Idiotypic vaccination against human B-cell lymphoma. Rescue of variable region gene sequences from biopsy material for assembly as single-chain Fv personal vaccines

Idiotypic determinants on neoplastic B cells could provide tumor antigens for vaccination of patients with B-cell tumors. Because this approach requires an individual vaccine for each patient, simple methods for obtaining idiotypic antigen are desirable. Using polymerase chain reaction (PCR) with family-based V-gene and J-region primers, the variable region genes of heavy and light chains (VH and VL) of Ig have been obtained from biopsy material from 13 patients with B-cell tumors. In each case, analysis of random clones derived from the PCR product showed repeated, clonally-related sequences, whereas normal lymphoid tissue generated no repeated sequences. In 3/3 cases, the repeated sequences were found to be the same as those in a tumor-derived hybridoma. Mutational patterns in the V-genes differed among the tumors, with follicular lymphoma tending to be more highly mutated. The individual VH and VL sequences have been assembled with a flexible linker sequence to encode single-chain Fv (scFv). The scFv sequences can be cloned into bacterial expression vectors to produce protein, or into vectors suitable for direct vaccination using naked DNA. In a model system, expressed scFv protein retained all idiotypic determinants defined by a panel of five anti-idiotypic monoclonal antibodies (MoAbs). Similarly, expressed scFv proteins from two patients were shown to react with anti-idiotypic antibodies. This approach allows production of potential vaccines from surgical biopsies within 2 to 3 weeks.

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Idiotypic vaccination against human B-cell lymphoma. Rescue of variable region gene sequences from biopsy material for assembly as single-chain Fv personal vaccines

Blood ◽

10.1182/blood.v83.11.3279.3279 ◽

1994 ◽

Vol 83 (11) ◽

pp. 3279-3288 ◽

Cited By ~ 117

Author(s):

RE Hawkins ◽

D Zhu ◽

M Ovecka ◽

G Winter ◽

TJ Hamblin ◽

...

Keyword(s):

B Cell ◽

Tumor Antigens ◽

Variable Region ◽

B Cell Lymphoma ◽

Repeated Sequences ◽

Expression Vectors ◽

Single Chain ◽

Region Gene ◽

Biopsy Material ◽

Single Chain Fv

Abstract Idiotypic determinants on neoplastic B cells could provide tumor antigens for vaccination of patients with B-cell tumors. Because this approach requires an individual vaccine for each patient, simple methods for obtaining idiotypic antigen are desirable. Using polymerase chain reaction (PCR) with family-based V-gene and J-region primers, the variable region genes of heavy and light chains (VH and VL) of Ig have been obtained from biopsy material from 13 patients with B-cell tumors. In each case, analysis of random clones derived from the PCR product showed repeated, clonally-related sequences, whereas normal lymphoid tissue generated no repeated sequences. In 3/3 cases, the repeated sequences were found to be the same as those in a tumor-derived hybridoma. Mutational patterns in the V-genes differed among the tumors, with follicular lymphoma tending to be more highly mutated. The individual VH and VL sequences have been assembled with a flexible linker sequence to encode single-chain Fv (scFv). The scFv sequences can be cloned into bacterial expression vectors to produce protein, or into vectors suitable for direct vaccination using naked DNA. In a model system, expressed scFv protein retained all idiotypic determinants defined by a panel of five anti-idiotypic monoclonal antibodies (MoAbs). Similarly, expressed scFv proteins from two patients were shown to react with anti-idiotypic antibodies. This approach allows production of potential vaccines from surgical biopsies within 2 to 3 weeks.

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Acquisition of potential N-glycosylation sites in the immunoglobulin variable region by somatic mutation is a distinctive feature of follicular lymphoma

Blood ◽

10.1182/blood.v99.7.2562 ◽

2002 ◽

Vol 99 (7) ◽

pp. 2562-2568 ◽

Cited By ~ 167

Author(s):

Delin Zhu ◽

Helen McCarthy ◽

Christian H. Ottensmeier ◽

Peter Johnson ◽

Terry J. Hamblin ◽

...

Keyword(s):

Follicular Lymphoma ◽

B Cell ◽

Somatic Mutation ◽

Germinal Center ◽

Variable Region ◽

B Cell Lymphoma ◽

Single Chain ◽

Single Chain Fv ◽

Glycosylation Sites ◽

Vh Genes

Most patients with follicular lymphoma (FL) have somatically mutated V genes with intraclonal variation, consistent with location in the germinal center site. Using our own and published sequences, we have investigated the frequency of potential N-glycosylation sites introduced into functional VH genes as a consequence of somatic mutation. FL cells were compared with normal memory B cells or plasma cells matched for similar levels of mutation. Strikingly, novel sites were detected in 55 of 70 (79%) patients with FL, compared to 7 of 75 (9%) in the normal B-cell population (P < .001). Diffuse large B-cell lymphoma (DLCL) showed an intermediate frequency (13 of 32 [41%] patients). Myeloma and the mutated subset of chronic lymphocytic leukemia showed frequencies similar to those of normal cells in 5 of 64 (8%) patients and 5 of 40 (13%) patients, respectively. In 3 of 3 random patients with FL, immunoglobulin was expressed as recombinant single-chain Fv inPichia pastoris, and glycosylation was demonstrated. These findings indicate that N-glycosylation of the variable region may be common in FL and in a subset of DLCL. Most novel sites are located in the complementarity-determining regions. VH sequences of nonfunctional VH genes contained few sites, arguing for positive selection in FL. One possibility is that the added carbohydrate in the variable region contributes to interaction with elements in the germinal center environment. This common feature of FL may be critical for tumor behavior.

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An Updated Perspective on Current Prognostic and Predictive Biomarkers in Chronic Lymphocytic Leukemia in the Context of Chemoimmunotherapy and Novel Targeted Therapy

Cancers ◽

10.3390/cancers12040894 ◽

2020 ◽

Vol 12 (4) ◽

pp. 894 ◽

Cited By ~ 3

Author(s):

Jared A. Cohen ◽

Riccardo Bomben ◽

Federico Pozzo ◽

Erika Tissino ◽

Andrea Härzschel ◽

...

Keyword(s):

Targeted Therapy ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Predictive Biomarkers ◽

Variable Region ◽

B Cell Lymphoma ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

Region Gene ◽

Novel Biomarkers

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a variable clinical course. Novel biomarkers discovered over the past 20 years have revolutionized the way clinicians approach prognostication and treatment especially in the chemotherapy-free era. Herein, we review the best established prognostic and predictive biomarkers in the setting of chemoimmunotherapy (CIT) and novel targeted therapy. We propose that TP53 disruption (defined as either TP53 mutation or chromosome 17p deletion), unmutated immunoglobulin heavy chain variable region gene status (UM IGHV), NOTCH1 mutation, and CD49d expression are the strongest prognosticators of disease progression and overall survival in the field of novel biomarkers including recurrent gene mutations. We also highlight the predictive role of TP53 disruption, UM IGHV, and NOTCH1 mutation in the setting of CIT and TP53 disruption and CD49d expression in the setting of novel targeted therapy employing B-cell receptor (BCR) and B-cell lymphoma-2 (BCL2) inhibition. Finally, we discuss future directions in the field of biomarker development to identify those with relapsed/refractory disease at risk for progression despite treatment with novel therapies.

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Single chain Fv cytokine fusion proteins as vaccines for B cell lymphoma

Immunotechnology ◽

10.1016/s1380-2933(97)89084-7 ◽

1996 ◽

Vol 2 (4) ◽

pp. 300

Keyword(s):

B Cell ◽

Fusion Proteins ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Single Chain ◽

Single Chain Fv

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Dissemination of a Sjögren's syndrome–associated extranodal marginal-zone B cell lymphoma: Circulating lymphoma cells and invariant mutation pattern of nodal Ig heavy- and light-chain variable-region gene rearrangements

Arthritis & Rheumatism ◽

10.1002/art.21558 ◽

2005 ◽

Vol 54 (1) ◽

pp. 127-137 ◽

Cited By ~ 15

Author(s):

A. Hansen ◽

K. Reiter ◽

A. Pruss ◽

C. Loddenkemper ◽

O. Kaufmann ◽

...

Keyword(s):

B Cell ◽

Light Chain ◽

Marginal Zone ◽

Cell Lymphoma ◽

Variable Region ◽

B Cell Lymphoma ◽

Gene Rearrangements ◽

Region Gene ◽

Variable Region Gene ◽

Mutation Pattern

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Cell-free production of scFv fusion proteins: an efficient approach for personalized lymphoma vaccines

Blood ◽

10.1182/blood-2006-07-030593 ◽

2006 ◽

Vol 109 (8) ◽

pp. 3393-3399 ◽

Cited By ~ 83

Author(s):

Gregory Kanter ◽

Junhao Yang ◽

Alexei Voloshin ◽

Shoshana Levy ◽

James R. Swartz ◽

...

Keyword(s):

B Cell ◽

Expression System ◽

Specific Antigen ◽

Therapeutic Vaccine ◽

Antibody Fragment ◽

B Cell Lymphoma ◽

Keyhole Limpet Hemocyanin ◽

Single Chain ◽

Single Chain Fv ◽

Fv Antibody

Abstract The unique immunoglobulin (Ig) idiotype on the surface of each B-cell lymphoma represents an ideal tumor-specific antigen for use as a therapeutic vaccine. We have used an Escherichia coli—based, cell-free protein-expression system to produce a vaccine within hours of cloning the Ig genes from a B-cell tumor. We demonstrated that a fusion protein consisting of an idiotypic single chain Fv antibody fragment (scFv) linked to a cytokine (GM-CSF) or to an immunostimulatory peptide was an effective lymphoma vaccine. These vaccines elicited humoral immune responses against the native Ig protein displayed on the surface of a tumor and protected mice against tumor challenge with efficacy equal to that of the conventional Ig produced in a mammalian cell and chemically coupled to keyhole limpet hemocyanin. The cell-free E coli system offers a platform for rapidly generating individualized vaccines, thereby allowing much more efficient application in the clinic.

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Secondary Rearrangements and Hypermutation Generate Sufficient B Cell Diversity to Mount Protective Antiviral Immunoglobulin Responses

Journal of Experimental Medicine ◽

10.1084/jem.189.11.1791 ◽

1999 ◽

Vol 189 (11) ◽

pp. 1791-1798 ◽

Cited By ~ 18

Author(s):

Constantino López-Macías ◽

Ulrich Kalinke ◽

Marilia Cascalho ◽

Matthias Wabl ◽

Hans Hengartner ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Gene Replacement ◽

Antibody Responses ◽

Single Chain ◽

Cell Repertoire ◽

Region Gene ◽

B Cell Repertoire ◽

Single Chain Fv ◽

Cell Diversity

Variable (V) region gene replacement was recently implicated in B cell repertoire diversification, but the contribution of this mechanism to antibody responses is still unknown. To investigate the role of V gene replacements in the generation of antigen-specific antibodies, we analyzed antiviral immunoglobulin responses of “quasimonoclonal” (QM) mice. The B cells of QM mice are genetically committed to exclusively express the anti-(4-hydroxy-3-nitrophenyl) acetyl specificity. However, ∼20% of the peripheral B cells of QM mice undergo secondary rearrangements and thereby potentially acquire new specificities. QM mice infected with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus, or poliovirus mounted virus-specific neutralizing antibody responses. In general, kinetics of the antiviral immunoglobulin responses were delayed in QM mice; however, titers similar to control animals were eventually produced that were sufficient to protect against VSV-induced lethal disease. VSV neutralizing single-chain Fv fragments isolated from phage display libraries constructed from QM mice showed VH gene replacements and extensive hypermutation. Thus, our data demonstrate that secondary rearrangements and hypermutation can generate sufficient B cell diversity in QM mice to mount protective antiviral antibody responses, suggesting that these mechanisms might also contribute to the diversification of the B cell repertoire of normal mice.

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