (Ig), heavy/light chain (HLC) pairs (IgG-κ and-λ, IgA-κ and -λ HLC) and the ratio of monoclonal involved-HLC (i-HLC) to polyclonal uninvolved (u-HLC) Ig concentrations assessed by the Hevylite(TM) method with the free light chain κ/λ ratio (FLC-r), selected prognostic laboratory parameters i.e. Hb, platelets, albumin, β2-microglobulin (β2-M), Ca, lactate dehydrogenase (LDH), creatinine and the Durie-Salmon (D-S) and International Staging System (ISS), stages (1-3) for MM. Hevylite assays were done on the sera of 132 MM patients at the time of diagnosis (IgG 94, IgA 38). HLC-r was calculated in the case of i-HLC-κ from the i-HLC-κ/u-HLC-λ ratio and for i-HLC-λ from the i-HLC-λ/u-HLC-κ ratio. D-S and ISS stages were evenly distributed. Md IgG-κ HLC-r was 64.8 (2.7-2222) and of IgG-λ HLC-r 49.6 (0.7-465.1), in the case of IgA-κ, Md HLC-r was 408.9 (3.4-3966) and for IgA-λ HLC-r the Md was 180.0 (0.1-3110). Normal levels of HLC pairs and HLC-r did not always rule out the diagnosis of MM. HLC-r correlated with FLC-r in IgG (r = 0.244, P = 0.018), but not in the IgA type. For IgG, HLC-r values were significantly different in patients with abnormal vs normal levels of Hb (P < 0.0001), albumin (P < 0.043), β2-M (P < 0.0001) and creatinine (P = 0.034) but not thrombocyte count, Ca or LDH. For the IgA isotype, we found a significant difference in HLC-r values only for thrombocyte count (P = 0.026) and β2-M (P = 0.016) but not for Hb, albumin, Ca, LDH or creatinine. For the IgG isotype there was a significant relationship of HLC-r index to stages 1-3 (P = 0.038) and substage A vs B (P = 0.048) according to D-S, and with high significance to stages 1-3 according to ISS (P = 0.005) and between stages 1 vs 3 (P = 0.001). For the IgA isotype, we found significant differences in HLC-r only between stages 1-3 (P = 0.025) according to D-S but not in the case of ISS. There were no significant correlations between i-HLC Ig levels and D-S or ISS stages in both IgG-κ and λ and IgA-κ and λ. Exceptions were significant differences for stages 1 vs 3 (P = 0.012) and 2 vs 3 (P = 0.017) for the IgG-λ isotype. There were no correlations of the HLC-r and u-HLC levels for either D-S or ISS stratifications in all HLC isotypes. We found a significant positive contribution of HLC-r using the i-HLC/u-HLC ratio even in the case of i-HLC-λ i.e. i-HLC-λ/u-HLC-κ. Variable results for the relationship of important laboratory parameters and D-S and ISS stratifications (stage 1-3) to HLC-r values in IgG and IgA isotypes make separate interpretation of the Hevylite method results necessary in clinical practice.

Immunoglobulin Heavy/Light Chain Measurements Provide Prognostic Information in Multiple Myeloma Patients Undergoing Autologous Stem Cell Transplantation

Introduction. Both reduction in M-protein and reconstitution of the immune system are associated with increased survival in multiple myeloma (MM) patients receiving an autologous stem cell transplant (ASCT). Heavy/light chain (HLC) pair analysis allows discrimination between Igκ and Igλ and as a result allows measurement of both the monoclonal involved and polyclonal uninvolved HLC pair. Abnormal HLC ratios and suppression of the uninvolved HLC pair correlate with shorter overall survival in MM patients. We evaluated HLC analysis for predicting progression free survival in MM patients receiving an ASCT.Methods. We prospectively evaluated 28 intact immunoglobulin MM (IIMM) patients (12 Female, 16 Male; 20 IgG, and 8 IgA; median age: 54 years; range 37-67 years) who received ASCT subsequent to high dose melphalan. Median follow up time from ASCT was 391 days (range: 61-603 days). Patient responses were assigned according to international response criteria guidelines prior to ASCT. Serum samples were analysed with Hevylite®, prior to ASCT (median: 2 days; range 0-77 days) and following ASCT (median: 50 days; range 22-119 days), on a SPAPLUS turbidimeter. HLC concentrations and ratios were compared to normal ranges (IgGk: 3.85-12.07 g/L; IgGl: 1.91-6.74 g/L; IgAk: 0.57-2.08 g/L; IgAλ: 0.44-2.04 g/L HLC ratio reference range: IgGk /IgGλ: 1.12-3.21; IgAk / IgAλ: 0.78-1.94). Progression free survival (PFS) was estimated by Kaplan Meier analysis and compared using a log rank test (Graphpad Prism).Results. Prior to ASCT, 1 patient achieved CR, 13 obtained VGPR, 9 obtained PR, 2 obtained (MR), 2 PD and 1 patient had no response assigned. An abnormal HLC ratio (18/28 patients) prior to ASCT was associated with significantly poorer PFS (17.1 months versus median not reached p=0.036). In comparison, achievement of ≥VGPR (14/28 patients) prior to ASCT was not associated with increased PFS (p=0.74). Addition of a normal HLC ratio to the assignment of response (in 8/14 patients achieving a ≥VGPR) added significant prognostic information and was significantly associated with improved PFS (median not reached versus 11.5 months, p=0.025). Suppression of the uninvolved HLC pair only (concentration below the normal reference range) prior to ASCT (15/28 patients) had no significant impact on PFS (p=0.32). Post-ASCT, an abnormal HLC ratio (11/28 patients) was not significantly associated with poorer PFS (p=0.18). However, suppression of the uninvolved HLC pair (6/28 patients) was significantly associated with poorer PFS (10.6 months versus median not reached p=0.013).Discussion. HLC analysis may provide early prognostic markers for PFS in MM patients receiving an ASCT. Warranting larger patient cohort studies, this preliminary data indicates that a normalised HLC ratio prior to ASCT and the absence of HLC paired suppression post-ASCT may be significant markers of improved PFS, adding prognostic information to current response criteria.Acknowledgments: This work was partially supported by the National Science Fund (D02-35/2009). DisclosuresGuenova:Novartis Pharma Sevices Bulgaria: Consultancy, Research Funding, Speakers Bureau; Roche Bulgaria: Consultancy, Research Funding, Speakers Bureau; Amgen Bulgaria: Consultancy, Research Funding, Speakers Bureau; Sanofi-Aventis Bulgaria: Consultancy, Research Funding, Speakers Bureau.

Comparison of IgA Heavy/Light Chain Immunoassays with Capillary Zone Electrophoresis and Total Immunoglobulin Measurements

Introduction: Capillary zone electrophoresis (CZE) and measurement of total immunoglobulins (total Ig) are standard techniques for the identification and quantification of monoclonal immunoglobulins (M-Ig). Heavy/light chain (HLC) pair analysis allows discrimination between Igκ and Igλ and as a result allows measurement of both the monoclonal involved and polyclonal uninvolved HLC pair. We compared measurement of M-Ig by CZE and total Ig to HLC levels.Methods: 93 samples from 8 patients with IgA intact immunoglobulin multiple myeloma (MM) were analysed. M-Ig was measured using CZE Sebia Capillarys 2 system, total IgA (tIgA) and IgAκ and IgAλ HLC concentrations on a SPAPLUS analyser. IgA HLC levels were measured with Hevylite® and compared to published normal ranges (IgAκ (g/L): 0.57-2.08, IgAλ (g/L): 0.44-2.04, IgAκ/IgAλ: 0.78-1.94). Passing and Bablok fit analysis was used to determine correlation between the assays.Results: Measurement of the involved HLC pair (iHLC) (median: 12.45g/L; range: 0.64-44.71g/L) compared well with CZE (n=34; median: 11.04g/L; range: 1.24-37.71g/L.; y= 1.2x + -2.65, R2= 0.94). Measurement of iHLC (median: 0.88. range: 0.05-21.55g/L) also compared well with tIgA measurement (n=65 median=1.44g/L; range: 0.227-21.11g/L, y=0.85x + -0.26, R2=0.98). Percent changes in iHLC concentrations from baseline through treatment compared well with CZE (n=28; y=1.59x + 0.15 R2=0.93) and tIgA (n=57; y=1.06x + 0.01, R2=0.96). Of the 34 samples with quantifiable M-protein by CZE, 32(94%) had an abnormal HLC ratio. The two discrepant samples were follow up samples from the same patient, where HLC normalised alongside total IgA entering the normal reference range. In addition, all 15 samples (15/65; 23%) where tIgA concentration was above the normal reference range all had abnormal HLC ratios. M-Ig was not detected by CZE in 48/82 (57%) samples, 46/48 of the samples (96%) had a normalised HLC ratio. In 38/65 (58%) samples, tIgA concentrations were within the normal reference range, and 34 (90%) had a normalised HLC ratio. HLC ratio for all patient samples normalised in subsequent samples following treatment.Conclusion: The measurement of M-Ig is comparable between Hevylite® and both CZE and tIgA. The presented data indicate that Hevylite® is a more sensitive test for detecting residual disease and warrants prospective studies on larger cohorts of patients.Acknowledgments:This work was partially supported by the National Science Fund (D02-35/2009). DisclosuresGuenova:Novartis Pharma Sevices Bulgaria: Consultancy, Research Funding, Speakers Bureau; Roche Bulgaria: Consultancy, Research Funding, Speakers Bureau; Amgen Bulgaria: Consultancy, Research Funding, Speakers Bureau; Sanofi-Aventis Bulgaria: Consultancy, Research Funding, Speakers Bureau.

Prognostic Impact Of Serum Heavy/Light Chain Pairs In Patients With MGUS and Smoldering Myeloma: Long-Term Results From a Single Institution

BackgroundAsymptomatic monoclonal gammopathies, such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM), are clinical conditions that usually precede symptomatic multiple myeloma (MM). However, risk stratification is crucial due to the heterogeneous progression rate among patients with these entities, particularly nowadays when chemoprevention trials are encouraged in high risk patients. In this sense, biomarkers and prognostic index based on tumoral load, M-protein behaviour (evolving vs. non-evolving) and/or immunological status have been developed. In MGUS, serum heavy/light chain (HLC) pairs have allowed the identification of abnormal ratios of involved and non-involved immunoglobins for each specific heavy-chain isotype (IgG-kappa/IgG-lambda, IgA-kappa/IgA-lambda and IgM-kappa/IgM-lambda). The aim of the present study was to investigate the prognostic impact on progression of the isotype-specific suppression of the uninvolved HLC-pair in a series of patients with MGUS and SMM with long follow up. Patients and MethodsWe retrospectively evaluated 114 patients (median age 61 years; 44M/70F) with SMM (35) and MGUS (79). Median follow up for alive patients was 13 years (range 3 to 27 years). Only 12 patients with SMM accomplished both diagnostic criteria for high risk (bone marrow plasma cell infiltration ≥10% and M-protein ≥30 g/L). Heavy isotype distribution was mainly IgG (71%), IgA (15.8%) and IgM (11.4%); only two patients (1.8%; 1 MGUS and 1 SMM) had only light-chain M-protein. Median bone marrow plasma cell infiltration was 4% and 15% in MGUS and SMM, respectively. 13 patients (11.4%) showed an “evolving” pattern of their serum M-protein.All patients had an available initial frozen serum sample. Three serum HLC pairs (IgG, IgM and IgA) were evaluated by immunonephelometry (Hevylite; gently provided by The Binding Site, Ltd); HLC kappa/lambda ratio were calculated for each one. Normal values were obtained from 95% normal values reported in healthy donors. ResultsProgression to malignant symptomatic gammopathies was observed in 15 patients (13.2%; 9 SMM and 6 MGUS), mainly to MM (13) with exception of AL amyloidosis and Waldenström's macroglobulinemia in one case each. Risk of progression was 4 times higher in patients with SMM than in those with MGUS (p=0.009), being 8 times higher for high risk SMM (p<0.001). An “evolving” pattern of serum M-protein (p=0.004; HR 4.93, IC 95% 1.7 to 14.8) and a serum M-protein greater than 15 g/L (p=0.01; HR 5.2, IC 95% 1.5 to 18.5) were also associated with higher risk of progression.Patients with SMM had lower IgG-lambda, IgA-lambda and HLC IgM ratio (p<0.05) than patients with MGUS. IgG HLC ratio was positively associated with bone marrow plasma cell infiltration (r=0.555; p<0.0001). Normal or lower than normal HLC ratios for IgG and IgM were associated with longer time to progression to symptomatic disease (TTP) than higher values (p=0.008 and p=0.013, respectively) (Figure 1). This difference in HLC IgG ratio were even more evident in SMM patients (p=0.002), where only 1 patient with normal or low HLC IgG ratio has progressed. There was also a trend for HLC IgM ratio in this sense (p=0.08). In a multivariate analysis taking into account SMM vs. MGUS diagnosis, “evolving” pattern and HLC IgM and IgG ratio, only the first three variables remained statistically significant for predicting TTP. Suppression of uninvolved HLC pair was more evident in IgG patients, both MGUS and SMM. Hence, any reduction in IgA-kappa or IgA-lambda isotypes was associated with higher risk of progression (p=0.04) (Figure 2). [Display omitted] [Display omitted] ConclusionHLC ratios seem to be a valuable tool in the risk stratification of patients with SMM and MGUS. Suppression of the uninvolved isotype (i.e. uninvolved IgA or HLC IgM ratio in IgG MGUS or SMM) is particularly interesting since this phenomenon has not been previously recognized. The picture of differential clonal suppression of heavy-chain isotypes across risk groups is an issue to be prospectively explored. Disclosures:Fernández de Larrea:The Binding Site Ltd: Consultancy, Honoraria.

Mutation of Y407 in the CH3 domain dramatically alters glycosylation and structure of human IgG

Antibody engineering is increasingly being used to influence the properties of monoclonal antibodies to improve their biotherapeutic potential. One important aspect of this is the modulation of glycosylation as a strategy to improve efficacy. Here, we describe mutations of Y407 in the CH3 domain of IgG1 and IgG4 that significantly increase sialylation, galactosylation, and branching of the N-linked glycans in the CH2 domain. These mutations also promote the formation of monomeric assemblies (one heavy-light chain pair). Hydrogen-deuterium exchange mass spectrometry was used to probe conformational changes in IgG1-Y407E, revealing, as expected, a more exposed CH3–CH3 dimerization interface. Additionally, allosteric structural effects in the CH2 domain and in the CH2–CH3 interface were identified, providing a possible explanation for the dramatic change in glycosylation. Thus, the mutation of Y407 in the CH3 domain remarkably affects both antibody conformation and glycosylation, which not only alters our understanding of antibody structure, but also reveals possibilities for obtaining recombinant IgG with glycosylation tailored for clinical applications.

Isotype-Specific Heavy/Light Chain (HLC) Suppression as a Predictor of Myeloma Development in Monoclonal Gammopathy of Undetermined Significance (MGUS).

Abstract 1788Poster Board I-814 BackgroundClinically available prognostic factors for progression of MGUS to myeloma include the size of the M-spike, heavy chain isotype, and free light chain (FLC) K/L ratio. The combined use of these variables provides approximately a 10-fold difference in risk for progression. The highest risk group, however, still has only a 27% twenty year risk of progression. Therefore, additional predictors of progression are needed. Suppression of uninvolved immunoglobulins has been an inconsistent predictor of progression. The novel Hevylite” assay measures concentrations of each isotype-specific immunoglobulin heavy light chain (HLC) pair (i.e., IgG kappa, IgG lambda, IgA kappa, IgA lambda) and enables us for the first time to determine if suppression of isotype-specific Ig of the opposite HLC is occurring and if it is predictive of progression of MGUS to myeloma. We studied the prognostic value of isotype specific HLC suppression of uninvolved immunoglobins as a predictor of progression in MGUS. Methods: Total immunoglobulins were quantitated by immunonephelometry, and suppression was defined as concentrations less than the lower limit of the reference range. HLC concentrations were quantitated with Hevylite reagents (The Binding Site, Ltd). The antibodies used in this investigational nephelometric assay span epitopes in the junctional areas between the HLC constant regions. Based on reference ranges for GK (407-1130 mg/dL), GL (188-583 mg/dL), AK (55-292 mg/dL), and AL (39-249 mg/dL) that we derived from 276 normal samples, we compared the proportion of patients who had isotype specific suppression of the opposite HLC pair among 30 patients with MGUS who progressed to myeloma versus 36 patients who had not progressed over a similar follow-up period. To confirm the relationship of isotype specific HLC pair suppression compared to suppression of uninvolved immunoglobulins of other heavy chain classes, we tested a second cohort of stored samples from 50 IgG and 14 IgA MGUS patients prior to the development of MM. ResultsIn the initial samples from 36 IgG MGUS patients who had stable disease, 6% had suppression of uninvolved intact immunoglobulins of IgA and/or IgM, while 22% had isotype specific suppression of the opposite HLC pair (i.e., IgG lambda suppressed in IgG kappa MGUS, and vice versa). In contrast, in baseline samples from 30 IgG MGUS patients who eventually progressed to myeloma or related malignancy, 7% had suppression of IgA and/or IgM, while 40% had isotype specific suppression of the opposite IgG HLC pair. Thus, HLC pair suppression was more frequent than suppression of Igs from other heavy chain classes, and MGUS patients who eventually progressed had a 2-fold higher rate of isotype specific HLC pair suppression than stable MGUS patients. These findings were confirmed independently on a second cohort of patients who progressed to myeloma 5-10 years after blood collection. In samples from 50 IgG MGUS patients, 42% had suppression of Igs of other heavy chain classes, while 80% had suppression of the isotype specific HLC by the Hevylite assay. In the last samples prior to progression, the suppression of heavy chain classes increased to 60%, while isotype specific HLC suppression rose to 90%. Interestingly, the 14 IgA patients had equivalent suppression of gamma or mu heavy chain classes as compared to the HLC pairs, with 50% of the patients having suppression in the initial sample and 79% in the last sample prior to MM. ConclusionOur data suggest that suppression of the opposite IgG HLC pair is a predictor of patients destined for progression. Isotype specific HLC pair suppression of this nature is more frequent in these patients (more sensitive) than suppression of other heavy chain classes. We were not able to demonstrate a similar effect in IgA MGUS. Larger studies are underway to determine if HLC pair suppression is independent of M-spike quantitation, heavy chain isotype, and FLC ratio. Biologically, our findings suggest that clonal IgG plasma cells are able to suppress other IgG producing non-clonal plasma cells far more effectively than IgA or IgM secreting cells, and this occurs more frequently with cells that will eventually undergo malignant transformation. DisclosuresBradwell:The Binding Site Group Ltd: Equity Ownership.

Anti-Inflammatory Activity of Human IgG4 Antibodies by Dynamic Fab Arm Exchange

Antibodies play a central role in immunity by forming an interface with the innate immune system and, typically, mediate proinflammatory activity. We describe a novel posttranslational modification that leads to anti-inflammatory activity of antibodies of immunoglobulin G, isotype 4 (IgG4). IgG4 antibodies are dynamic molecules that exchange Fab arms by swapping a heavy chain and attached light chain (half-molecule) with a heavy-light chain pair from another molecule, which results in bispecific antibodies. Mutagenesis studies revealed that the third constant domain is critical for this activity. The impact of IgG4 Fab arm exchange was confirmed in vivo in a rhesus monkey model with experimental autoimmune myasthenia gravis. IgG4 Fab arm exchange is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 antibodies.

A B-cell receptor-specific selection step governs immature to mature B cell differentiation.

Seventy percent of peripheral immature conventional (B2) B cells fail to develop into mature B cells. The nature of this cell loss has not been characterized; the process that governs which immature B cells develop into long-lived peripheral B cells could be either stochastic or selective. Here, we demonstrate that this step is in fact selective, in that the fate of an immature B cell is highly dependent on its Ig receptor specificity. A significant skewing of the B cell receptor repertoire occurs by the time cells enter the mature B cell fraction, which indicates that there is selection of only a minority of immature B cells to become mature B cells. Because only a few heavy-light chain pairs are enhanced of the diverse available repertoire, we favor the idea that selection is positive for these few heavy-light chain pairs rather than negative against nearly all others. Because most immature B cells are lost at this transition, this putative positive selection event is likely to be a major force shaping the mature B cell receptor repertoire available for adaptive immune responses.