CD52 Expression Patterns in Normal and Neoplastic B-Cells: Myeloma Is an Unlikely Target for Single Agent Alemtuzumab Therapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4883-4883
Author(s):  
A. C. Rawstron ◽  
G. Laycock-Brown ◽  
F. E. Davies ◽  
R. G. Owen ◽  
P. Hillmen ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Single Agent ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Normal Controls

Abstract Alemtuzumab is a highly effective monoclonal antibody therapy for some B-cell disorders, and has been suggested as a possible therapeutic agent for treatment of myeloma. Monoclonal antibody therapeutic efficacy is closely associated with the expression level of the therapeutic target, as demonstrated by the lack of efficacy of single-agent rituximab in CLL. However, there are conflicting reports about the expression levels of CD52, the target for alemtuzumab, in plasma cell disorders. The aim of this study is to assess a large series of cases of plasma cell and B-cell disorders, utilising a standard approach to allow comparison of the target molecule. Plasma cells were assessed from patients with myeloma at presentation or relapse (n=106), monoclonal gammopathy of undetermined significance (MGUS, n=34), and from normal controls (n=19). In addition, B-cells were assessed from patients with chronic lymphocytic leukaemia (CLL, n=87), diffuse large B-cell lymphoma (DLBCL, n=10), follicular lymphoma (FCL, n=9), Waldenstroms macroglobulinaemia (WM, n=20), and also from normal bone marrow (n=37). Normal and neoplastic B-cells showed expression of CD52 (>20% of cells above the CD3-control levels) in all patients except for 1/10 DLBL. B-CLL and WM are known to show responses to single-agent alemtuzumab therapy, and these two disorders had the highest levels of expression. In contrast, B-progenitor cells in normal bone marrow are unaffected by alemtuzumab, and proliferate during alemtuzumab treatment in CLL patients. The levels of CD52 expression by normal B-progenitors were 3-fold lower than CLL/WM. In DLBL and FCL, the B-cells showed very similar levels of CD52 expression to normal B-progenitors, on average 2.8-fold lower than CLL. All plasma cells, whether neoplastic (CD19− or CD19+56+) or normal (CD19+56−), showed much lower levels of expression than normal and neoplastic B-cells. Plasma cell CD52 expression was detectable in 68% of normal controls (13/19), 50% of MGUS patients (17/34), and only 43% of myeloma patients (46/106). Expression was uni-modal in all cases. There was significantly lower expression of CD52 by myeloma plasma cells than by their normal counterparts (median 2.4-fold decrease, P=0.03). Neoplastic plasma cell CD52 expression showed a high degree of inter-patient variation, but fewer than 10% of myeloma patients (7/106) had CD52 expression at a similar level to CLL cells. Neoplastic plasma cell CD52 expression was approximately 6-fold lower than that of normal B-progenitors, and nearly 20-fold lower than that of CLL cells. In summary, CD52 expression is not detectable above control levels in a significant proportion of myeloma patients. In cases with detectable CD52 expression, the antigen is at a much lower level than is present on normal B-progenitors, which actively proliferate during alemtuzumab therapy. The risk of immunosuppression due to depletion of residual normal B/T-cells must also be considered. As alemtuzumab efficacy appears to correlate with CD52 expression levels, myeloma is highly unlikely to respond to alemtuzumab as a single agent except in rare cases. However, alemtuzumab is more likely to be effective in the IgM immunosecretory disorders which show strong CD52 expression.

scholarly journals Complement receptors regulate differentiation of bone marrow plasma cell precursors expressing transcription factors Blimp-1 and XBP-1

2005 ◽  
Vol 201 (6) ◽  
pp. 993-1005 ◽  
Author(s):  
Dominique Gatto ◽  
Thomas Pfister ◽  
Andrea Jegerlehner ◽  
Stephen W. Martin ◽  
Manfred Kopf ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Cell Activation ◽  
Plasma Cells ◽  
Antibody Responses ◽  
Complement Receptors ◽  
Essentially Normal ◽  
Bone Marrow Plasma

Humoral immune responses are thought to be enhanced by complement-mediated recruitment of the CD21–CD19–CD81 coreceptor complex into the B cell antigen receptor (BCR) complex, which lowers the threshold of B cell activation and increases the survival and proliferative capacity of responding B cells. To investigate the role of the CD21–CD35 complement receptors in the generation of B cell memory, we analyzed the response against viral particles derived from the bacteriophage Qβ in mice deficient in CD21–CD35 (Cr2−/−). Despite highly efficient induction of early antibody responses and germinal center (GC) reactions to immunization with Qβ, Cr2−/− mice exhibited impaired antibody persistence paralleled by a strongly reduced development of bone marrow plasma cells. Surprisingly, antigen-specific memory B cells were essentially normal in these mice. In the absence of CD21-mediated costimulation, Qβ-specific post-GC B cells failed to induce the transcriptional regulators Blimp-1 and XBP-1 driving plasma cell differentiation, and the antiapoptotic protein Bcl-2, which resulted in failure to generate the precursor population of long-lived plasma cells residing in the bone marrow. These results suggest that complement receptors maintain antibody responses by delivery of differentiation and survival signals to precursors of bone marrow plasma cells.

scholarly journals A monoclonal antibody that recognizes B cells and B cell precursors in mice.

1981 ◽  
Vol 153 (2) ◽  
pp. 269-279 ◽  
Author(s):  
R L Coffman ◽  
I L Weissman
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Bone Marrow Cells ◽  
Cell Lineage ◽  
Hematopoietic Stem ◽  
B Cell Precursors

The monoclonal antibody, RA3-2C2, appears to be specific for cells within the B cell lineage. This antibody does not recognize thymocytes, peripheral T cells, or nonlymphoid hematopoietic cells in the spleen or bone marrow. Nor does it recognize the pluripotent hematopoietic stem cells, the spleen colony-forming unit, All sIg+ B cells and most plasma cells are RA3-2C2+. In addition, approximately 20% of nucleated bone marrow cells are RA3-2C2+ but sIg-. This population contains B cell precursors that can give rise to sIg+ cells within 2 d in vitro.

scholarly journals High levels of circulating triiodothyronine induce plasma cell differentiation

2013 ◽  
Vol 220 (3) ◽  
pp. 305-317 ◽  
Author(s):  
Flavia Fonseca Bloise ◽  
Felipe Leite de Oliveira ◽  
Alberto Félix Nobrega ◽  
Rita Vasconcellos ◽  
Aline Cordeiro ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Cell Physiology ◽  
White Pulp ◽  
Plasma Cell Differentiation ◽  
Circulating Levels

The effects of hyperthyroidism on B-cell physiology are still poorly known. In this study, we evaluated the influence of high-circulating levels of 3,5,3′-triiodothyronine (T3) on bone marrow, blood, and spleen B-cell subsets, more specifically on B-cell differentiation into plasma cells, in C57BL/6 mice receiving daily injections of T3for 14 days. As analyzed by flow cytometry, T3-treated mice exhibited increased frequencies of pre-B and immature B-cells and decreased percentages of mature B-cells in the bone marrow, accompanied by an increased frequency of blood B-cells, splenic newly formed B-cells, and total CD19+B-cells. T3administration also promoted an increase in the size and cellularity of the spleen as well as in the white pulp areas of the organ, as evidenced by histological analyses. In addition, a decreased frequency of splenic B220+cells correlating with an increased percentage of CD138+plasma cells was observed in the spleen and bone marrow of T3-treated mice. Using enzyme-linked immunospot assay, an increased number of splenic immunoglobulin-secreting B-cells from T3-treated mice was detectedex vivo. Similar results were observed in mice immunized with hen egg lysozyme and aluminum adjuvant alone or together with treatment with T3. In conclusion, we provide evidence that high-circulating levels of T3stimulate plasmacytogenesis favoring an increase in plasma cells in the bone marrow, a long-lived plasma cell survival niche. These findings indicate that a stimulatory effect on plasma cell differentiation could occur in untreated patients with Graves' disease.

The Parthenolide Derivative LC-1 Is An Effective Single Agent and Is Highly Synergistic with Existing Therapies in Multiple Myeloma.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1708-1708
Author(s):  
Elisabeth J Walsby ◽  
Saman Hewamana ◽  
Alan Burnett ◽  
Steven Knapper ◽  
Chris Fegan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Nuclear Localization ◽  
Plasma Cells ◽  
Bone Marrow Cells ◽  
Binding Capacity ◽  
Single Agent ◽  
Normal Bone Marrow ◽  
Normal Bone

Abstract Multiple myeloma (MM) remains incurable with conventional therapeutic agents and has a median survival of only 3–5 years. Therefore, there is clearly a need for novel treatment strategies that can change the natural pathology of this condition. The nuclear factor κB (NF-κB) family of transcription factors is constitutively activated in MM cell lines and the majority of MM patients. Since NF-κB has known oncogenic activity in a number of human malignancies, targeted inhibition of this family of proteins may be useful in the treatment of MM. We and others have recently shown that the parthenolide derivative LC-1 has activity in acute myeloid leukaemia (AML) and chronic lymphocytic leukaemia (CLL) cells. Unusually, it induces apoptosis via the activation of both the intrinsic and extrinsic pathways and apoptosis is preceded by marked inhibition of NF-κB. Importantly, LC-1 is more potent against primary AML blasts and CLL lymphocytes than normal bone marrow progenitors and normal B-cells and T-cells. In this study we set out to evaluate LC-1 in MM cell lines and plasma cells derived from MM patients. LC-1 was cytotoxic to MM cell lines H929, U266 and JJN3 and induced apoptosis in a dosedependent manner resulting in an overall LD50 of 3.6mM (±1.8) after 48 hours in culture. Primary myeloma plasma cells, identified by CD38 and CD138 positivity, had a mean LD50 for LC-1 of 5.4mM (±1.6) after 48 hours of in vitro culture. Normal bone marrow cells were significantly less sensitive to the effects of LC-1 under the same conditions (P = 0.0007). Treatment of MM cell lines with LC-1 resulted in a decrease in the nuclear localization of NF-κB, as evidenced by a dose-dependent decrease in the DNA binding capacity of the NF-κB subunit RelA after 4 hours of treatment. To demonstrate whether synergy exists between LC-1 and existing MM therapies, the H929 cell line was treated for 48 hours with LC-1 and doxorubicin (32:1), melphalan (1:1) or bortezimib (1:500) and the combination indices (CI) calculated using the median effect method. A combination index of less than 1 denotes synergy. LC-1 did not show synergy with doxorubicin (CI >1) but was synergistic with melphalan and bortezimib (CI values of 0.53 and 0.59 respectively). Taken together our data clearly demonstrate that LC-1 has activity in MM cell lines and primary MM cells. Its ability to inhibit the nuclear localization of NF-κB is important to its cytotoxic effects. Furthermore, it may also provide an explanation for the synergy demonstrated with melphalan and bortezimib. These results provide a rationale for exploring the potential of LC-1 in clinical studies.

Identifying Phenotypically and Clinically Distinct Entities within the Spectrum of Marginal Zone Lymphoma and Lymphoplasmacytic Lymphoma/Waldenstrom Macroglobulinemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2652-2652
Author(s):  
Ruth M de Tute ◽  
Eve Roman ◽  
Andrew Jack ◽  
Roger G Owen ◽  
Andy Rawstron
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Clinical Features ◽  
Marginal Zone ◽  
Marginal Zone Lymphoma ◽  
Cell Populations ◽  
Normal Bone Marrow ◽  
Lymphoplasmacytic Lymphoma ◽  
Normal Bone

Abstract Abstract 2652 The mature B-cell neoplasms marginal zone lymphoma (MZL), lymphoplasmacytic lymphoma (LPL) and Waldenstrom macroglobulinemia (WM) display considerable overlap in clinic presentation, morphology, immunophenotype and genetic features. There is known to be poor reproducibility in the classification of these disorders and often only minimal diagnostic material is available. Multiparameter flow cytometry is a valuable tool for diagnosis and monitoring of B-cell disorders that can be applied to low cellularity samples or when the neoplastic cells represent a minority of total leucocytes. However, even when using large numbers of markers it is not possible to discriminate between MZL, LPL and WM and the aberrant expression of certain markers appears to occur with similar frequency in the different disease categories. The aim of this study was to determine whether there are phenotypically distinct entities within the spectrum of marginal zone and lymphoplasmacytic lymphomas, and to correlate these entities with clinical features. Samples from 146 people with a diagnosis of MZL, LPL or WM (122 bone marrow, 13 peripheral blood, 10 tissue biopsies), 8 with hairy cell leukemia, 10 reactive lymph node biopsies and 10 normal bone marrow samples were analysed using a panel of 39 markers. A minimum of 50,000 cells were acquired and analysed using a BD FACSCanto II with Diva software. Median fluorescence intensities were recorded for neoplastic CD19+ B-cell populations as well as for germinal centre (GC) and non-GC lymph node B-cells, B-progenitors, mature B-cells and plasma cell populations. Unsupervised cluster analysis was performed using dChip software. Clinical features and outcome data were collected and recorded centrally (www.hmrn.org.uk). Cluster analysis identified 5 entities with significant differences in their immunophenotypic profile. Neoplastic cells in all the MZL/LPL/WM groups typically expressed CD31, CD39 and CD49d with lack of CD10, CD23 and weaker expression of LAIR1 (CD305), CXCR5 (CD185) and CD22 compared to normal B-cells. CD5 and CD43 were expressed in a subset of cases but co-expression occurred in <1% of cases. The groups are characterised as follows: (1) clustered with normal mature B-cells, characterised by weaker CD20/CD95 and stronger CXCR5 expression relative to other MZL/LPL/WM cases, shorter survival, anemia and lymphadenopathy, typically requiring therapeutic intervention at diagnosis; (2) clustered with HCL, characterised by CD25 and CD11c expression and a higher degree of extranodal involvement; (3) did not cluster with other B-cell types, characterised by very strong CD79b and surface IgM expression often with anemia requiring supportive care; (4) characterised by strong CD24 expression with relatively good clinical course; and (5) clustered with normal bone marrow plasma cells, characterised by weak CD20, CD24 and IgM expression and also with better clinical features.Group2 year survivalAnemia (Hb <10.5g/dL)Lympho-cytosis (>4 × 10^9/L)Raised B2M (>2.7mg/L)Lymph-adenopathy (≥2 nodes)Extranodal involvementTherapeutic intervention at diagnosis115/24 (63%)10/19 (53%)9/19 (47%)8/10 (80%)12/19 (63%)4/19 (21%)14/19 (74%)228/37 (76%)8/25 (32%)11/25 (44%)6/11 (55%)9/23 (39%)10/23 (43%)9/25 (36%)325/31 (81%)14/24 (58%)5/23 (22%)11/18 (61%)4/19 (21%)5/19 (26%)13/24 (54%)422/27 (81%)5/22 (23%)5/22 (23%)10/17 (59%)5/20 (25%)4/20 (20%)8/22 (36%)522/26 (85%)5/16 (31%)5/16 (31%)6/8 (75%)8/14 (57%)5/14 (36%)5/16 (31%) The results indicate that the current diagnostic categories of MZL and LPL/WM have a great degree of overlap and comprise a number of phenotypically distinct entities which also appear to have different clinical features and outcomes. Enhancing the classification is likely to facilitate diagnosis, monitoring and identification of the underlying features of these B-cell malignancies. Disclosures: Rawstron: BD Biosciences: Patents & Royalties.

Variable Expression of Therapeutic Antibody Targets May Have Implications for Efficacy of Therapy in Myeloma and Waldenstrom Macroglobulinaemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3425-3425
Author(s):  
Ruth M de Tute ◽  
Jane Shingles ◽  
Andy C. Rawstron ◽  
Roger G Owen
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Surface Expression ◽  
Cell Populations ◽  
Expression Data ◽  
Expression Levels ◽  
Normal Bone ◽  
Cd38 Expression

Abstract Recent advances in the treatment of myeloma have included the development of immunotherapies using monoclonal antibodies targeted against plasma cell specific antigens. Elotuzumab is a therapeutic antibody directed against the SLAM family member CS1, also known as CD319, SLAMF7 or CRACC. Expression of this antigen has been investigated extensively using immunohistochemistry and gene expression profiling and has been demonstrated on normal and malignant plasma cells. Clinical trials using Elotuzumab in myeloma have shown promising results, especially in combination with other therapeutic agents, such as lenalidomide and dexamethasone. Daratumumab, a humanised antibody to CD38, has also shown encouraging responses in a percentage of refractory patients in Phase I and II trials, both as a single agent and in combination with lenalidomide. Despite this progress a significant number of patients fail to respond to these therapies for reasons which remain unclear. Monoclonal antibody-based therapy in Waldenstrom macroglobulinemia (WM) has traditionally targeted the B cell component. We have previously demonstrated that WM plasma cells are not depleted with either rituximab or alemtuzumab resulting in delayed IgM responses. Plasma cell specific antibodies may be applicable to WM and may be particularly suited to those instances when the clinical features are a consequence of the M protein such as hyperviscosity and neuropathy. There are no published data correlating quantitative surface expression data with outcome and it is possible that variability in the surface expression levels of the targets could affect efficacy of these therapeutic antibodies. The aim of this study was to evaluate the expression of CD319 and CD38 in patients with a range of plasma cell dyscrasias using multi-parametric flow cytometry. Bone marrow aspirates from patients with myeloma, MGUS or WM along with normal staging bone marrows were analysed using 8-colour flow cytometry. Leucocytes were isolated using ammonium chloride lysis and cells were then incubated with a cocktail of surface antibodies containing CD319, CD19, CD38, CD138, CD45 and CD20. Following fixation and permeabilisation cells were then incubated with Kappa and Lambda. Plasma cells and B-cells were enumerated and monoclonal B-cell and plasma cell populations were assessed. Expression of CD319 was seen on all plasma cell populations and was absent from all B-cell populations (Median fluorescent intensity (MFI) 12088 vs 114, p<0.001). There was definite heterogeneity in both CD319 and CD38 expression on plasma cells (CD319 MFI range 1871-21865; CD38 MFI range 4393-156258) and this was as a result of differences between diagnostic groups. There were significantly lower levels of expression seen in the clonal plasma cells in myeloma cases compared to normal bone marrows (CD319 MFI 7743 (1871-13880) vs 11118 (8412-15685), p=0.188; CD38 MFI 10113 (4665-28627) vs 54650 (32078-91906), p=0.003). CD319 expression was below normal levels in 33% of myeloma cases and CD38 expression was below normal in all myeloma samples. Lower levels of expression were also seen in myeloma relative to WM cases (CD319 MFI 7743 (1871-13880) vs 14488 (7056-21865), p=0.079; CD38 MFI 10113 (4665-28627) vs 47695 (15174-156258), p=0.003). There was no significant difference in CD319 or CD38 expression for WM cases when compared to normal bone marrow samples. Although CD319 and CD38 expression was seen in all plasma cell populations, there were differences in expression levels between myeloma plasma cells and those from MGUS, WM or normal bone marrow samples. The heterogeneity in surface expression seen could potentially affect efficacy of antibody treatment and may offer some explanation for the non-responders that have been seen in early trials of Elotuzumab and Daratumumab. We have also shown that the clonal plasma cells in WM have higher levels of surface expression of both targets than those in myeloma. Following the encouraging results shown in the myeloma setting, this expression data suggests that Elotuzumab and Daratumumab may also be highly effective for eradication of the plasma cell component of WM. Prospective studies in both myeloma and WM correlating surface expression levels to outcome would be of interest. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals Evidence for a bone marrow B cell transcribing malignant plasma cell VDJ joined to C mu sequence in immunoglobulin (IgG)- and IgA-secreting multiple myelomas.

1993 ◽  
Vol 178 (3) ◽  
pp. 1091-1096 ◽  
Author(s):  
P Corradini ◽  
M Boccadoro ◽  
C Voena ◽  
A Pileri
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Bone Marrow Cells ◽  
Indirect Evidence ◽  
Specific Marker

Multiple myeloma is a B cell malignancy characterized by the expansion of plasma cells producing monoclonal immunoglobulins (Ig). It has been regarded as a tumor arising at the B, pre-B lymphocyte, or even stem cell level. Precursor cells are presumed to proliferate and differentiate giving rise to the plasma cell clonal expansion. Antigenic features and specific Ig gene rearrangement shared by B lymphocytes and myeloma cells have supported this hypothesis. However, the existence of such a precursor is based upon indirect evidence and is still an open question. During differentiation, B cells rearrange variable (V) regions of Ig heavy chain genes, providing a specific marker of clonality. Using an anchor polymerase chain reaction assay, these rearranged regions from five patients with multiple myeloma were cloned and sequenced. The switch of the Ig constant (C) region was used to define the B cell differentiation stage: V regions are linked to C mu genes in pre-B and B lymphocytes (pre-switch B cells), but to C gamma or C alpha in post-switch B lymphocytes and plasma cells (post-switch B cells). Analysis of bone marrow cells at diagnosis revealed the presence of pre-switch B cells bearing plasma cell V regions still joined to the C mu gene. These cells were not identified in peripheral blood, where tumor post-switch B cells were detected. These pre-switch B cells may be regarded as potential myeloma cell precursors.

scholarly journals A novel membrane antigen selectively expressed on terminally differentiated human B cells

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1922-1930 ◽  
Author(s):  
T Goto ◽  
SJ Kennel ◽  
M Abe ◽  
M Takishita ◽  
M Kosaka ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Peripheral Blood ◽  
Plasma Cells ◽  
Affinity Constant ◽  
Target Antigen ◽  
Specific Marker

Abstract A monoclonal antibody (MoAb) that defines a novel terminal B-cell- restricted antigen, termed HM1.24, was developed against a human plasma cell line. The MoAb, designated anti-HM1.24, reacted with five different human myeloma cell lines, as well as with monoclonal neoplastic plasma cells obtained from the bone marrow or peripheral blood of patients with multiple myeloma or Waldenstrom's macroglobulinemia. The HM1.24 antigen was also expressed by mature Ig- secreting B cells (plasma cells and lymphoplasmacytoid cells) but not by other cells contained in the peripheral blood, bone marrow, liver, spleen, kidney, or heart of normal individuals or patients with non- plasma-cell-related malignancies. The anti-HM1.24 MoAb bound to human myeloma RPMI 8226 cells with an affinity constant of 9.2 x 10(8) M-1, indicating approximately 84,000 sites/cell. By immunoprecipitation assay under reducing conditions, this MoAb identified a membrane glycoprotein that had a molecular weight of 29 to 33 kD. Our studies indicate that the HM1.24-related protein represents a specific marker of late-stage B-cell maturation and potentially serves as a target antigen for the immunotherapy of multiple myeloma and related plasma cell dyscrasias.

scholarly journals A novel membrane antigen selectively expressed on terminally differentiated human B cells

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1922-1930 ◽  
Author(s):  
T Goto ◽  
SJ Kennel ◽  
M Abe ◽  
M Takishita ◽  
M Kosaka ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Peripheral Blood ◽  
Plasma Cells ◽  
Affinity Constant ◽  
Target Antigen ◽  
Specific Marker

A monoclonal antibody (MoAb) that defines a novel terminal B-cell- restricted antigen, termed HM1.24, was developed against a human plasma cell line. The MoAb, designated anti-HM1.24, reacted with five different human myeloma cell lines, as well as with monoclonal neoplastic plasma cells obtained from the bone marrow or peripheral blood of patients with multiple myeloma or Waldenstrom's macroglobulinemia. The HM1.24 antigen was also expressed by mature Ig- secreting B cells (plasma cells and lymphoplasmacytoid cells) but not by other cells contained in the peripheral blood, bone marrow, liver, spleen, kidney, or heart of normal individuals or patients with non- plasma-cell-related malignancies. The anti-HM1.24 MoAb bound to human myeloma RPMI 8226 cells with an affinity constant of 9.2 x 10(8) M-1, indicating approximately 84,000 sites/cell. By immunoprecipitation assay under reducing conditions, this MoAb identified a membrane glycoprotein that had a molecular weight of 29 to 33 kD. Our studies indicate that the HM1.24-related protein represents a specific marker of late-stage B-cell maturation and potentially serves as a target antigen for the immunotherapy of multiple myeloma and related plasma cell dyscrasias.

Humoral Immunity and Plasma Cell Changes in Patients Responding to CD19-Specific Chimeric Antigen Receptor (CAR)-Modified T-Cell Adoptive Immunotherapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1110-1110
Author(s):  
Vijay Bhoj ◽  
Michael C Milone ◽  
Carl H. June ◽  
David Porter ◽  
Stephan A. Grupp ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Humoral Immunity ◽  
Research Funding ◽  
Plasma Cells ◽  
Flow Cytometric ◽  
Antibody Titers

Abstract Introduction: T cells engineered to express chimeric antigen receptors (CARs) recognizing CD19 (CART19) can eliminate malignant cells in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). We and other groups have shown that persistent tumor eradication by CD19-specific T cell immunotherapy is accompanied by normal B-cell aplasia. It is assumed that responding patients cannot make new antibody responses post-successful CART19 treatment; however, the status of previously established humoral immunity in these patients is currently unknown. Understanding the consequence of successful CART19 therapy on established humoral immunity has implications for both the clinical management of CART19-treated patients as well as the potential application of this therapy to non-malignant diseases such as autoimmunity and transplantation. Methods: We performed a prospective, observational study of adult and pediatric patients with ALL and adults with relapsed/refractory CLL, who were enrolled in clinical trials of CART19 at our institution. Serum antibody titers to previously-generated vaccine or vaccine-related pathogens (Streptococcus pneumoniae, Tetanus toxoid, Hemophilus influenza type-B (HIB), Measles, Mumps, and Rubella) were determined along with a quantitative assessment of B-cell and plasma cell frequencies in blood and bone marrow aspirates. Specimens were collected during pre-established study assessments or additional time points when collected as required for clinical management. Due to the challenges of assessing plasma cells, multiple methods were employed for their quantification in fresh specimens including flow cytometry and immunohistochemistry (IHC). Flow cytometric assessment of plasma cells was performed on freshly obtained marrow samples. Only patients with at least 3 months of B-cell aplasia in the absence of regular intravenous immunoglobulin (IVIg) infusions were included in the study. Results: All patients had no evidence of leukemia or peripheral B cells post-CART19 infusion at the time of this study. Compared to pre-CART19 serum titers, antibodies to S. pneumoniae remained stable or increased in 9 of 12 patients despite lack of circulating B-cells. Antibody titers to Tetanus toxoid were stable or increased in 13 of 14 patients. Anti-HIB levels were stable or increased in 9 of 11 patients and antibodies to Measles, Mumps and Rubella were stable or increased in 12 of 13, 11 of 13, and 12 of 13 patients, respectively. Flow cytometric analysis of bone marrow aspirates after CART19 infusion revealed three patients with persistence of CD38+ CD138+ plasma cells (at 1, 3 and 9 months post infusion, respectively) despite a complete absence of peripheral CD19+ B cells. In 9 patients, CD20 and CD138 IHC analysis of bone marrow core biopsies revealed a decrease in plasma cell (ranges: 1-5% pre-CART19, 0-<1% post-CART19), consistent with our previously published data. Finally, in another subset of patients, neither B cells nor plasma cells were detectable by flow cytometry of aspirate material or IHC of core biopsies collected either pre- or post-CART19 treatment. Conclusions: The stable or increased titers of antibodies to previous vaccines are surprising and may, in part, reflect improved marrow function as a result of leukemia eradication. The demonstration of plasma cells in a subset of patients in the absence of detectable tumor or normal B cells provides strong evidence for the existence of a population of plasma cells that are resistant to lysis by CART19 cells. This is consistent with antibody titers to previously generated vaccine antigens, which remain stable despite effective CART19 treatment. The additional finding of a decrease in CD138+ cells in several patients by IHC suggests that some populations of plasma cells are either targeted directly by CART19 or have a short half-life (e.g. plasmablasts); CD138 is not sufficient to distinguish these populations. Overall, these results indicate that long-lived plasma cells are resistant to CART19, likely due to a loss of CD19 during plasma cell differentiation. Continued analysis of remaining plasma cells in the absence of ongoing B-cell maturation as a result of CART19 persistence may provide important information on turnover rates of these long-lived cells in humans. Disclosures Bhoj: Novartis: Research Funding. Milone:Novartis: Patents & Royalties, Research Funding. June:Novartis: Research Funding, Royalty income Patents & Royalties. Porter:Novartis: Patents & Royalties, Research Funding. Grupp:Novartis: Research Funding. Melenhorst:Novartis: Research Funding. Lacey:Novartis: Research Funding. Mahnke:Novartis: Research Funding.

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