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1.
Sci Rep ; 9(1): 13471, 2019 09 17.
Article in English | MEDLINE | ID: mdl-31530876

ABSTRACT

Preclinical models and clinical studies have shown that anti-CD20-based treatment has multifaceted consequences on T-cell immunity. We have performed a prospective study of peripheral T-cell compartment in FL patients, all exhibiting high tumor burden and receiving rituximab-chemotherapy-based regimen (R-CHOP). Before treatment, FL patients harbor low amounts of peripheral naive T cells, but high levels of CD4+ TEM, CD4+ Treg and CD8+ TEMRA subsets and significant amounts of CD38+ HLA-DR+ activated T cells. A portion of these activated/differentiated T cells also expressed PD-1 and/or TIGIT immune checkpoints. Hierarchical clustering of phenotyping data revealed that 5/8 patients with only a partial response to R-CHOP induction therapy or with disease progression segregate into a group exhibiting a highly activated/differentiated T cell profile and a markedly low proportion of naive T cells before treatment. Rituximab-based therapy induced a shift of CD4+ and CD8+ T cells toward a central memory phenotype and of CD8+ T cells to a naive phenotype. In parallel, a decrease in the number of peripheral T cells expressing both PD-1 and TIGIT was detected. These observations suggest that the standard rituximab-based therapy partially reverts the profound alterations observed in T-cell subsets in FL patients, and that blood T-cell phenotyping could provide a better understanding of the mechanisms of rituximab-based treatment.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Immunity, Cellular , Immunologic Memory/immunology , Lymphoma, Follicular/drug therapy , Lymphoma, Follicular/immunology , T-Lymphocyte Subsets/immunology , Aged , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Biomarkers , Cyclophosphamide , Doxorubicin , Female , Humans , Immunologic Memory/drug effects , Immunophenotyping , Lymphocyte Activation/drug effects , Lymphocyte Count , Lymphoma, Follicular/diagnosis , Male , Middle Aged , Neoplasm Grading , Neoplasm Staging , Prednisone , Rituximab/administration & dosage , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/metabolism , Treatment Outcome , Vincristine
2.
Leukemia ; 29(4): 947-57, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25231744

ABSTRACT

The long-lasting clinical response by lymphoma patients to anti-CD20 therapy has been attributed to the induction of an anti-tumor adaptive immunity. We previously demonstrated that a CD4-dependent mechanism is responsible for the long-term protection of CD20(+) tumor-bearing mice by anti-CD20 treatment. Here, we compare tumor immunity in tumor-bearing animals that did or did not receive anti-CD20 treatment. Splenic CD4(+)FoxP3(+) regulatory T cells (Tregs) expanded substantially in untreated mice that exhibited then a reduced survival, whereas Tregs depletion led to long-term survival of the animals, suggesting the establishment of a Treg-dependent immunosuppressive environment after tumor injection. Strikingly, anti-CD20 therapy reversed the initial expansion of Tregs, and was accompanied by a marked increase in the number of Th1 cells, with no detectable change in Th2 and Th17 cell numbers. Interleukin-12 serum level was also increased by the anti-CD20 treatment, and activated myeloid dendritic cells producing interleukin-12 could be detected in lymph nodes of treated animals, while interferon-γ blockade strongly reduced survival. Also, CD4(+) effector memory T cells were evidenced in surviving animals, and the transfer of CD4(+) T cells induced long-term protection. Thus, anti-CD20 therapy promotes strong anti-tumor adaptive immunity, opposes Treg expansion and inhibits tumor cells from maintaining an immunosuppressive environment.


Subject(s)
Antibodies, Monoclonal, Murine-Derived/pharmacology , Antineoplastic Agents/pharmacology , Gene Expression Regulation, Leukemic , Interferon-gamma/immunology , Interleukin-12/immunology , Lymphoma, B-Cell/drug therapy , T-Lymphocytes, Regulatory/drug effects , Adaptive Immunity/drug effects , Animals , CD4 Antigens/genetics , CD4 Antigens/immunology , Cell Proliferation/drug effects , Female , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/immunology , Immunologic Memory , Interferon-gamma/genetics , Interleukin-12/genetics , Lymph Nodes/drug effects , Lymph Nodes/immunology , Lymph Nodes/pathology , Lymphoma, B-Cell/immunology , Lymphoma, B-Cell/mortality , Lymphoma, B-Cell/pathology , Mice , Mice, Inbred C57BL , Rituximab , Signal Transduction , Survival Analysis , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathology , Th1 Cells/drug effects , Th1 Cells/immunology , Th1 Cells/pathology , Th17 Cells/drug effects , Th17 Cells/immunology , Th17 Cells/pathology , Th2 Cells/drug effects , Th2 Cells/immunology , Th2 Cells/pathology
3.
Mucosal Immunol ; 7(1): 46-56, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23591718

ABSTRACT

Definition of antibody (Ab) functions capable of preventing mucosal HIV transmission may be critical to both effective vaccine development and the prophylactic use of monoclonal Abs. Although direct antibody-mediated neutralization is highly effective against cell-free virus, increasing evidence suggests an important role for immunoglobulin G (IgG) Fcγ receptor (FcγR)-mediated inhibition of HIV replication. Thus, a panel of well-known neutralizing (NAbs) and nonneutralizing Abs (NoNAbs) were screened for their ability to block HIV acquisition and replication in vitro in either an independent or FcγR-dependent manner. Abs displaying the highest Fc-mediated inhibitory activity in various in vitro assays were selected, formulated for topical vaginal application in a microbicide gel, and tested for their antiviral activity against SHIVSF162P3 vaginal challenge in non-human primates (NHPs). A combination of three NAbs, 2G12, 2F5, and 4E10, fully prevented simian/human immunodeficiency virus (SHIV) vaginal transmission in 10 out of 15 treated NHPs, whereas a combination of two NoNAbs, 246-D and 4B3, although having no impact on SHIV acquisition, reduced plasma viral load. These results indicate that anti-HIV Abs with distinct neutralization and inhibitory functions differentially affect in vivo HIV acquisition and replication, by interfering with early viral replication and dissemination. Therefore, combining diverse Ab properties may potentiate the protective effects of anti-HIV-Ab-based strategies.


Subject(s)
Antibodies, Monoclonal/immunology , HIV Antibodies/immunology , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/prevention & control , Simian Immunodeficiency Virus/immunology , Vagina/immunology , Vagina/virology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Antibody-Dependent Cell Cytotoxicity , Female , HIV Antibodies/administration & dosage , HIV Antibodies/metabolism , Immunoglobulin Fc Fragments/immunology , Immunoglobulin Fc Fragments/metabolism , Macaca fascicularis , Macrophages/immunology , Macrophages/virology , Neutralization Tests , Protein Binding/immunology , Receptors, IgG/metabolism , Simian Acquired Immunodeficiency Syndrome/virology , Virus Replication/immunology
4.
Leukemia ; 25(1): 101-9, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20975664

ABSTRACT

Although anti-CD20 monoclonal antibodies (mAbs) show promise for the treatment of chronic lymphocytic leukemia (CLL), the success of the anti-CD20 mAb rituximab in CLL treatment has been limited. Novel anti-CD20 mAbs with more potent cytotoxic activity have recently been engineered, but so far most have only been tested in vitro with natural killer (NK) cells from healthy donors. Because it is still unclear whether these optimized cytotoxic mAbs will improve NK-cell killing of tumor cells in CLL patients, we characterized the relevant phenotypic and functional features of NK cells from CLL patients in detail. Expression of inhibitory and activating NK-cell receptors and of Fc gamma receptor IIIA (FcγRIIIA) is well preserved in CD16(+)CD56(dim) cytotoxic NK cells from these patients, independently of disease progression. These cells are fully functional following cytokine stimulation. In addition, the FcγRIIIA-optimized LFB-R603 anti-CD20 mAb mediates 100 times greater antibody-dependent cell-mediated cytotoxicity by NK cells from CLL patients and healthy donors than rituximab. Enhanced degranulation against autologous B-CLL cells is observed at lower concentrations of LFB-R603 than rituximab, regardless of CLL prognostic factors. These findings strongly justify further clinical development of anti-CD20 mAbs optimized for FcγR engagement in CLL patients.


Subject(s)
Antibodies, Monoclonal, Murine-Derived/therapeutic use , Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/therapeutic use , CD56 Antigen/analysis , Killer Cells, Natural/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Receptors, IgG/analysis , Adult , Aged , Aged, 80 and over , Antibody-Dependent Cell Cytotoxicity , Female , GPI-Linked Proteins/analysis , Humans , Immunophenotyping , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Male , Middle Aged , Rituximab
5.
Transfus Clin Biol ; 12(2): 114-22, 2005 Jun.
Article in French | MEDLINE | ID: mdl-15907389

ABSTRACT

Thirty years after their discovery by Milstein and Köhler, monoclonal antibodies have now come of age as therapeutics. Nineteen monoclonal antibodies are on the market and/or have got authorization to be used for the treatment of severe diseases. Many technical efforts have been devoted over the last two decades to the generation of second generation mAbs with better affinities, decreased immunogenicity and optimized effector functions. The development of molecular engineering techniques applied to antibody molecules has also made it possible to design bi-specific antibodies and fusion molecules exhibiting different modules with bi-functional activities. The use of proteomics and genomics combined with phage display allows now the rapid selection of antibodies directed against new targets at a high rate. Many efforts are currently focused on the selection of high-responder patients, the optimization of antibody delivery, schemes of infusion, antibody pharmaco-kinetics and bio-distribution, as well as on a better control of the severe side-effects generated by some antibody treatments.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Immunotherapy/trends , Animals , Antibodies, Bispecific/immunology , Antibodies, Bispecific/therapeutic use , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/adverse effects , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibody Affinity , Antigen-Antibody Reactions , Cell Line, Transformed/immunology , Clinical Trials as Topic , Herpesvirus 4, Human , History, 20th Century , Humans , Hybridomas/immunology , Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/immunology , Immunotherapy/history , Immunotherapy/methods , Mice , Peptide Library , Protein Engineering , Rats , Recombinant Fusion Proteins/therapeutic use , Species Specificity
6.
Faraday Discuss ; 126: 289-302; discussion 303-11, 2004.
Article in English | MEDLINE | ID: mdl-14992414

ABSTRACT

It has become increasingly clear that synchrotron infrared micro-spectroscopy is an extremely valuable analysis tool when determining the chemical composition of biological and biomedical samples, at the diffraction-limited spatial resolution. Highly resolved infrared micro-spectroscopy, together with the high signal-to-noise level of the recorded spectra, is essential in generating chemical and statistical (multivariate) images. This is illustrated in the case of individual cell and hair section studies. Unprecedented chemical images of lipid distribution and secondary structure relative concentration have been achieved using the synchrotron source. A comparison with a Focal plane Array imaging system, on the same hair section, shows that, despite the fast imaging processing and improved quality achieved with the focal plane array detectors, spectral quality is markedly superior in the case of the synchrotron source. It is clear that the two approaches could be very complementary if combined on the same sample area, in a synchrotron facility.


Subject(s)
Diagnostic Imaging/instrumentation , Diagnostic Imaging/methods , Spectrophotometry, Infrared/methods , Synchrotrons , Algorithms , Cell Differentiation , Hair/chemistry , Hair/ultrastructure , Humans
7.
Hum Antibodies ; 12(4): 113-21, 2003.
Article in English | MEDLINE | ID: mdl-15156099

ABSTRACT

Tumor specific peptides recognized by T lymphocytes infiltrating solid tumors, as well as the corresponding T cell receptor (TcR) repertoire usage, have been extensively investigated. By contrast, tumor infiltrating B cells and their immunoglobulin (Ig) repertoire have been studied only in a limited number of tumors. The objective of the present study was to determine, whether DNA sequence analysis of the expressed immunoglobulin variable regions of B cells that infiltrate breast cancer, could be used to reveal a potential specific tumor binding capacity of the antibodies. To answer this question, about 200 expressed Ig heavy (VH) and light chain variable gene (VL) regions were cloned, sequenced and comparatively analysed from a typical medullary beast carcinoma (MBC), where the massive B and plasma cell infiltration correlates with favourable prognosis despite of its high grade. The tumor infiltrating B cell Ig heavy and light chain sequences could be classified into clusters, families and subgroups, based on the identity level to germline, showing a pattern of oligoclonality. Some overrepresented clusters could be determined. In the course of a detailed analysis and search in Blastn database, a number of VH and VL sequences showed more than 99% homology to DNA sequences of Ig VH region, with proved tumor antigen binding capacity. Our data suggest, that potential tumor binder Ig VH and VL sequences might be selected using a detailed immunoglobulin variable region analysis. This new approach might have a benefit for further antibody engineering, as difficulties in search for tumor binders by phage library selection might be reduced and the time for selection shortened.


Subject(s)
B-Lymphocytes/immunology , Breast Neoplasms/immunology , Carcinoma, Medullary/immunology , Genes, Immunoglobulin , Immunoglobulin Variable Region/genetics , Lymphocytes, Tumor-Infiltrating/immunology , Female , Humans , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/chemistry , Immunoglobulin Light Chains/genetics , Immunoglobulin Variable Region/chemistry , Sequence Analysis, DNA
8.
Br J Haematol ; 114(3): 671-80, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11552997

ABSTRACT

Clinical grade ex vivo-generated antigen-presenting cells, macrophage-dendritic cells (MAC-DCs) or macrophage-activated killers (MAKs) were derived from peripheral blood mononuclear cells (PBMCs). Cultures (7 d) were performed in non-adherent conditions in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and either interleukin 13 (IL-13) or dihydroxy-vitamin D3 respectively. MAKs were activated during the last 24 h with interferon gamma (IFNgamma). Reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analyses indicated that IL-1beta and tumour necrosis factor alpha (TNFalpha) were produced by both cells. Higher pro-inflammatory cytokine (IL-1beta and TNFalpha) amounts were detected on average in MAK supernatants. In contrast, IL-12 p40 was found only in MAC-DC supernatants, but the biologically active IL-12 form (p70) was never detected. T-cell cytokines (IL-2, IL-4, IL-10) were not produced in culture conditions in which T cells were nevertheless present. At d 7, TNFalpha or lipopolysaccharide (LPS) upregulated IL-12 p40 production by MAC-DCs, while IL-12 p70 remained undetectable. LPS stimulation also increased TNFalpha production by these cells. Allogeneic mixed lymphocyte reactions (MLR) showed that MAKs are poor stimulatory cells compared with MAC-DCs. The MAC-DC stimulatory capacity was enhanced by LPS, although the expression of HLA class II, CD83, CD80 and CD86 was unmodified. Thus, MAC-DCs represent a tool for triggering adaptative immunity, while MAK should be primarily used as effector killer cells.


Subject(s)
Cytokines/biosynthesis , Dendritic Cells/immunology , Lymphocyte Activation , Macrophages/immunology , T-Lymphocytes/immunology , 24,25-Dihydroxyvitamin D 3/pharmacology , Cells, Cultured , Enzyme-Linked Immunosorbent Assay/methods , Fluorescent Antibody Technique, Direct , Fluorescent Antibody Technique, Indirect , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Immunotherapy, Adoptive , Interleukin-1/biosynthesis , Interleukin-1/genetics , Interleukin-12/biosynthesis , Interleukin-12/genetics , Interleukin-13/pharmacology , Lipopolysaccharides/pharmacology , Macrophage Activation , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Stimulation, Chemical , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/genetics
9.
J Immunol ; 166(6): 3882-9, 2001 Mar 15.
Article in English | MEDLINE | ID: mdl-11238632

ABSTRACT

To evaluate the potential role of human placental endothelial cells in the transport of IgG from maternal to fetal circulation, we studied Fc gamma receptor (Fc gamma R) expression by immunohistology and immunoblotting. Several pan-Fc gamma RII Abs that label the placental endothelium displayed a distribution pattern that correlated well with transport functions, being intense in the terminal villus and nil in the cord. In contrast, the MHC class 1-like IgG transporter, FcRn, and the classical Fc gamma RIIa were not expressed in transport-related endothelium of the placenta. Our inference, that Fc gamma RIIb was the likely receptor, we confirmed by analyzing purified placental villi, enriched in endothelium, by immunoblotting with a new Ab specific for the cytoplasmic tail of Fc gamma RIIb. These experiments showed that the Fc gamma RII expressed in villus endothelium was the b2 isoform whose cytoplasmic tail is known to include a phosphotyrosyl-based motif that inhibits a variety of immune responses. We suggest that this receptor is perfectly positioned to transport IgG although as well it may scavenge immune complexes.


Subject(s)
Antigens, CD/biosynthesis , Chorionic Villi/immunology , Chorionic Villi/metabolism , Endothelium, Vascular/immunology , Endothelium, Vascular/metabolism , Receptors, IgG/biosynthesis , Antibodies, Monoclonal/metabolism , Antibody Specificity , Antigens, CD/immunology , Antigens, CD/metabolism , Chorionic Villi/blood supply , Endothelium, Vascular/cytology , Female , Glycosylation , Humans , Microscopy, Fluorescence , Pregnancy , Protein Isoforms/biosynthesis , Protein Isoforms/immunology , Protein Isoforms/metabolism , Receptors, IgG/immunology , Receptors, IgG/metabolism , Reproducibility of Results , Tumor Cells, Cultured , U937 Cells , Umbilical Cord/blood supply , Umbilical Cord/immunology , Umbilical Cord/metabolism
10.
J Immunol ; 166(1): 531-7, 2001 Jan 01.
Article in English | MEDLINE | ID: mdl-11123333

ABSTRACT

Immune complex-mediated inflammatory responses are initiated by Fc gamma R on phagocytes. We report in this study that an inhibitory receptor, Fc gamma RIIb2, is expressed on circulating human monocytes, and when co-cross-linked with stimulatory Fc gamma R it down-regulates effector function. Fc gamma RIIb2 expression is increased by IL-4 and decreased by IFN-gamma, in contrast to the activating receptor, Fc gamma RIIa, which is increased by IFN-gamma and decreased by IL-4. Thus, Th1 and Th2 cytokines differentially regulate the opposing Fc gamma R systems, altering the balance of activating and inhibiting Fc gamma R. The detection and cytokine modulation of Fc gamma RIIb2 in human myeloid cells provide evidence of a negative regulator of immune complex-mediated responses in human phagocytes and offer a new approach to limit Ab-triggered inflammation in autoimmune disease.


Subject(s)
Adjuvants, Immunologic/biosynthesis , Cytokines/physiology , Monocytes/immunology , Monocytes/metabolism , Receptors, IgG/biosynthesis , Th1 Cells/immunology , Th2 Cells/immunology , Adjuvants, Immunologic/antagonists & inhibitors , Adjuvants, Immunologic/genetics , Adjuvants, Immunologic/physiology , Antigens, CD/genetics , Antigens, CD/metabolism , Blotting, Western , Cells, Cultured , Humans , Interferon-gamma/pharmacology , Interleukin-4/pharmacology , Neutrophils/immunology , Neutrophils/metabolism , Phagocytosis/immunology , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Protein Isoforms/metabolism , Receptor Aggregation/immunology , Receptors, IgG/antagonists & inhibitors , Receptors, IgG/genetics , Receptors, IgG/metabolism , Receptors, IgG/physiology , Reverse Transcriptase Polymerase Chain Reaction , Th1 Cells/metabolism , Th2 Cells/metabolism , Transcription, Genetic/immunology , Tumor Cells, Cultured
11.
Gene Ther ; 8(22): 1721-8, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11892840

ABSTRACT

We have developed a novel immunostimulatory molecule against tumor cells, composed of an anti-FcgammaRIII (CD16) scFv fused to the platelet-derived growth factor receptor (PDGFR) transmembrane region. This fusion molecule was stably expressed on the tumor cell surface and retained the ability of the parental antibody to bind soluble CD16. Tumor cells expressing anti-CD16 scFv triggered the release of IL-2 by Jurkat-CD 16/gamma cells and of TNFalpha by monocytes when co-cultured with these cells. Furthermore, NK cells could kill scFv-transfected HLA+ class I H1299 lung carcinoma tumor cells, but not the parental cells, indicating that anti-CD16 scFv tumor expression prevents the killer inhibitory receptor (KIR)-mediated inhibition of NK cell cytotoxicity. This anti-CD16 scFv tumor expression also enhanced tumor phagocytosis by IFNgamma-activated macrophages, a mechanism known to induce a protective long-term adaptative immunity to tumors. In vivo Winn tests performed in SCID mice showed that the expression of anti-CD16 scFv on tumor cells, but not of the negative control anti-phOx scFv, prevented tumor cell growth. Thus, expression of FcR antibodies or other FcR-specific ligands on tumor cells represents a novel and potent antibody-based gene therapy approach, which may have clinical applications in cancer


Subject(s)
Carcinoma, Non-Small-Cell Lung/therapy , Genetic Therapy/methods , Lung Neoplasms/therapy , Receptors, IgG/genetics , Receptors, Platelet-Derived Growth Factor/genetics , Recombinant Fusion Proteins/immunology , Animals , Carcinoma, Non-Small-Cell Lung/immunology , Female , Humans , Interleukin-2/metabolism , Jurkat Cells/immunology , Killer Cells, Natural/immunology , Lung Neoplasms/immunology , Macrophage Activation , Macrophages/immunology , Mice , Mice, SCID
12.
Prep Biochem Biotechnol ; 31(4): 341-54, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11765899

ABSTRACT

The present study describes the methodology used to purify human recombinant low-affinity FcgammaRIIa2 produced in E. coli and to evaluate its binding to surface IgG. The recombinant molecule was purified by a two-step chromatographic procedure, including affinity chromatography using IV.3 anti-FcgammaRIIa1/2 immunosorbent, followed by gel filtration chromatography. Using this method, the purified recombinant FcgammaRIIa2 was 99% pure. It exhibited an isoeletric point of 5.2. Binding studies demonstrated a specific binding of the purified recombinant molecule to surface IgG expressed by human B cells. Thus, we have set up a method which allows to purify functional human recombinant FcgammaRIIa2 for further characterization of its biological activities.


Subject(s)
Chromatography, Affinity/methods , Chromatography, Gel/methods , Escherichia coli/genetics , Receptors, IgG/isolation & purification , B-Lymphocytes/immunology , Binding Sites, Antibody/immunology , Humans , Immunoglobulin G/immunology , Receptors, IgG/immunology , Recombinant Proteins/isolation & purification , Solubility
13.
Bull Cancer ; 87(11): 777-91, 2000 Nov.
Article in French | MEDLINE | ID: mdl-11125287

ABSTRACT

Following 15 years of experimental studies, tumor immunotargeting using monoclonal antibodies directed against tumor associated antigens shows now important monoclonal antibodies directed against tumor associated antigens shows now important clinical developments. This is mainly due to encouraging therapeutic results which have obtained using humanized antibodies such as the anti-CD20 rituximab in follicular B lymphomas and the anti-DrbB2 herceptin in breast carcinomas. Thanks to genetic engineering it is possible to graft variable or hypervariable regions from murine antibodies to human IgG, and even to obtain fully human antibodies by using either transgenic mice containing a large part of the human repertoire of human IgG, or selection of human antibody fragments expressed by phages. Radiolabeling of antibodies played a major role to demonstrate the tumor immunotargeting specificity and remains attractive for the diagnosis by immunoscintigraphy as well as for the treatment by radioimmunotherapy of some cancers. In this review, the current results and the prospects of diagnostic and therapeutic uses of anti-tumor antibodies and their fragments will be described. Concerning diagnosis, 123-iodine or 99m-technetium labeled Fab fragments allowed very demonstrative tumor images but this technique has a limited effect upon the therapeutic attitude. Immuno-PET (positron emission tomography) could enhance the sensitivity of this imaging method. Radio-immunoguided surgery and immunophotodetection are attractive techniques still under evaluation. Concerning therapy, 131-iodine labeled anti-CD20 antibodies gave spectacular results in non-Hodgkin's B lymphomas. In solid tumors which as less radiosensitive, radioimmunotherapy could concern small tumors and need the use of two-steps targeting and/or alpha emitters radioisotopes. Some other strategies will be described such as bispecific antibodies directed against tumors and immune effector cells, some antibody fragments expressed on T cells called T-bodies or some biological studies using intrabodies. Published data and works in progress demonstrate that immunotargeting of tumors will have a growing place in the treatments of cancer patients.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Immunotoxins/therapeutic use , Neoplasms/radiotherapy , Radiopharmaceuticals/therapeutic use , Technology Transfer , Animals , Antibodies, Bispecific/therapeutic use , Antibodies, Monoclonal/chemistry , Bacteriophages/genetics , Genetic Engineering/methods , Humans , Immunoglobulin Fragments/genetics , Immunotoxins/chemistry , Interprofessional Relations , Liposomes , Mice , Neoplasms/diagnostic imaging , Neoplasms/surgery , Radioimmunotherapy/methods , Tomography, Emission-Computed/methods
15.
Pathol Biol (Paris) ; 47(8): 771-5, 1999 Oct.
Article in French | MEDLINE | ID: mdl-10573694

ABSTRACT

Intracellular immunization is a novel therapeutic approach based on intracellular expression of recombinant antibody fragments, either Fab or single chain Fv (scFv generated by the assembly of the VH with the VL region), targeted to the desired cell compartment (cytosol, nucleus, endoplasmic reticulum ...) using appropriate targeting sequences. Due to their exquisite specificity, these intracellular antibodies can be used to neutralize or modulate the functional activity of the target molecule. Intracellular immunization strategies currently under investigation in the field of oncology are directed against mutated oncogenic molecules such as ErbB-2, p21ras, and p53, as well as against apoptosis-inhibiting molecules such as Bcl-2. The first Phase I clinical trials on intracellular immunization are under way in the United States.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Immunization , Amino Acid Sequence , Antibody Specificity , Cell Compartmentation , Humans , Immunoglobulin Fab Fragments/immunology , Molecular Sequence Data , Recombinant Proteins/therapeutic use
16.
Cancer Detect Prev ; 23(6): 506-10, 1999.
Article in English | MEDLINE | ID: mdl-10571661

ABSTRACT

Intracellular expression of recombinant antibodies allows one to interfere with the functions of oncogenic molecules expressed in various cell compartments and has therefore a vast clinical potential in cancer therapy. We inhibited the functions of oncogenic Ras mutant forms by intracellular expression of a neutralizing single-chain antibody (scFv). In vitro studies indicated that the scFv is expressed in the cytosol of Xenopus laevis oocytes and of tumor cells, blocks ras-mediated activation processes, and induces tumor cell death. In vivo studies performed using scFv cDNA inserted into an adenoviral vector showed that the scFv dramatically affects tumor growth. Second, intracellular expression of scFvs directed against p53 indicated that these antibody fragments can be successfully targeted to cell nucleus, bind p53, and partially restore the transcriptional activity of p53 mutants in human tumor cells. Thus, intracellular scFvs directed against oncogenic molecules may represent a new class of antitumor agents.


Subject(s)
Antibodies, Neoplasm/therapeutic use , Immunoglobulin Fragments/immunology , Intracellular Fluid/metabolism , Neoplasms, Experimental/therapy , Proto-Oncogene Proteins p21(ras)/immunology , Tumor Suppressor Protein p53/immunology , Animals , Immunoglobulin Fragments/genetics , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/immunology , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Proto-Oncogene Proteins p21(ras)/biosynthesis , Proto-Oncogene Proteins p21(ras)/genetics , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Tumor Suppressor Protein p53/biosynthesis , Tumor Suppressor Protein p53/genetics
17.
Immunol Lett ; 68(2-3): 347-53, 1999 Jun 01.
Article in English | MEDLINE | ID: mdl-10424442

ABSTRACT

The signaling events induced upon cross-linking of the human FcgammaRIIa1 (CD32) which contains an immune receptor tyrosine-based activation motif (ITAM) in its intracellular region, were investigated in the promyelocytic HL-60 cells. It is shown here that the FcgammaRIIa1 engagement recruits the Ras pathway in these cells, as evidenced by the tyrosine-phosphorylation of the Shc adaptator protein and of the extracellular signal-regulated kinase 2 (ERK2). However, p95vav, a molecule able to interact with Rac-1 and to regulate cytoskeletal reorganization, was also found to be phosphorylated. In addition, co-immunoprecipitation experiments demonstrated that Vav is associated with SLP-76 upon FcgammaRIIa1 activation. A strong phosphorylation of p120cbl was also observed. The phosphorylation of molecules such as p95vav, SLP-76 and p120cbl suggests that FcgammaRIIa1 triggering also activates signaling pathways other than the Ras pathway.


Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , Antigens, CD/metabolism , Cell Cycle Proteins , Leukemia, Promyelocytic, Acute/metabolism , Phosphoproteins/metabolism , Proto-Oncogene Proteins/metabolism , Receptors, IgG/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , HL-60 Cells , Humans , Mitogen-Activated Protein Kinase 1 , Phosphorylation , Protein Binding , Proteins/metabolism , Proto-Oncogene Proteins c-vav , Shc Signaling Adaptor Proteins , Signal Transduction , Src Homology 2 Domain-Containing, Transforming Protein 1
19.
Exp Hematol ; 27(4): 751-61, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10210333

ABSTRACT

Phagocytic cells with macrophage or dendritic cell phenotype, able to capture and ingest tumor cells, were derived in large numbers from peripheral blood mononuclear cells using two different activation procedures. Peripheral blood mononuclear cells were stimulated in nonadherent conditions in the presence of human AB serum with either granulocyte-macrophage colony-stimulating factor and dihydroxy-vitamin D3 for 7 days and with interferon-gamma for the last 18 hours to obtain activated macrophages (MAK) or with granulocyte-macrophage colony-stimulating factor and interleukin-13 for 7 days (with fresh interleukin-13 added on day 4) to obtain macrophage-dendritic cells (MAC-DC). A strong ability of MAC-DC to phagocytose yeasts was observed, in contrast to a low-intermediate phagocytosis capacity by MAK. Both CD14+ FCgammaR+ (FcgammaRI/CD64, FcgammaRII/CD32, FcgammaRIII/CD16) MAK and CD1a+/CD86+, CD14- MAC-DC were able to phagocytose whole tumor cells. However, only MAK phagocytosis was enhanced by FcgammaR engagement. MAK but not MAC-DC could lyse tumor cell in antibody-dependent cell cytotoxicity assays, via FcgammaRI. Thus, MAK as well as MAC-DC may represent valuable tools for different in vivo therapy strategies that do or do not include the use of monoclonal antibodies.


Subject(s)
Dendritic Cells/cytology , Macrophages/cytology , Antibody-Dependent Cell Cytotoxicity , Antigens, CD/biosynthesis , Calcitriol/pharmacology , Cell Differentiation/drug effects , Cell Differentiation/immunology , Cells, Cultured , Cytotoxicity Tests, Immunologic , Dendritic Cells/drug effects , Dendritic Cells/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Immunophenotyping , Interleukin-13/pharmacology , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/drug effects , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Macrophages/drug effects , Macrophages/immunology , Phagocytosis/drug effects , Saccharomyces cerevisiae
20.
Immunol Lett ; 65(3): 143-51, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10065736

ABSTRACT

Breast medullary carcinoma are heavily infiltrated by B-lymphocytes and associated with a good prognosis despite their high histological grade. We investigated the Ig repertoire of B-lymphocytes infiltrating one such tumour. A single cell suspension was obtained from a tumor specimen by enzymatic digestion. VH, Vkappa, and Vlambda regions were amplified by RT-PCR using mixtures of primers optimized to maximize the diversity of the PCR products. They were then cloned and sequenced. Analysis of 9 VH, 5 Vkappa, and 10 Vlambda sequences using the Kabat database indicated that several VH and VL region subgroups (I, II and III) are expressed by B-lymphocytes infiltrating this tumor. The analysis of CDR3 regions also showed a variability, although some VH and VL clones exhibited identical or nearly identical sequences. Thus, the B-cell infiltration observed in this breast medullary carcinoma does not reflect a monoclonal proliferation and represents an oligoclonal or a polyclonal B-cell proliferation.


Subject(s)
B-Lymphocytes/immunology , Breast Neoplasms/immunology , Carcinoma, Medullary/immunology , Immunoglobulin Variable Region/genetics , Lymphocytes, Tumor-Infiltrating/metabolism , Amino Acid Sequence , Antibody Diversity/genetics , B-Lymphocytes/metabolism , Base Sequence , Breast Neoplasms/genetics , Carcinoma, Medullary/genetics , Cloning, Molecular , Gene Rearrangement, B-Lymphocyte, Heavy Chain , Gene Rearrangement, B-Lymphocyte, Light Chain , Humans , Immunoglobulin Heavy Chains/biosynthesis , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/biosynthesis , Immunoglobulin Light Chains/genetics , Immunoglobulin Variable Region/biosynthesis , Molecular Sequence Data , Multigene Family/immunology , Tumor Cells, Cultured
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