Anti-tumour effect of monoclonal antibodies against selected antigens expressed by B-cells in Chronic Lymphocytic Leukaemia : strategies to enhance the efficacy of immunotherapy

Résumé : Les anticorps monoclonaux ont une place de plus en plus prépondérante dans le traitement des lymphomes et leucémies. Dans cette étude, trois anticorps monoclonaux murins, dirigés contre les antigènes CDS, CD71 et HLA-DR exprimés à la surface des cellules de leucémies lymphoïdes chroniques (LLC), ont été évalués. In vitro, les anticorps radiomarqués ont montrés des bonnes liaisons spécifiques sur les différentes cellules cibles. L'anti-CD71 inhibait la prolifération de la plupart des lignées cellulaires testées avec une accumulation des cellules en phase S précoce du cycle cellulaire. L'anti-HLA-DR inhibait aussi la prolifération des lignées leucémique JOK1-5.3 et lymphoïde Daudi. Cette inhibition était associée à une agrégation des cellules. Aucune induction d'apoptose n'a pu être clairement observée avec ces anticorps. L'anti-CD5 n'a montré aucun effet d'inhibition de croissance in vitro. In vivo, l'injection des anticorps individuellement augmentait significativement la survie médiane de souris SCID greffées avec des cellules JOK1-5.3 en i.p. De plus, l'anticorps antiCD5 combiné à l'anti-HLA-DR ou l'anti-CD71, sous certaines conditions, inhibait complètement le développement tumoral dans la quasi totalité des souris traitées avec une augmentation significative de l'efficacité comparée aux anticorps seuls. L'augmentation de l'efficacité thérapeutique des anticorps monoclonaux par les cytokines, dont l'IL-2, a déjà été montrée dans la littérature. Au regard du meilleur comportement de l'IL-2 sous la forme complexée à un anticorps anti-IL-2, nous avons évalué l'efficacité de l'IL-2/anti-IL-2 seul ou combinés au rituximab chez différents modèles tumoraux s.c. (BL60.2, Daudi, Ramos) ou i.p. (JOK15.3) de souris SCID. Le complexe IL-2/anti-IL-2 a montré un effet anti-tumoral dans les souris greffées avec BL60.2 et Daudi. Le traitement IL-2/anti-IL-2 combiné au rituximab a montré une efficacité accrue chez des souris avec BL60.2 par rapport au rituximab seul. En revanche, nous n'avons pas observé de différence avec IL-2/anti-IL-2 seul.Aussi, nous avons évalué l'utilisation de l'agent couplant tri-fonctionnel TMEA pour produire des anticorps bispecifiques. Les expériences préliminaires avec les anticorps rituximab et herceptine, ont mis en évidence sur gel SDS-Page la formation de dimers (~100kDa) et de trimers (~150kDa). Les anticorps bispecifiques sont composés d'un fragment Fab' d'une spécificité et de un ou deux fragments Fab' de l'autre spécificité permettant de moduler la capacité de liaison. Nous avons enfin montré qu'une construction anti-CD5/anti-CD20 était capable de se lier indépendamment ou simultanément à ses antigènes cibles. En conclusion, ce travail a montré l'efficacité thérapeutique des trois anticorps monoclonaux étudiés dans un model de LLC in vivo, et plus particulièrement l'intérêt de certaines combinaisons. D'autre part, nous avons montré l'efficacité anti-tumorale du complexe IL-2/anti-IL-2 in vivo. Des études futures devront permettre de définir un régime favorable pour augmenter l'efficacité de la thérapie avec les anticorps monoclonaux. Enfin, nous avons montré la faisabilité d'utiliser l'agent couplant TMEA pour produire des anticorps bispécifiques fonctionnels.Abstract : Monoclonal antibody (mAb) therapy has become an integral part in different treatments of lymphomas and leukaemias. In this study, we describe three murine mAbs directed against the CD5, CD71 and HLA-DR antigens expressed on chronic lymphocytic leukaemia cells (CLL). In vitro, radiolabeled purified mAbs showed good specific binding on live target cells. Anti-CD71 mAb inhibited proliferation of most cell lines with an accumulation of responding cells in early S-phase of the cell cycle, but without induction of apoptosis. Anti-HLA-DR mAb showed proliferation inhibition of leukaemia JOK1-5.3 and lymphoid Daudi cells, associated with cell aggregation, but again no specific sign of apoptosis was observed. Anti-CD5 mAb did not show any growth inhibitory effect in vitro. In vivo, in a model of SCID mice grafted i.p. with JOK1-5.3 cells, injection of individual mAbs induced significant prolongation of median survival, up to complete inhibition of tumour growth in some mice. Antibody combination of anti-CD5 with anti-HLA-DR or anti-CD71, evaluated in an early treatment, completely inhibited tumour growth in most mice, with a significant efficacy enhancement as compared to mAb used as single agents. Previous reports described the improved efficacy of mAb therapy when combined with cytokines such as IL-2. Relying further on the improved efficacy of IL-2 when administered as an immune complex with anti-IL-2 mAb, we evaluated the anti-tumour effect of the IL-2/anti-IL-2 complex alone or combined with rituximab in subcutaneous (BL60.2, Daudi, Ramos) or i.p. (JOK1-5.3) tumour models in SCID mice. The IL-2/anti-IL-2 complex demonstrated an anti-tumour effect in BL60.2 and Daudi grafted SCID mice. Combination of IL-2/anti-IL-2 treatment with rituximab showed increased efficacy as compared to rituximab alone in BL60.2 grafted mice. However, no difference was observed with IL-2/anti-IL-2 complex alone in these experiments. Finally, we evaluated the feasibility of producing bispecific antibodies (bsAbs) using a trifunctional coupling agent, called TMEA. In preliminary experiments coupling rituximab with herceptine Fab' fragments we obtained the formation of dimers (~100kDa) and trimers (~150kDa) as observed on SDS-Page gel. This method allowed us to produce bsAb with one Fab' fragments of one specificity and one or two Fab' fragments of the second specificity. An anti-CD5/anti-CD20 bsAb was shown to bind targeted antigen either independently or simultaneously. In conclusion, these data show that the three mAbs were all able to induce significant growth inhibition of the JOK1-5.3 cell line in vivo, and efficacy was enhanced when used in combination. IL2/anti-IL-2 complex displayed anti-tumour efficacy in vivo. Further evaluation is necessary to define the most favourable combination to improve mAb therapy. BsAb were produced using the tri-functional agent allowing antibody fragments with relatively good binding. The poor yield obtained with such chemical couplings limited the use of these constructs in preclinical experiments.

Improved immunogenicity against HIV-1 clade C antigens by using NYVAC-C with restored replication competence.

In order to improve the immunogenicity of currently available non-replicating pox virus HIV vaccine vectors, NYVAC was genetically modified through re-insertion of two host range genes (K1L and C7L), resulting in restored replicative capacity in human cells. In the present study these vectors, expressing either a combination of the HIV-1 clade C antigens Env, Gag, Pol, Nef, or a combination of Gal, Pol, Nef were evaluated for safety and immunogenicity in rhesus macaques, which were immunized at weeks 0, 4 and 12 either by scarification (conventional poxvirus route of immunization), intradermal or by intramuscular injection (route used in previous vaccine studies).Replication competent NYVAC-C-KC vectors induced higher HIV-specific responses, as measured by IFN- ELISpot assay, than the replication defective NYVAC-C vectors. Application through scarification only required one immunization to induce maximum HIV-specific immune responses. This method simultaneously induced relatively lower anti-vector responses. In contrast, two to three immunizations were required when the NYVAC-C-KC vectors were given by intradermal or intramuscular injection and this method tended to generate slightly lower responses. Responses were predominantly directed against Env in the animals that received NYVAC-C-KC vectors expressing HIV-1 Env, Gag, Pol, Nef, while Gag responses were dominant in the NYVAC-C-KC HIV-1 Gag, Pol, Nef immunized animals.The current study demonstrates that NYVAC replication competent vectors were well tolerated and showed increased immunogenicity as compared to replication defective vectors. Further studies are needed to evaluate the most efficient route of immunization and to explore the use of these replication competent NYVAC vectors in prime/boost combination with gp120 protein-based vaccine candidates. This studies was performed within the Poxvirus T-cell Vaccine Discovery Consortium (PTVDC) which is part of the CAVD program.

Is carbohydrate-deficient transferrin a specific marker for alcohol abuse? A study in patients with chronic viral hepatitis.

Carbohydrate-deficient transferrin, a transferrin isoform, is hailed as a new marker of chronic alcohol abuse, but its specificity is, however, not unequivocally accepted. The aim of the present study was therefore to determine carbohydrate-deficient transferrin levels in patients with chronic hepatitis B and C with or without documented chronic alcohol intake. Carbohydrate-deficient transferrin was measured using a double-antibody radioimmunoassay (CDTect, Pharmacia) in serum samples from 66 patients (45 males and 21 females; mean age: 39 years) with chronic viral hepatitis B (n = 20) or C (n = 46). Diagnosis of the underlying liver disease was established by liver biopsy. Carbohydrate-deficient transferrin levels were raised in 15 patients [23%; hepatitis B (n = 2) and hepatitis C (n = 13)]. In patients with chronic hepatitis B, the carbohydrate-deficient transferrin level was raised in two abstainers. In the 46 patients with chronic hepatitis C, 10 (22%) patients with an alcohol consumption of < 60 g/day for the men and 30 g/day for the women had raised carbohydrate-deficient transferrin levels. The overall specificity of carbohydrate-deficient transferrin for chronic alcohol abuse was thus 78%, suggesting an association between elevated carbohydrate-deficient transferrin levels and the presence of chronic viral hepatitis. Carbohydrate-deficient transferrin levels were not correlated with the histological grading or staging of chronic hepatitis B and C, or with biological markers of hepatic synthesis and cellular damage. Thus, an increased carbohydrate-deficient transferrin level may occur in patients with chronic viral hepatitis in the absence of chronic alcohol abuse. This fact should be kept in mind by physicians when using this marker to detect alcohol abuse.

Potential target antigens for immunotherapy in human pancreatic cancer.

BACKGROUND: To be effective and selective, immunotherapy ideally targets specifically tumor cells and spares normal tissues. Identification of tumor specific antigens is a prerequisite to establish an effective immunotherapy. Still very little is known about the expression of tumor-related antigens in pancreatic neoplasms. Cancer Testis antigens (CT) are antigens shared by a variety of malignant tumors, but not by normal tissues with the exception of germ cells in testis. Restricted expression in neoplastic tissues and inherent immunogenic features make CT antigens ideal for use in immunotherapy. We analyzed the expression of a selected panel of nine CT antigens that have been proven to elicit an efficient immunogenic response in other malignancies. In addition we analyzed the expression of HERV-K-MEL, an immunogenic antigen of viral origin. METHODS: Pancreatic adenocarcinoma tumor samples (n=130) were obtained intraoperatively, control tissues (n=23) were collected from cadaveric donor and from patients with chronic pancreatitis. Tumor-associated antigen expression of MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A10, LAGE-1, NY-ESO-1, SCP-1, SSX-2, SSX-4 and HERV-K-MEL was assessed by PCR. Sequencing of PCR products were performed to assess the expression of SSX-4 in neoplastic and normal pancreatic tissues. RESULTS: Three of 10 tested antigens were expressed in over 10% of malignant pancreatic tissue samples. SSX-4 was found positive in 30% of cases, SCP-1 in 19% and HERV-K-MEL in 23% of cases. No expression of CT antigens was found in non-malignant pancreatic tissue with the exception of SSX-4 and and SSX-2. CONCLUSIONS: Fifty two percentage of the analyzed tissues expressed at least one CT antigen. The concomitant expression of SSX-4 in both malignant and non-malignant pancreatic tissue is a new finding which may raise concerns for immunotherapy. However, HERV-K-MEL is expressed with a relatively high prevalence and may be a candidate for specific immunotherapy in a large subgroup of pancreatic cancer patients. This study advocates the analysis of patients with regard to their immunogenic profile before the onset of antigen-specific immunotherapy.

Lymphocyte specificity to protein antigens. III. Capacity of low responder mice to beef cytochrome c to respond to a peptide fragment of the molecule.

Lymph node cells derived from A.TH or A.TL mice primed with beef cytochrome c show striking patterns of reactivity when assayed in vitro for antigen-induced T cell proliferation. Whereas cells from A.TH mice respond specifically to beef cytochrome c or peptides composed of amino acids 1-65 and 81-104, cells from A.TL mice respond neither to beef cytochrome c nor to peptide 1-65, but proliferate following exposure to either peptide 81-104 or to a cytochrome c hybrid molecule in which the N-terminal peptide of beef (1-65) was substituted by a similar peptide obtained from rabbit cytochrome c. Thus, T cells from mice phenotypically unresponsive to beef cytochrome may, in fact, contain populations of lymphocytes capable of responding to a unique peptide, the response to which is totally inhibited when the same fragment is presented in the sequence of the intact protein.

The adjustment of energy expenditure and oxidation to energy intake: the role of carbohydrate and fat balance.

Evidence is accumulating that total body mass and its relative composition influence the rate of fat utilization in man. This effect can be explained by two factors operating in concert: (i) the effect of the size of the tissue mass and (ii) the nature of the fuel mix oxidized, i.e. the proportion of energy derived from fat vs. carbohydrate. In a cross-sectional study of 307 women with increasing degrees of obesity, we observed that the respiratory quotient (RQ) in post-absorptive conditions became progressively lower with increased body fatness, indicating a shift in substrate utilization. However, the RQ is known to be also influenced by the diet commonly ingested by the subjects. A short-term mixed diet overfeeding in lean and obese women has also demonstrated the high sensitivity of RQ to changes in energy balance. Following a one-day overfeeding (2500 kcal/day in excess of the previous 24 h energy expenditure), the magnitude of increase in RQ was identical in lean and obese subjects and the net efficiency of substrate utilization and storage was not influenced by the state of obesity.

Lymphocyte specificity to protein antigens. V. Conformational dependence of activation of cytochrome c-specific T cells.

Variations in the immunogenic and antigenic properties of native and denatured forms of cytochrome c were observed depending on the strain of mouse tested. In C57BL/6 and (C57BL/6 X DBA/2)F1 (BDF1) mice, priming with either native or denatured cytochrome c (apocytochrome c) gave rise to T cell blasts responding in a similar fashion to the two forms of the antigen and to different peptides derived from CNBr cleavage of the protein when tested for proliferation in the presence of C57BL/6 or BDF1 accessory cells. A different pattern of proliferation was observed when apocytochrome c-specific DBA/2 or BDF1 T cell blasts were tested with DBA/2 accessory cells. In this case, no response was obtained to heme peptide 1-65. This was not due to an inability of DBA/2 macrophages to process and present heme peptide 1-65, as they were able to present this antigen to native cytochrome c-specific BDF1 T cell blasts. Thus, it seems that different sets of clones are generated upon priming BDF1 mice with denatured cytochrome c which are able to recognize different sets of peptides depending on the nature of the accessory cells. The results obtained are consistent with the hypothesis that degradation and presentation of native and denatured cytochrome c by macrophages is dependent on the three-dimensional conformation of the protein.

Therapeutic cancer vaccines based on molecularly defined human tumor antigens.

The results of numerous phases I and II clinical trials testing the safety and immunogenicity of various cancer vaccine formulations based on cytolytic T lymphocytes (CTLs)-defined tumor antigens have been reported recently. Specific T cell responses can be detected in only a fraction of immunized patients. A smaller but significant fraction of these patients have objective tumor responses. Efficient therapeutic vaccination should aim at boosting naturally occurring anti-tumor responses and at sustaining a large contingent of tumor antigen-specific and fully functional effector T cells at tumor sites.

An overview of how rubisco and carbohydrate metabolism may be regulated at elevated atmospheric [CO2] and temperature

Selostus: Hiilihydraatti- ja proteiiniaineenvaihdunnan säätely kohonneen hiilidioksidipitoisuuden ja lämpötilan vallitessa

Protein turnover and thermogenesis in response to high-protein and high-carbohydrate feeding in men.

The rates of energy expenditure and wholebody protein turnover were determined during a 9-h period in a group of seven men while they received hourly isocaloric meals of high-protein (HP) or high-carbohydrate (HC) content. Their responses to feeding were compared with those to a short period of fasting (15-24 h). The 9-h thermic response to the repeated feeding of HP meals was found to be greater than that to the HC meals (9.6 +/- 0.6% vs 5.7 +/- 0.4% of the energy intake, respectively, means +/- SEM, p less than 0.01). The rate of whole-body nitrogen turnover over 9 h increased from 17.6 +/- 2.2 g on the fasting day to 27.4 +/- 1.4 g during HC feeding (NS) and there was a further increase to 58.2 +/- 5.3 g resulting from HP feeding (p less than 0.001). By using theoretical estimates (based upon ATP requirements) of the metabolic cost of protein synthesis, 36 +/- 9% of the thermic response to HC feeding and 68 +/- 3% of the response to HP feeding could be accounted for by the increases in protein synthesis compared with the fasting state.

HLA class II antigens (DR, DQ LOCI) and peripheral arthritis in ankylosing spondylitis.

Fifty one patients with ankylosing spondylitis (AS) were typed for HLA-A, B, C, DR, and DQ antigens. The antigen frequencies were compared with those of a normal population and with a B27 positive control group. All but one of the patients with AS were HLA-B27 positive. A positive linkage disequilibrium between Cw1, Cw2, DR1, and the B27 antigen was observed. Patients with AS showed a significant increase in DQw2 antigen compared with the B27 positive control group. No differences in antigenic frequencies were observed in patients having peripheral arthritis and patients with only axial involvement. Seven out of nine patients (78%) with an erosive peripheral arthritis were DR7 positive, suggesting that DR7 or genes closely linked could be related with a more aggressive peripheral joint involvement in patients with AS.

Expression hierarchy of T cell epitopes from melanoma differentiation antigens: unexpected high level presentation of tyrosinase-HLA-A2 Complexes revealed by peptide-specific, MHC-restricted, TCR-like antibodies.

Peptide Ags presented by class I MHC molecules on human melanomas and that are recognized by CD8(+) T cells are the subjects of many studies of antitumor immunity and represent attractive candidates for therapeutic approaches. However, no direct quantitative measurements exist to reveal their expression hierarchy on the cell surface. Using novel recombinant Abs which bind these Ags with a peptide-specific, MHC-restricted manner, we demonstrate a defined pattern of expression hierarchy of peptide-HLA-A2 complexes derived from three major differentiation Ags: gp100, Melan-A/Mart-1, and tyrosinase. Studying melanoma cell lines derived from multiple patients, we reveal a surprisingly high level of presentation of tyrosinase-derived complexes and moderate to very low expression of complexes derived from other Ags. No correlation between Ag presentation and mRNA expression was found; however, protein stability may play a major role. These results provide new insights into the characteristics of Ag presentation and are particularly important when such targets are being considered for immunotherapy. These results may shed new light on relationships between Ag presentation and immune response to cancer Ags.