A Crucial Cytotoxic Role of Anti-Idiotypic Antibody in Immunotherapy for B-Cell Neoplasms with Tumor Cell-Derived Heat Shock Protein 70.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1642-1642
Author(s):  
Kazuya Sato ◽  
Junko Jimbo ◽  
Naoka Okamura ◽  
Takaaki Hosoki ◽  
Motohiro Shindo ◽  
...  
Keyword(s):  
Heat Shock ◽  
Immune Responses ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Variable Region ◽  
Autologous Tumor ◽  
Antigenic Peptides ◽  
Control Peptide ◽  
Specific Antigens ◽  
Idiotypic Antibody

Abstract Abstract 1642 Poster Board I-668 Background Tumor-derived heat shock proteins (HSPs), which bind to tumor-specific antigenic peptides, can be used for cancer immunotherapy. In this meeting, we previously reported that vaccination with mouse B-cell leukemia/lymphoma cell line, A20, -derived HSP70 (A20-HSP) against A20 cells in mice induces tumor-specific cellular immune responses (Sato et al. Blood 2001;98:1852-1857; Iuchi et al. Int J Hematol 2006;84:449-458) as well as A20-specific humoral immunity through complement dependent cell-cytotoxicities (CDCs) (Sato K et al., 2007, Blood 110:682a, Abstruct#2302). In the B-cell neoplasms, idiotype is known as one of the important tumor specific antigens which induce anti-idiotype cellular and humoral immune responses. A20-secreted monoclonal IgG (A20-Ig) and A20-idiotypic epitope peptide (A20-IP: DYWGQGTEL), which is derived from variable region of the heavy chain of the A20-Ig, are known as the A20-specific antigens (J Immunol. 2002; 168: 3983-91). Further analysis using A20-Ig and A20-IP enables to establish novel HSP70-based therapeutic strategies for B-cell neoplasms. The present study investigated whether immunization with A20-HSP induces anti-idiotypic antibodies, and also evaluated whether the antibodies showed CDCs. Methods A20 and syngeneic Balb/c mice (H-2Kd) were used in this study. HSP70 was purified from either A20 cells or healthy mouse liver tissue. A20-Ig was purified from A20 culture supernatants. After the subcutaneous immunization with A20-HSP, liver-derived HSP70 (control) to the healthy mice, the sera were harvested for the following experiments. To detect anti-A20-IP antibodies, the sera were assayed by ELISA to detect the specific IgG against A20-HSP, or A20-Ig. To confirm that immunization with A20-HSP induces anti-idiotypic antibodies, we analyzed the inhibitory effect (% inhibition) of A20-IP on the A20-HSP-mice sera reactivity against A20-HSP by preincubation of the sera with A20-IP or H-2Kd biding control peptide (mouse influenza hemagglutinin peptide: IYSTVASSL) by ELISA. To confirm that A20-HSP mice sera contains CDC type antibody directed to A20-IP, the CDC activity was determined by the trypan blue uptake of A20 cells after incubation with the complement and A20-HSP mice sera preincubated without or with A20-IP or control peptide. Results The IgG level in the A20-HSP mice against either A20-HSP or A20-Ig was significantly increased in comparison to that in control mice. % inhibition of A20-IP (45.7%) was significantly higher than that of control peptide (1.0%), indicating that almost half of IgG in the A20-HSP mice sera which reacts to A20-HSP recognizes A20-IP. The CDC activity of A20-HSP mice sera against A20 was markedly inhibited by preincubation of the sera with A20-IP but with control peptide. Conclusions: Immunization with A20-HSP70 induces anti-idiotype antibody and the antibody contributes a crucial role in eradication of A20 by CDC activity in mice. These findings enable to establish a novel and advantageous therapeutic strategy against B-cell neoplasms utilizing the anti-idiotypic antibody in HSP-based autologous tumor immunotherapy. Disclosures No relevant conflicts of interest to declare.

Evidence for Autostimulatory Mechanisms in Chronic Lymphocytic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2880-2880
Author(s):  
Martin Trepel ◽  
Fabian Muller ◽  
Mareike Frick ◽  
Janina Rahlff ◽  
Claudia Wehr ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Phage Display ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Specific Binding ◽  
Variable Region ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Fab Fragment

Abstract Abstract 2880 Background: The development and / or course of chronic lymphocytic leukemia (CLL) may be driven by the recognition of antigens through the B cell receptor (BCR). While it has been recognized that the diversity of epitope recognition may be astonishingly confined in CLL, knowledge on antigens recognized by CLL BCRs is still limited. Here, we identified and characterized an epitope recognized by a defined CLL BCR which may broaden our view on potential mechanisms of antigenic drive in CLL. Methods: The B- cell receptor of a random CLL-patient was cloned and expressed as Fab fragment in E.coli. Random phage display reptile litanies we skeletal on the immobilized Fab and landed peptides were tested for specific binding. Specific clones we sequenced and sequences were analyzed for homology to known proteins. Recognition of candidate proteins was verified in brooding assays or recombinant proteins. Results: Screening random phage display peptide libraries, we identified a CLL BCR epitope mimic that displayed a high degree of homology to a conserved peptide string in the variable region of immunoglobulin heavy and light chains. CLL BCR binding to this epitope as well as binding to full length heavy and light immunoglobulin chains was verified by binding assays and a protein array screening. Interestingly, the CLL BCR also interacted with itself, as the identified epitope was also present in its own primary amino acid sequence. Conclusions: These findings suggest the possibility of self-recognition of BCRs within the CLL cell membrane or BCR interactions between neighboring CLL cells. This may potentially result in autostimulation of the leukemic cell independent of “exogenous” antigens and may account for self-sufficient signaling of some CLL-BCRs in driving disease progression. As the peptide mimicking this immunoglobulin epitope is known to be recognized by BCRs of other CLL cases in addition to the index case investigated here, such autostimulatory mechanisms may be relevant to a large number of CLL patients. Disclosures: No relevant conflicts of interest to declare.

Immunotherapy using heat-shock protein preparations of leukemia cells after syngeneic bone marrow transplantation in mice

Blood ◽  
2001 ◽  
Vol 98 (6) ◽  
pp. 1852-1857 ◽  
Author(s):  
Kazuya Sato ◽  
Yoshihiro Torimoto ◽  
Yasuaki Tamura ◽  
Motohiro Shindo ◽  
Hitoshi Shinzaki ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Heat Shock ◽  
T Lymphocytes ◽  
Immune Responses ◽  
Marrow Transplantation ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Antigenic Peptides ◽  
Cd8 T Lymphocytes

Abstract Heat-shock proteins (HSPs) act as molecular chaperones binding endogenous antigenic peptides and transporting them to major histocompatibility complexes. HSPs chaperone a broad repertoire of endogenous peptides including tumor antigens. For the immunotherapy of tumors, a strategy using HSPs may be more advantageous than other procedures because the identification of each tumor-specific antigen is not necessary. In this study, the efficacy of immunotherapy against minimal residual leukemia cells using HSP preparations was evaluated. HSP70 and GP96 were purified from syngeneic leukemia cell line A20 and immunized into BALB/c mice during the reconstitution period of the immune system after syngeneic bone marrow transplantation. In this procedure, all mice not immunized were dead within 60 days of A20 inoculation, whereas the survival times of HSP-immunized mice were significantly prolonged. In addition, the depletion of either CD4+ or CD8+ T lymphocyte significantly abrogated this efficacy, indicating that both CD4+ and CD8+ T lymphocytes were required for tumor cell rejection. Moreover, the vaccination of HSPs elicited a specific response of potent CD8+ T lymphocytes cytotoxic against A20 in vitro. These observations suggest that immunization of the complex of HSPs and peptides derived from leukemia cells leads to immune responses. These immune responses are sufficient to reject minimal amounts of leukemia cells for relatively immunocompromised mice after syngeneic bone marrow transplantation.

Idiotype-pulsed dendritic cell vaccination for B-cell lymphoma: clinical and immune responses in 35 patients

Blood ◽  
2002 ◽  
Vol 99 (5) ◽  
pp. 1517-1526 ◽  
Author(s):  
John M. Timmerman ◽  
Debra K. Czerwinski ◽  
Thomas A. Davis ◽  
Frank J. Hsu ◽  
Claudia Benike ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
B Cell ◽  
Tumor Regression ◽  
B Cell Lymphoma ◽  
Active Immunotherapy ◽  
Dendritic Cell Vaccination ◽  
Molecular Response ◽  
Autologous Tumor ◽  
Dc Vaccine

Tumor-specific clonal immunoglobulin expressed by B-cell lymphomas (idiotype [Id]) can serve as a target for active immunotherapy. We have previously described the vaccination of 4 patients with follicular lymphoma using dendritic cells (DCs) pulsed with tumor-derived Id protein and now report on 35 patients treated using this approach. Among 10 initial patients with measurable lymphoma, 8 mounted T-cell proliferative anti-Id responses, and 4 had clinical responses—2 complete responses (CRs) (progression-free [PF] for 44 and 57 months after vaccination), 1 partial response (PR) (PF for 12 months), and 1 molecular response (PF for 75+ months). Subsequently, 25 additional patients were vaccinated after first chemotherapy, and 15 of 23 (65%) who completed the vaccination schedule mounted T-cell or humoral anti-Id responses. Induction of high-titer immunoglobulin G anti-Id antibodies required coupling of Id to the immunogenic carrier protein keyhole limpet hemocyanin (Id-KLH). These antibodies could bind to and induce tyrosine phosphorylation in autologous tumor cells. Among 18 patients with residual tumor at the time of vaccination, 4 (22%) had tumor regression, and 16 of 23 patients (70%) remain without tumor progression at a median of 43 months after chemotherapy. Six patients with disease progression after primary DC vaccination received booster injections of Id-KLH protein, and tumor regression was observed in 3 of them (2 CRs and 1 PR). We conclude that Id-pulsed DC vaccination can induce T-cell and humoral anti-Id immune responses and durable tumor regression. Subsequent boosting with Id-KLH can lead to tumor regression despite apparent resistance to the primary DC vaccine.

LRPAP1 Is a Frequent B-Cell Receptor Antigen in Mantle Cell Lymphoma (MCL)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2685-2685 ◽  
Author(s):  
Lorenz Thurner ◽  
Sylvia Hartmann ◽  
Klaus-Dieter Preuss ◽  
Natalie Fadle ◽  
Maria Kemele ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Variable Region ◽  
Cell Receptor ◽  
Healthy Controls ◽  
Cell Of Origin ◽  
Vh Genes

Abstract Introduction: Chronic antigenic stimulation may play an important role in the pathogenesis of malignant lymphomas. Although most MCL cases are believed to have an antigen-naive B cell as cell of origin, overrepresentation of certain VH genes has been reported. Therefore we screened BCRs from MCLs for possible antigens. Methods: BCRs were expressed as recombinant Fabs based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA of snap-frozen MCL specimens and established MCL cell lines. The purified BCR-Fabs were checked for binding to proteins expressed on macroarrays of human cDNA expression libraries and on bacterial lysates. In addition, sera from patients with MCL were screened for antibodies against respective BCR antigens. Results: The recombinant MCL-BCR derived Fabs from 9 patients and of four established MCL cell lines were tested on protein arrays. Recombinant lymphoma-BCR-derived Fabs from 4/9 patients and from 1/4 MCL cell lines reacted with human low density lipoprotein receptor-related protein associated protein 1 (LRPAP1). Specific secondary modifications of LRPAP1 explaining its autoimmunogenicity were not found. 8/30 patients with MCL had anti-LRPAP1-antibodies in their serum, which was the case in only 1/200 healthy controls. Finally, LRPAP1 specifically induced proliferation of Maver1 cells that express a BCR with specificity for LRPAP1. Conclusions: LRPAP1 is the first molecularly defined antigenic target of MCL-BCRs. The high frequency of LRPAP1-reactive BCRs in MCL suggests a role of LRPAP1 in the pathogenesis of MCL, even in cases with unmutated VH genes. The prevalence of LRPAP1-antibodies in MCL patients and healthy controls identifies LRPAP1-antibody as the first serologic risk factor for MCL (odds ratio: 72.36) Supported by Wilhelm-Sander-Stiftung Disclosures No relevant conflicts of interest to declare.

The Repertoire of Heavy Chain Immunoglobulin Genes in B-Cell Chronic Lymphocytic Leukemia in Russia and Belarus

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4579-4579
Author(s):  
Bella Biderman ◽  
Eugene Nikitin ◽  
Tatiana Sergienko ◽  
Alexandra Bakun ◽  
Irina Taras ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Heavy Chain ◽  
Conflicts Of Interest ◽  
Variable Region ◽  
Lymphocytic Leukemia ◽  
Antigen Receptors ◽  
Mutation Status ◽  
The Uk ◽  
Cell Chronic Lymphocytic Leukemia

Abstract Abstract 4579 Introduction. Mutation status of the heavy chain variable region genes is known as an important factor in long-term prognosis in B-cell chronic lymphocytic leukemia (B-CLL). A more detailed study of the gene sequences of immunoglobulin heavy chain (IgVH) led to the discovery of stereotyped antigen receptors (SAR) - receptors that have the same set of VH-, D- and JH- genes used. SARs have been found almost in a quarter of all B-CLL cases. Since this study there have been no data available concerning VH-gene usage and SARs in Russian and Belarusian B-CLL patients. Patients and methods. DNA or cDNA was amplified in 6 separated reactions using primers specific for VH-families, and a consensus JH primer [Campbell et al. 1992] or primer sets recommended by the BIOMED-2 [van Dongen et al. 2003]. PCR products were sequenced using family-specific primers and Big Dye Terminator v3.1 kit (Applied Biosystems). Sequences were analyzed with IgBlast (http://www.ncbi.nlm.nih.gov/igblast). 98% homology cutoff was used to discriminate between mutated and unmutated cases. Results. Total of 547 patients with B-CLL where analyzed; 192 patients with mutated IgVH-genes (35%) and 355 patients with unmutated ones (65%). We have identified 65 stereotyped receptors (SARs) in 198 of 491 Russian patients (40%). Twenty one SARs (confirmed) appeared in more than 3 patients (110 out of 198, 55%), 44 SARs (potential) found in 2 patients (88 out of 198 cases, 45%). The vast majority of confirmed SARs were found in the subgroup of patients without mutations in IgVH genes (95%). Among the potential SARs 15 pairs were from patients with and without mutations (34%), seven pairs from patients with mutations (16%) and 22 pairs from patients without IgVH mutations (50%). The most common SARs were: VH1–69/D3-3/JH6 (24 patients, 5%); VH1–69/D3–16/JH3 (8 patients); VH1–69/D2-2/JH6 (8 patients); VH1–69/D3–10/JH6 (6 patients). Among 56 Belarusian patients we have identified only 4 SARs, one confirmed SAR (4 cases) and 3 potential ones. Confirmed (VH1–69/D3-3/JH6) and one potential (VH1–69/D3–16/JH3) were also found in Russia while other two potential (VH1–69/D2–15/JH6 and VH2–5/D2-2/JH6) were not observed in Russia. Discussion. In Russia and Belarus, VH1–69 gene is found in 20% of all cases of B-CLL, and almost always (95%) in unmutated cases. This finding well correlates to the data obtained by [Kryachok et al. 2012] concerning high VH1–69 usage in Ukrainian patients. In other European countries, this gene is less common: about 14% of cases in Sweden, France and Spain, 11% in the UK and about 6–7% in Greece and Italy [Ghia et al. 2005; Tobin et al. 2004; Duke et al. 2003]. In all these countries, this gene is also prevalent in patients with unmutated VH-genes. Nordic countries are characterized by very frequent use of gene VH3–21 - 9%, while in Russia and the countries of central and southern Europe, it occurs at least 3 times less (about 3%), and was not found in Belarusian samples. This gene is also associated with poor prognosis of B-CLL, regardless of mutation status of IgVH genes. Interestingly, in Asia (China, Japan, Iraq) VH1–69 and VH3–21 genes are almost not observed [Nakamura et al. 1999; Farsangi et al. 2007; Lijuan Chen et al. 2008]. Narrowing of the repertoire of IgVH genes - specific feature of B-CLL indicatites that influence of antigen (at least in some cases) occurs during the development of the disease. Also, factors of genetic background as well as geographical environment could be important. It is possible that future treatment decisions will be based not only on the IgVH mutation status, but also on the characteristics of certain antigen receptor. Disclosures: No relevant conflicts of interest to declare.

Origin of (sporadic) Plasma Cell Myeloma: Lysolipids Are Not the Target of Clonal Immunoglobulins

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2055-2055
Author(s):  
Michael Pfreundschuh ◽  
Natalie Fadle ◽  
Evi Regitz ◽  
Maria Kemele ◽  
Klaus-Dieter Preuss
Keyword(s):  
B Cell ◽  
Conflicts Of Interest ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Antigen Specificity ◽  
Western Blots ◽  
Antibody Reactivity ◽  
Specific Antigens ◽  
Competition Assays

Abstract Background: Lysolipids have been claimed to be involved in the origin of sporadic monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) and of Gaucher's associated MGUS/MM because lyso-glucosylceramide (LGL1) and lysophosphatidylcholine (LPC) were purported to be the target of the clonal immunoglobulin in 31% of patients with sporadic and 85% with Gaucher's associated MGUS/MM(Nair et al. N Engl J Med 2016;374:555-61). The low titers (1:250) of the reported anti-lysolipid reactivity raised doubts as to whether these reactivities were indeed mediated by the clonal immunoglobulin (paraprotein). Patients and Methods: We analyzed the sera from 96 patients with MGUS/MM for the presence of anti-lysolipid reactivity by means of immunofixation, ELISA, serum protein electrophoresis (SPSP) before and after absorption with sphingosine beads to remove antibodies against lysolipids and paraprotein-specific antigens (paratarg-7 and sumoylated HSP90), respectively, and Western blots. Moreover, the BCR from MGUS/MM with a paraprotein-mediated reactivity against paratarg-7 and HSP90 were cloned and expressed as Fab fragments and used to determine antigen specificity by direct antigen binding and antigen-competition assays. Results: The presence of antibody reactivity against LGL1 and LPC was demonstrated in 28/96 (29%) MGUS/MM patients, confirming the rate observed in Nair's study (31%). Seven of these 28 sera also contained reactivity against paratarg-7 and 2 against HSP90 (always at titers >1x106). In none of the 28 lysolipd-reactive cases was the anti-lysolipd reactivity mediated by the clonal immunoglobulin as demonstrated by low antibody titers (<1:500), immunoglobulin subclasses different from the clonal immunoglobulin (as shown by immunofixation), inability of lysolipids to compete with specific antigens for binding with the clonal immunoglobulin or the recombinant B-cell receptor and demonstration by immunoglobulin affinity chromatography, that separated LGL1 reactivity from the monoclonal immunoglobulin. Absorption with sphingosine-beads completely removed the anti-lipd reactivity from the respective sera and the LGL1 immunoreactive bands after SPEP, but did not remove the monoclonal peak from the serum electrophoresis, while this was always the case with paratarg-7 (see figure) and HSP90 when they were the antigenic targets of the paraproteins. Anti-lysolipid reactivities were rare in 140 healthy controls (6%), but more frequent in 140 patients with autoimmune diseases (19%; p=0.002). Conclusions: Our data disprove a role of lysolipids in the origin of sporadic MGUS/MM. While we had no access to sera from patients with Gaucher'sassociated MGUS/MM, the report that lysolipds are the targets of the paraproteins in 85% of these cases and hence play a role in the pathogenesis of these diseases must also be met with caution. To prove that an observed antibody reactivity is mediated by the clonal immune globulin all of the following prerequisites must be met: 1st, the clonal immunoglobulin and the antibody mediating the reactivity against the antigen have the identical light and heavy chains; 2nd, the reaction has a serum titer >1x106; 3rd, absorption with the antigen removes the monoclonal peak in the serum electrophoresis; 4th, a specific reactivity of the expressed (clonal) B-cell receptor with the antigen is demonstrated; and 5th, convincing data is provided by competition assays that the paraprotein and the B-cell receptor recognize the same antigen and epitope of the antigen under investigation. Not a single one of these prerequisites has been met in the study of Nair et al. A role of lysolipids in the pathogenesis of any form of MGUS/MM cannot be assumed until the complete set of the 5 prerequisites is demonstrated for these autoantigens. A request for exchange of sera has been forwarded to Nair et al., and if granted, results will be reported. Supported by Wilhelm-Sander-Stiftung. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Disease-associated dendritic cells respond to disease-specific antigens through the common heat shock protein receptor

Blood ◽  
2003 ◽  
Vol 102 (5) ◽  
pp. 1806-1814 ◽  
Author(s):  
Justin Stebbing ◽  
Brian Gazzard ◽  
Simon Portsmouth ◽  
Frances Gotch ◽  
Louise Kim ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Heat Shock ◽  
Immune Responses ◽  
Cytotoxic T Cells ◽  
Kaposi Sarcoma ◽  
Antigenic Peptides ◽  
Protein Receptor ◽  
Functionally Impaired ◽  
The Common ◽  
Hiv 1

AbstractThe most abundant intracellular proteins, heat shock proteins (HSPs), serve as molecular chaperones for regulatory and maturation pathways. Diverse families of HSPs have been shown to bind antigenic peptides and to play major roles in innate and adaptive immune responses through the common HSP receptor, CD91. HIV-1+ patients with Kaposi sarcoma (KS) were matched for CD4 count and HIV-1 RNA viral load to HIV-1+ patients without Kaposi sarcoma (and negative for Kaposisarcoma–associated herpesvirus). We then investigated the pathways used by tumor lysates, viral lysates, and viral particles in their activation. In particular, we observed immune responses after HSP depletion using antitumor antibiotics and blockade of the common HSP receptor, CD91. Despite the impaired functional capacity of dendritic cells (DCs) derived from patients with KS, DCs retain the ability to prime the adaptive arm of the immune system through the common HSP receptor, leading to phenotypic activation and stimulation of tetramer-positive CD8+ cytotoxic T cells. We also show that interferon-producing plasmacytoid DCs are selectively depleted in KS-positive compared with matched KS-negative HIV-1–infected patients. Functionally impaired DCs can effectively cross-present immune responses through the common HSP receptor. These results have important implications for the etiopathogenesis of KS and for the development and design of any compounds, including vaccines, derived from cellular lysates.

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