Helper or cytolytic functions can be selectively induced in bifunctional T cell clones.

By using bifunctional T cell populations, we have shown in this report that elicitation of helper versus cytolytic function depends on the stimulatory signal at the membrane. Interestingly enough, the transduction of these signals is likely to be achieved via different metabolic pathways. Thus, helper function is associated with intracellular Ca2+ mobilization and PLC activation, while cytolysis can occur even in the absence of detectable levels of these second messengers. These results indicate that selective activation through the same membrane-transducing molecule may orientate T cell function through qualitatively or quantitatively different second messengers. This would be an important part of immune regulation.

Monoclonal antibodies reveal the structural basis of antibody diversity.

Hybridoma technology has made it possible to introduce into continuous culture normal antibody-forming cells and to obtain large amounts of the immunoglobulin produced by each of these cells. Examination of the structure of a number of monoclonal antibodies that react with a single antigen has provided new information on the structural basis of the specificity and affinity of antibodies. Comparisons of families of monoclonal antibodies derived from a single germ line gene revealed the importance of somatic mutation in generating antibody diversity. Monoclonal antibodies that react with variable regions of other monoclonals allow the further dissection and modulation of the immune response. Finally, the continued somatic instability of immunoglobulin genes in cultured antibody-forming cells makes it possible to determine the rate of somatic mutation and to generate mutant monoclonal antibodies that may be more effective serological reagents.

Recovery of central memory and naive peripheral T cells in Follicular Lymphoma patients receiving rituximab-chemotherapy based regimen.

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.

Effect of doxorubicin on mouse hybridoma B cells: stimulation of immunoglobulin synthesis and secretion.

The purpose of these studies was to investigate whether the cell-differentiating effect of anthracyclines can trigger an over-production and secretion of molecules that may interfere with the tumor-host relationship. We exposed mouse hybridoma B-cells, which are devoted to immunoglobulin production, to doxorubicin (10-40 ng/ml). We found that most doxorubicin-treated cells secreted 3- to 5-fold higher amounts of immunoglobulin than untreated cells, along with an accumulation of 50% of them in the G2+M phase of the cell cycle. The antigenic specificity of the immunoglobulin and its size pattern as determined by polyacrylamide gel electrophoresis were similar whether or not cells were treated with doxorubicin. The enhancement of immunoglobulin secretion by doxorubicin was associated with an increase of the intracellular pool of heavy and light chains of the immunoglobulin. Furthermore, an elevated synthesis of immunoglobulin was observed. The synthesis of other proteins also appeared to be modified in these circumstances. These data suggest that doxorubicin can potentiate the biological functions of target cells when used at low concentrations, elevating the production and secretion of effector molecules that interfere with the tumor-host relationship.

In vivo acquisition of Fc gamma RII expression on polyoma virus-transformed cells derived from tumors of long latency.

BALB/c 3T3 cells transformed in vitro with polyoma virus were cloned and passaged once in syngeneic mice. Resulting tumors from each clone were explanted and recultured. Expression of receptor for Fc of IgG (Fc gamma RII) in the original in vitro maintained clones and in cells derived from tumors elicited by the respective cells was measured at the protein level as well as at the mRNA level. Clones were assayed in pairs. The ancestor in vitro maintained clones [designated cultured cells (C)] were compared with cells derived from the same clones after a single passage in vivo followed by explantation and reculturing [designated cultured-tumor-cultured cells (CTC)]. C cells of any of the tested clones did not express Fc gamma RII. On the other hand, certain CTC cells were positive. The Fc gamma RII-positive cells were derived from tumors appearing after a long precancer latency period (greater than 140 days). CTC cells derived from tumors that appeared after shorter latency periods (less than 80 days) were Fc gamma RII negative. These results were obtained both by using radioimmunoassay and monoclonal antibodies against mouse Fc gamma RII as well as by Northern blot analysis using the Fc gamma RII complementary DNA probe. The involvement of macrophages as the Fc gamma RII-expressing cells in CTC cells was excluded. Fc gamma RII expression was down-regulated in CTC cells as a function of time following their explantation into culture. Fc gamma RII expression could be up-regulated in these cells and induced on C cells by maintaining the cultured cells in the presence of normal mouse serum or recombinant interferon. We also tested the expression of Fc gamma RII on CTC cells following their inoculation into syngeneic mice for a second time (CTCx2 cells). The results showed a positive correlation between Fc gamma RII expression in the inoculated ancestor CTC cells and on the CTCx2 cell progeny.

Intracellular expression of an antibody fragment-neutralizing p21 ras promotes tumor regression.

Mutated ras genes are found in a large number of human tumors and, therefore, constitute one of the primary targets for cancer treatment. Microinjection of the neutralizing anti-Ras monoclonal antibody Y13-259 was previously reported to induce transient phenotypic reversion of ras-transformed rodent fibroblasts in vitro. We have prepared a single-chain Fv fragment (scFv) derived from Y13-259, and here, we show that intracellular expression of the scFv led to the specific inhibition of the Ras signaling pathway in Xenopus laevis oocytes and NIH3T3 fibroblasts. Moreover, neutralizing Ras with the scFv specifically promoted apoptosis in vitro in human cancer cells but not in untransformed cells. As a step toward cancer gene therapy, we finally demonstrated that intratumor transduction of HCT116 colon carcinoma cells with the anti-Ras scFv using an adenoviral vector elicited sustained tumor regression in nude mice.

Restoration of transcriptional activity of p53 mutants in human tumour cells by intracellular expression of anti-p53 single chain Fv fragments.

We report here the production and the properties of single chain Fv fragments (scFvs) derived from the anti-p53 monoclonal antibodies PAb421 and 11D3. 11D3 is a newly generated monoclonal antibody which exhibits properties very comparable to those of PAb421. The scFvs PAb421 and 11D3 are able to stably associate with p53 and to restore the DNA binding activity of some p53 mutants in vitro. When expressed in p53 -/-human tumour cells, the scFv421 is essentially localized in the cytoplasm in the absence of p53, and in the nucleus when exogenous p53 is present. Thus, p53 is also able to stably associate with an anti-p53 scFv in cells. Cotransfection of p53 -/- human tumour cells with expression vectors encoding the His273 p53 mutant and either scFv leads to restoration of the p53 mutant deficient transcriptional activity. These data demonstrate that, in human tumour cells, these scFvs are able to restore a function essential for the tumour suppressor activity of p53 and may represent a novel class of molecules for p53-based cancer therapy.

A tumor specific single chain antibody dependent gene expression system.

The design of conditional gene expression systems restricted to given tissues or cellular types is an important issue of gene therapy. Systems based on the targeting of molecules characteristic of the pathological state of tissues would be of interest. We have developed a synthetic transcription factor by fusing a single chain antibody (scFv) directed against p53 with the bacterial tetracycline repressor as a DNA binding domain. This hybrid protein binds to p53 and can interact with a synthetic promoter containing tetracycline-operator sequences. Gene expression can now be specifically achieved in tumor cells harboring an endogenous mutant p53 but not in a wild-type p53 containing tumor cell line or in a non-transformed cell line. Thus, a functional transactivator centered on single chain antibodies can be expressed intracellularly and induce gene expression in a scFv-mediated specific manner. This novel class of transcriptional transactivators could be referred as 'trabodies' for transcription-activating-antibodies. The trabodies technology could be useful to any cell type in which a disease related protein could be the target of specific antibodies.

p53 antibodies in patients with various types of cancer: assay, identification, and characterization.

Alteration of the p53 gene is the most frequent genetic alteration in human cancer and leads to the accumulation of mutant p53 in the nucleus of tumor cells. In addition, it has been shown that patients with various types of neoplasia have p53 antibodies in their sera which could be used as an indirect diagnostic procedure for p53 alteration. Using a new ELISA, we have analyzed the sera from more than 1000 patients with various types of cancer and from healthy blood donors. We demonstrate that p53 antibodies are detected mainly in cancer patients and are strictly proportional to the occurrence of p53 mutations. Using various immunological approaches, these antibodies were unambiguously demonstrated to be directed toward the human p53 protein. Isotyping analysis of these antibodies strongly suggested that they correspond to a humoral response to the p53 protein which accumulates in the tumor cell. This finding suggests that serological analysis, combined with histochemistry, is suitable for assessing the integrity of the p53 gene in cancer patients.

Soluble Fc gamma receptors.

Soluble Fc gamma receptors have been identified in biological fluids of mice and humans. They are produced either by alternative splicing of the exon encoding the transmembrane region of the receptor (Fc gamma RII) or by proteolytic cleavage at the cell membrane (Fc gamma RII and Fc gamma RIII). They inhibit B cell proliferation and immunoglobulin production. Their concentrations in plasma seem to be modified during the development of certain diseases, as for instance in multiple myeloma, where plasma concentrations of soluble Fc gamma RIII are correlated with the stage of the disease.

[Therapeutic monoclonal antibodies: a little history, a lot of engineering, and... some clinical successes]. / Anticorps monoclonaux à usage thérapeutique: un peu d'histoire, beaucoup d'ingénierie, et... quelques succès cliniques.

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.

Regulatory effects of IgG-BF on hybridoma B cells. molecular characterization of variant cell lines.

Immunoglobulin G-binding factors (IgG-BF) produced by mouse T cells or hybridoma T cells (T2D4) were used to manipulate in vitro mouse hybridoma B cells. Both IgG production by, and proliferation of, these cells was inhibited by IgG-BF, or during co-cultures with IgG-BF-producing T2D4 cells. Thus, treatment of tumor B cells, besides its potential therapeutic use, represents an invaluable model for studying the regulation of Ig production by IgG-BF at a molecular level. To further analyze the molecular events induced by IgG-BF in B cell hybridomas, a set of variant clones of a hybridoma cell line (UN2) was isolated and variants were characterized for their Ig production and their Fc gamma R expression.

Ion channels and B cell mitogenesis.

Given the presence of ionic channels at the membrane of lymphocytes, we have analyzed the effect of various channels blockers on B lymphocytes activation. TEA and 4-AP, two K+ channels blockers, quinine, a blocker of Ca2(+)-activated K+ channels, nickel and verapamil, two Ca2+ channels blockers, all inhibited LPS-induced B cell proliferation. However, these drugs neither inhibited the induction of Ia and Fc gamma RII expression nor cell enlargement and early RNA synthesis, indicating that the entry of B lymphocytes into G1 phase was not affected. In contrast, both late RNA synthesis and the induction of the TfR, which occur while the cell progress through G1, were inhibited by these blockers. These data show that TEA, quinine and verapamil block B lymphocyte activation during the G1 phase, probably between G1A and G1B. To question whether these effects were due to the block of voltage-activated K+ channels, we compared the ability of TEA, quinine, verapamil, 4-AP and nickel to block proliferation and K+ channels. A striking correlation was found for all the drugs but less for 4-AP. Moreover, TMA, a TEA analog unable to block K+ currents, did not affect B cell proliferation. Taken together, our data suggests that functional voltage-gated K+ channels are required at a precise stage of the G1 phase of the B cell cycle.

Intracellular expression and functional properties of an anti-p21Ras scFv derived from a rat hybridoma containing specific lambda and irrelevant kappa light chains.

A rat single-chain Fv (Y238 scFv) was derived from the Y13-238 monoclonal antibody, a non-neutralizing anti-Ras antibody. The Y13-238 hybridoma expresses two functional light chains. N-terminus microsequencing of these chains showed the presence of the Y3 Ag1.2.3 Vkappa chain derived from the rat fusion partner and of a rat Vlambda chain. Primers designed for rat Vlambda amplification allowed the cloning of a functional scFv that could bind p21Ras. The kinetics of interaction of purified Y238 scFv with the p21Ras protein was evaluated by BIAcore with a NTA sensor chip and gave an apparent affinity constant in the nanomolar range (K(D)=4.58+/-0.63 nM). Immunoprecipitation experiments of Y238 scFv expressed in Xenopus laevis oocytes confirmed the specificity of the scFv for the Ras protein. Y238 scFv could be intracellularly expressed in oocytes and in mammaliam cells without adverse effect on the Ras signalling cascade. This scFv was therefore used as control in experiments where another anti-Ras scFv (Y259 scFv, derived from the neutralizing anti-Ras mAb Y13-259) blocked the Ras pathway in vitro and led to tumor regression in a nude mouse model [Cochet, O., Kenigsberg, M., Delumeau, I., Virone-Oddos, A., Multon, M.C., Fridman, W.H., Schweighoffer, F., Teillaud, J.L., Tocqué, B., 1998. Intracellular expression of an antibody fragment-neutralizing p21 ras promotes tumor regression. Cancer Res. 58, 1170-1176.]. Finally, BIAcore analyses indicated that the epitopes recognized by Y238 and Y259 scFvs are not overlapping and allowed a more precise definition of the Y13-238 epitope.

Some cellular and molecular characteristics of high and low tumorigenicity variants of polyoma-virus transformed cells.

We analyzed several cellular and molecular properties of BALB/c 3T3 cellular clones transformed in vitro with polyoma virus and exhibiting a high or low tumorigenicity phenotype. We also analyzed the same clones after a single in vivo passage in syngeneic mice. This passage invariably induced and/or selected variants exhibiting a very high tumorigenicity phenotype. BALB/c mice bearing tumors induced by the inoculation of the above cells, regardless of their tumorigenicity phenotype, have a lower number of L3T4 positive splenocytes than appropriate controls. The response to Con-A of spleen cells from such mice was also suppressed. Concomitantly, an increase in Mac-1 positive splenocytes could be measured. In spite of the non-specific suppression of T cells, spleen cells from tumor-bearers showed a specific proliferative response to polyoma antigens. Molecular analysis of polyoma transformed cells showed no differences between the various cells with respect to integration of the polyoma viral genes or with respect to src, myc and fos proto-oncogenes. In vitro maintained cells and in vivo passaged cells seemed to differ, however, in the content of polyoma middle T. Whereas polyoma virus transformed cells maintained only in culture never expressed low affinity receptors for IgG (Fc gamma RII), certain in vivo passaged cells did. This expression could be measured both at the protein and the mRNA level. Those in vivo passaged cells which expressed F alpha RII gave tumors following a long latency period. Ongoing experiments will indicate whether or not Fc gamma RII expression is linked to long latency of tumor development.

Increased expression of Fc gamma receptor in cancer patients and tumor bearing mice.

In this study we report on some lines of ongoing research performed in our laboratory, in relation to the increased expression of FcR on tumor cells, as well as on cells present in the tumor-bearing host, and its possible role in tumor progression. In a previous study we have shown that a Polyoma virus (PyV)-induced anaplastic carcinoma (SEYF-a tumor) contained an FcR-expressing subpopulation of tumorigenic cells. We tested the effect of in vivo passaging of FcR-expressing and of non-FcR-expressing sub-populations of SEYF-a tumor cells on the expression of FcR, as revealed by the ability of these cells to bind the 2.4G2 monoclonal antibody, which is directed against mouse Fc gamma 2b/gamma 1R. It was found that upon in vivo passaging these two sub-populations became practically identical in their ability to bind anti-Fc gamma R antibody. On the other hand, in vitro passaging of FcR-expressing SEYF-a cells resulted in a gradual decrease in the expression of Fc gamma R. These results, indicating that the expression of Fc gamma R on tumor cells, per se, is dependent on a factor present in the in vivo environment were confirmed using 3T3 cells transformed in vitro by PyV (C) and forming tumors at first injection to mice (CTC). C cultures of various clones did not express Fc gamma R, while CTC cultures (cultures from tumors) became positive. We also detected an increase in the level of a soluble form of Fc gamma 2b/gamma 1R in the circulation of mice bearing PyV induced tumors. This increase paralleled the appearance of palpable tumors. A similar pattern of increase was observed in mice inoculated with the c-H-ras transformed tumorigenic clone 8/F/5, but not in mice inoculated with non-tumorigenic 3T3 cells. Data published by us show that metastatic breast cancer patients had significantly elevated Fc gamma R levels on their peripheral blood mononuclear cells (PBMC). Experiments presented here indicate a direct correlation between increased Fc gamma R levels on PBMC and tumor mass in colon, ovary and lung metastatic carcinoma patients. The possibility that malignantly transformed cells have the potential to cause proliferation of Fc gamma R expressing T cells was tested. It was found that extract derived from r-H-ras transformed 3T3 cells triggers the proliferation of a T cell hybridoma expressing Fc gamma R.

Generation of phagocytic MAK and MAC-DC for therapeutic use: characterization and in vitro functional properties.

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.

Anti-CD20 therapy induces a memory Th1 response through the IFN-γ/IL-12 axis and prevents protumor regulatory T-cell expansion in mice.

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.

Release of soluble Fc gamma RII/CD32 molecules by human Langerhans cells: a subtle balance between shedding and secretion?

Freshly isolated human Langerhans cells (LC) express two forms of Fc gamma RII: a membrane-associated form detected by monoclonal antibody (MoAb) anti-CD32, which recognize an extracytoplasmic epitope of the molecule, and a soluble secreted form, whose existence is suggested by reverse transcriptase-polymerase chain reaction (RT-PCR) experiments. Indeed, RT-PCR performed on total LC RNA reveals the presence of two Fc gamma RIIA mRNA, one encoding the FC gamma RIIA with a transmembrane region (membranous form) and the other without this region (soluble form). Densitometry studies performed on the two PCR products reveal that the ratio between the membranous form and the soluble secreted form is about 1.5. LC maintained in culture for 24-48 h lose the major part of their membrane Fc gamma RII expression (shown by flow cytometry) and release soluble Fc gamma RII molecules (revealed by dot-blot assay), but maintain the same ratio of the two Fc gamma RIIA mRNA. The disappearance of the membrane-associated Fc gamma RII may be explained either by modification of its recycling pathway or by proteolytic cleavage of the receptor at the cell surface. Thus, soluble Fc gamma RII molecules generated during LC culture may result from proteolytic cleavage of the cell-surface receptor and/or secretion of a soluble form derived from the translation of an alternate spliced mRNA. Interestingly, addition of TNF-alpha (10 ng/ml) to the culture medium i) maintains the expression of the membranous form, which can be detected on the LC surface at the same level as on freshly isolated LC, and ii) reverses the ratio (to 0.6) of the two Fc gamma RII mRNA, the mRNA encoding the soluble form becoming predominant. Thus, TNF-alpha seems to modify the expression of the Fc gamma RII at the mRNA level, favoring the secretion of soluble Fc gamma RII molecules, and changes the fate of the membranous Fc gamma RII.

In vitro inhibition of tumor B cell growth by IgG-BF-producing Fc gamma RII+ T cell hybridoma and by immunoglobulin G-binding factors.

The growth-modulating effect on mouse hybridoma B cells of IgG-BF-producing Fc gamma RII+ mouse T cell hybridomas and of the IgG-BF isolated from the culture supernatants of these cells has been examined. Cocultures of IgG-secreting hybridoma B cells with IgG-BF-producing T hybridomas or with partially purified IgG-BF demonstrated a reproducible inhibition of the tumor B cell growth. The inhibition was due to a cytostatic and not to a cytotoxic effect. Hybridoma B cells cultured in liquid medium in the presence of soluble IgG-BF, or cocultured in semisolid agarose assays with IgG-BF-producing hybridoma T cells did not undergo immediate cytolysis but were prevented from proliferating. Thus, our data indicate that IgG-BF-producing Fc gamma RII+ T cells interfere with the proliferation of transformed B cells, possibly through soluble IgG-BF.