Construction and characterisation of a functional CD19 specific single chain Fv fragment for immunotherapy of B lineage leukaemia and lymphoma

doi:10.1016/S0161-5890(97)00144-2

Molecular Immunology

Volume 34, Issues 16–17, November–December 1997, Pages 1157-1165

https://doi.org/10.1016/S0161-5890(97)00144-2 Get rights and content

Abstract

The B cell specific antigen CD19 is a target for the immunotherapy of B lineage leukaemias and lymphomas. We have engineered a single chain Fv (scFv) fragment from the mouse hybridoma cell line FMC63 which produces monoclonal antibody specific for CD19. The genes encoding the FMC63 heavy and light chain variable regions were amplified from cDNA and a scFv was constructed by splice overlap extension PCR. Analysis of staining of lymphoblastoid cell lines, peripheral blood lymphocytes and tonsil sections demonstrated that the monovalent scFv fragment has the same cellular specificity as the parent hybridoma antibody. Kinetic studies with radiolabelled material showed that the scFv binds target cells with a K_a of 2.3 × 10⁻⁹ for the parent antibody. This CD19 scFv will be used in experimental models to test its therapeutic efficacy and immunogenicity, with a view to application in the diagnosis and treatment of human B cell cancers.

References (46)

P. Antoniw et al.
Radioimmunotherapy of colorectal carcinoma xenografts in nude mice with yttrium-90 A33 IgG and Tri-Fab (TFM)
Br. J. Cancer
(1996)
C. Bebbington
Expression of antibody genes in mammalian cells
R.H. Begent et al.
Clinical evidence of efficient tumor targeting based on single-chain Fv antibody selected from a combinatorial library
Nat. Med.
(1996)
B.E. Bejcek et al.
Development and characterization of three recombinant single chain antibody fragments (scFvs) directed against the CD19 antigen
Cancer Res.
(1995)
A.E. Bolton et al.
The labelling of proteins to high specific radioactivities by conjugation to a ¹²⁵I-containing acylating agent
Biochem. J.
(1973)
W.L. Carroll et al.
Hybridoma fusion cell lines contain an aberrant κ transcript
Mol. Immunol.
(1988)
P. Carter et al.
Improved oligonucleotide site-directed mutagenesis using M13 vectors
Nucleic Acids Res.
(1985)
J.L. Casey et al.
Preparation, characterisation and tumour targeting of cross-linked divalent and trivalent anti-tumour Fab′ fragments
Br. J. Cancer
(1996)
T. Clackson et al.
General applications of PCR to gene cloning and manipulation
B. Dorken et al.
B cell antigens: CD19

G.I. Evan et al.

Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product

Mol. Cell Biol.

(1985)

J.V. Gavilondo-Cowley et al.

Specific amplification of rearranged immunoglobulin variable region genes from mouse hybridoma cells

Hybridoma

(1990)

M.A. Ghetie et al.

Anti-CD19 inhibits the growth of human B-cell tumor lines in vitro and of Daudi cells in SCID mice by inducing cell cycle arrest

Blood

(1994)

M.A. Ghetie et al.

Recent developments in immunotoxin therapy

Curr. Opin. Immunol.

(1994)

M.L. Grossbard et al.

Anti-B4-blocked ricin: a phase I trial of 7-day continuous infusion in patients with B-cell neoplasms

J. Clin. Oncol.

(1993)

G. Hale et al.

Remission induction in non-Hodgkin lymphoma with reshaped human monoclonal antibody CAMPATH-1H

Lancet

(1988)

F. Hartmann et al.

Treatment of refractory Hodgkin's disease with an $anti- CD16 CD30$ bispecific antibody

Blood

(1997)

A. Hekman et al.

Initial experience with treatment of human B cell lymphoma with anti-CD19 monoclonal antibody

Cancer Immunol Immunother

(1991)

H.R. Hoogenboom et al.

Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains

Nucleic Acids Res.

(1991)

R. Irving et al.

Superseding hybridoma technology with phage display libraries

B. Jansen et al.

Establishment of a human t(4; 11) leukemia in severe combined immunodeficient mice and successful treatment using anti-CD19 (B43)-pokeweed antiviral protein immunotoxin

Cancer Res.

(1992)

L.S. Jespers et al.

Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen

Biotechnology N.Y.

(1994)

F.T. Lee et al.

Technetium-99 m labelling of DD-3B6/22 antifibrin monoclonal antibody fragment Fab′ for thrombus imaging

Immunol. Cell Biol.

(1993)

Cited by (168)

Class I HDAC inhibitors enhance antitumor efficacy and persistence of CAR-T cells by activation of the Wnt pathway
2024, Cell Reports
Epigenetic modification shapes differentiation trajectory and regulates the exhaustion state of chimeric antigen receptor T (CAR-T) cells. Limited efficacy induced by terminal exhaustion closely ties with intrinsic transcriptional regulation. However, the comprehensive regulatory mechanisms remain largely elusive. Here, we identify class I histone deacetylase inhibitors (HDACi) as boosters of CAR-T cell function by high-throughput screening of chromatin-modifying drugs, in which M344 and chidamide enhance memory maintenance and resistance to exhaustion of CAR-T cells that induce sustained antitumor efficacy both in vitro and in vivo. Mechanistically, HDACi decrease HDAC1 expression and enhance H3K27ac activity. Multi-omics analyses from RNA-seq, ATAC-seq, and H3K27ac CUT&Tag-seq show that HDACi upregulate expression of TCF4, LEF1, and CTNNB1, which subsequently activate the canonical Wnt/β-catenin pathway. Collectively, our findings elucidate the functional roles of class I HDACi in enhancing CAR-T cell function, which provides the basis and therapeutic targets for synergic combination of CAR-T cell therapy and HDACi treatment.
Digital PCR Improves Sensitivity and Quantification in Monitoring CAR-T Cells in B Cell Lymphoma Patients
2024, Transplantation and Cellular Therapy
Chimeric antigen receptor T cells (CAR-T) has emerged as a promising therapy, over 60% of patients fail to sustain a long-term response. The underlying factors that leads to the effectiveness of this therapy are not completely understood, CAR-T cell persistence and monitoring seems to be pivotal for ensuring a successful response. Various monitoring methods such as multiparametric flow cytometry (MFC) or quantitative PCR (qPCR) have been applied. Our objective is to develop digital PCR (dPCR) assays for detection and quantification of CAR-T cells, comparing them with MFC and qPCR. Samples taken at different follow-up times from 45 patients treated with CAR-T therapy were analyzed to assess the correlation between the different methodologies. dPCR presented a high correlation with MFC and qPCR (r = 0.97 and r = 0.87, respectively), while offering a higher sensitivity (0.01%) compared to MFC (0.1%) and qPCR (1%). dPCR emerged as an alternative and highly sensitivity method for monitoring CAR-T cell dynamics. This technique is well-suited for implementation in clinical practice as a complementary technique to MFC.
Inhibition of CD38 enzymatic activity enhances CAR-T cell immune-therapeutic efficacy by repressing glycolytic metabolism
2024, Cell Reports Medicine
Chimeric antigen receptor (CAR)-T therapy has shown superior efficacy against hematopoietic malignancies. However, many patients failed to achieve sustainable tumor control partially due to CAR-T cell exhaustion and limited persistence. In this study, by performing single-cell multi-omics data analysis on patient-derived CAR-T cells, we identify CD38 as a potential hallmark of exhausted CAR-T cells, which is positively correlated with exhaustion-related transcription factors and further confirmed with in vitro exhaustion models. Moreover, inhibiting CD38 activity reverses tonic signaling- or tumor antigen-induced exhaustion independent of single-chain variable fragment design or costimulatory domain, resulting in improved CAR-T cell cytotoxicity and antitumor response. Mechanistically, CD38 inhibition synergizes the downregulation of CD38-cADPR -Ca²⁺ signaling and activation of the CD38-NAD⁺-SIRT1 axis to suppress glycolysis. Collectively, our findings shed light on the role of CD38 in CAR-T cell exhaustion and suggest potential clinical applications of CD38 inhibition in enhancing the efficacy and persistence of CAR-T cell therapy.
Signaling via a CD27-TRAF2-SHP-1 axis during naive T cell activation promotes memory-associated gene regulatory networks
2024, Immunity
The interaction of the tumor necrosis factor receptor (TNFR) family member CD27 on naive CD8⁺ T (Tn) cells with homotrimeric CD70 on antigen-presenting cells (APCs) is necessary for T cell memory fate determination. Here, we examined CD27 signaling during Tn cell activation and differentiation. In conjunction with T cell receptor (TCR) stimulation, ligation of CD27 by a synthetic trimeric CD70 ligand triggered CD27 internalization and degradation, suggesting active regulation of this signaling axis. Internalized CD27 recruited the signaling adaptor TRAF2 and the phosphatase SHP-1, thereby modulating TCR and CD28 signals. CD27-mediated modulation of TCR signals promoted transcription factor circuits that induced memory rather than effector associated gene programs, which are induced by CD28 costimulation. CD27-costimulated chimeric antigen receptor (CAR)-engineered T cells exhibited improved tumor control compared with CD28-costimulated CAR-T cells. Thus, CD27 signaling during Tn cell activation promotes memory properties with relevance to T cell immunotherapy.
CAR therapy: The role of academic institutions in its production
2024, Bulletin de l'Academie Nationale de Medecine
L’avènement des lymphocytes T à récepteur antigénique chimérique a transformé le traitement de nombreuses hémopathies malignes. La disponibilité de ces produits a amélioré la survie des patients, mais elle a également conduit à l’émergence d’inégalités dans l’Union européenne. Le prix élevé, associé à la capacité de production limitée des laboratoires pharmaceutiques, explique pourquoi une proportion très élevée de patients candidats à ces thérapies ne peuvent pas en bénéficier. En même temps, les institutions académiques à but non lucratif sont capables de développer ces produits de même qualité que les produits commercialisés et dont l’efficacité et la sécurité sont également comparables. Cet article explique les étapes nécessaires pour atteindre cet objectif.
The advent of T lymphocytes with chimeric antigen receptors has transformed the treatment of many haematological malignancies. The availability of these products has improved patient survival, but it has also led to the emergence of inequalities in the European Union. The high price, combined with the limited production capacity of pharmaceutical companies, explains why a very high proportion of patients who are candidates for these therapies cannot benefit from them. At the same time, not-for-profit academic institutions can develop products of the same quality as those on the market, with comparable efficacy and safety. This article explains the steps needed to achieve this objective.
CRISPR-Cas12a-integrated transgenes in genomic safe harbors retain high expression in human hematopoietic iPSC-derived lineages and primary cells
2023, iScience
Discovery of genomic safe harbor sites (SHSs) is fundamental for multiple transgene integrations, such as reporter genes, chimeric antigen receptors (CARs), and safety switches, which are required for safe cell products for regenerative cell therapies and immunotherapies. Here we identified and characterized potential SHS in human cells. Using the CRISPR-MAD7 system, we integrated transgenes at these sites in induced pluripotent stem cells (iPSCs), primary T and natural killer (NK) cells, and Jurkat cell line, and demonstrated efficient and stable expression at these loci. Subsequently, we validated the differentiation potential of engineered iPSC toward CD34⁺ hematopoietic stem and progenitor cells (HSPCs), lymphoid progenitor cells (LPCs), and NK cells and showed that transgene expression was perpetuated in these lineages. Finally, we demonstrated that engineered iPSC-derived NK cells retained expression of a non-virally integrated anti-CD19 CAR, suggesting that several of the investigated SHSs can be used to engineer cells for adoptive immunotherapies.

View all citing articles on Scopus

^§: Present Address: Department of Development and Genetics, Babraham Institute, Babraham, Cambridge, CB2 4AT, U.K.

View full text

Construction and characterisation of a functional CD19 specific single chain Fv fragment for immunotherapy of B lineage leukaemia and lymphoma

Abstract

Radioimmunotherapy of colorectal carcinoma xenografts in nude mice with yttrium-90 A33 IgG and Tri-Fab (TFM)

Br. J. Cancer

Expression of antibody genes in mammalian cells

Clinical evidence of efficient tumor targeting based on single-chain Fv antibody selected from a combinatorial library

Nat. Med.

Development and characterization of three recombinant single chain antibody fragments (scFvs) directed against the CD19 antigen

Cancer Res.

The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent

Biochem. J.

Hybridoma fusion cell lines contain an aberrant κ transcript

Mol. Immunol.

Improved oligonucleotide site-directed mutagenesis using M13 vectors

Nucleic Acids Res.

Preparation, characterisation and tumour targeting of cross-linked divalent and trivalent anti-tumour Fab′ fragments

Br. J. Cancer

General applications of PCR to gene cloning and manipulation

B cell antigens: CD19

Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product

Mol. Cell Biol.

Specific amplification of rearranged immunoglobulin variable region genes from mouse hybridoma cells

Hybridoma

Anti-CD19 inhibits the growth of human B-cell tumor lines in vitro and of Daudi cells in SCID mice by inducing cell cycle arrest

Blood

Recent developments in immunotoxin therapy

Curr. Opin. Immunol.

Anti-B4-blocked ricin: a phase I trial of 7-day continuous infusion in patients with B-cell neoplasms

J. Clin. Oncol.

Remission induction in non-Hodgkin lymphoma with reshaped human monoclonal antibody CAMPATH-1H

Lancet

Treatment of refractory Hodgkin's disease with an anti-CD16CD30 bispecific antibody

Blood

Initial experience with treatment of human B cell lymphoma with anti-CD19 monoclonal antibody

Cancer Immunol Immunother

Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains

Nucleic Acids Res.

Superseding hybridoma technology with phage display libraries

Establishment of a human t(4; 11) leukemia in severe combined immunodeficient mice and successful treatment using anti-CD19 (B43)-pokeweed antiviral protein immunotoxin

Cancer Res.

Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen

Biotechnology N.Y.

Technetium-99 m labelling of DD-3B6/22 antifibrin monoclonal antibody fragment Fab′ for thrombus imaging

Immunol. Cell Biol.

The labelling of proteins to high specific radioactivities by conjugation to a ¹²⁵I-containing acylating agent

Treatment of refractory Hodgkin's disease with an $anti- CD16 CD30$ bispecific antibody