Preclinical validation of AXL receptor as a target for antibody-based pancreatic cancer immunotherapy.

AXL receptor tyrosine kinase (RTK) is implicated in proliferation and invasion of many cancers, particularly in pancreatic ductal adenocarcinoma (PDAC), for which new therapeutic options are urgently required. We investigated whether inhibition of AXL activity by specific monoclonal antibodies (mAbs) is efficient in limiting proliferation and migration of pancreatic cancer cells. Expression of AXL was evaluated by immunohistochemistry in 42 PDAC. The AXL role in oncogenesis was studied using the short hairpin RNA approach in a pancreatic carcinoma cell line. We further generated antihuman AXL mAbs and evaluated their inhibitory effects and the AXL downstream signaling pathways first in vitro, in a panel of pancreatic cancer cell lines and then in vivo, using subcutaneous or orthotopic pancreatic tumor xenografts. AXL receptor was found expressed in 76% (32/42) of PDAC and was predominantly present in invasive cells. The AXL-knockdown Panc-1 cells decreased in vitro cell migration, survival and proliferation, and reduced in vivo tumor growth. Two selected anti-AXL mAbs (D9 and E8), which inhibited phosphorylation of AXL and of its downstream target AKT without affecting growth arrest-specific factor 6 (GAS6) binding, induced downexpression of AXL by internalization, leading to an inhibition of proliferation and migration in the four pancreatic cancer cell lines studied. In vivo, treatment by anti-AXL mAbs significantly reduced growth of both subcutaneous and orthotopic pancreatic tumor xenografts independently of their KRAS mutation status. Our in vitro and preclinical in vivo data demonstrate that anti-human AXL mAbs could represent a new approach to the pancreatic cancer immunotherapy.

Efficient CD4 binding and immunosuppressive properties of the 13B8.2 monoclonal antibody are displayed by its CDR-H1-derived peptide CB1.

A systematic exploration of the V(H)2/V(kappa)12-13 variable domains of the anti-CD4 monoclonal antibody (mAb) 13B8.2 was performed by the Spot method to screen for paratope-derived peptides (PDPs) demonstrating CD4 binding ability. Nine peptides, named CB1 to CB9, were identified, synthesized in a cyclic and soluble form and tested for binding to recombinant soluble CD4. Among them, CB1, CB2 and CB8 showed high anti-CD4 activity. Competition studies for CD4 binding indicated that PDPs CB1, CB8, and the parental mAb 13B8.2 recognized the same complementarity determining region (CDR)3-like loop region. PDP CB1 was shown to mimic the biological properties of 13B8.2 mAb in two independent cellular assays, demonstrating inhibitory activities in the micromolar range on antigen presentation and human immunodeficiency virus promoter activation. Our results indicate that the bioactive CDR-H1 PDP CB1 has retained a significant part of the parental 13B8.2 mAb properties and might be a lead for the design of anti-CD4 peptidomimetics of clinical interest.

The chimeric mouse-human anti-CD4 Fab 13B8.2 expressed in baculovirus inhibits both antigen presentation and HIV-1 promoter activation.

The anti-CD4 mAb 13B8.2, directed against the CDR3-like loop of the D1 domain of CD4, inhibits signal transduction pathways leading to both T cell activation and HIV replication. VH9/DSP2/JH2 and Vkappa12-13/Jkappa2 rearrangements, corresponding to genes encoding the heavy and light chain variable regions of the 13B8.2 mAb, were inserted into baculovirus cassettes upstream from pre-installed human Fdgamma1 and Ckappa genes, respectively. After expression in insect cells, a complete correctly-processed Fab was secreted into the culture medium; it was protein-G immunopurified with a yield of 5 mg/L. The chimeric Fab 13B8.2 showed anti-CD4 binding activity with an affinity value of 3.3 nM and recognized the same region on the CDR3-like loop as the parental mAb. The mouse-human Fab inhibited IL2 secretion following antigen presentation and displayed a strong capacity to prevent HIV-1 promoter activation. Taken together, these results indicate that the chimeric Fab retained a major part of the parental 13B8.2 mAb properties and suggest that it might be a valuable therapeutic tool.

A constitutive nitric oxide synthase modulates insulin secretion in the INS-1 cell line.

We provide immunocytochemical evidence that the neuronal isoform of constitutive NO synthase (cNOS) is expressed in the rat insulinoma cell line INS-1. Furthermore, using N omega-nitro-L-arginine methyl ester (L-NAME), a pharmacological inhibitor of cNOS activity, we show that this enzyme is implicated in the modulation of insulin secretion in INS-1 cells. Indeed, in the presence of 2.8 mM glucose, L-NAME induced a specific and dose-dependent increase in insulin release, suggesting that cNOS exerts an inhibitory tone on basal insulin secretion. Moreover, L-arginine, the physiological substrate of cNOS, significantly reduced the marked enhancing effect of L-NAME on insulin release and to a lesser extent, at low concentrations, that of 10 mM KCl. L-NAME also potentiated the insulin secretion stimulated by 5.5 and 8.3 mM glucose, but in this case, its effect was not reduced by L-arginine. In conclusion, our data show that the neuronal isoform of cNOS exerts a negative modulation on insulin secretion in INS-1 cells, confirming the previous results obtained in the isolated perfused rat pancreas or pancreatic islets.

Thyroid peroxidase autoantibodies obtained from random single chain FV libraries contain the same heavy/light chain combinations as occur in vivo.

Three combinatorial libraries were constructed from unpurified, CD19(+), and antithyroid peroxidase (anti-TPO) B cells extracted from thyroid tissue of Graves' disease patients. Fifteen of the 41 randomly derived anti-TPO single chain variable region fragments (scFvs), showed VH1-3/V lambda 1-51 or VH1-69/V lambda 1-40 heavy/light chain pairing similar to that obtained with TPO-specific scFv derived from an in-cell library. One VH1-3/V lambda 1-51 scFv, A16, showed exactly the same nucleotide sequence as in-cell scFv ICB7, demonstrating that in vivo rearrangement can be obtained from a random combinatorial library. The majority of the scFvs used a heavy chain gene derived from the VH1-3 gene segment, whereas the light chain gene segments used were more heterogeneous, with dominance of the V kappa 1-39 and V lambda 1-51 gene segments. The anti-TPO scFvs showed high affinities to TPO, with values between 0.77 and 12.3 nM, and defined seven antigenic regions on the TPO molecule. The anti-TPO fragments, particularly VH1-3/V lambda 1-51 randomly associated scFv B4, which mimic natural H/L pairing, and VH1-3/V lambda 1-40 in-cell-derived scFv ICA5, efficiently displaced the TPO binding of serum autoantibodies from 20 Graves' disease patients. Our study directly demonstrates that antibodies derived from combinatorial libraries are likely to represent in vivo pairing, leading to high affinity antibody fragments mimicking the binding of serum autoantibodies to TPO.

A neuronal isoform of nitric oxide synthase expressed in pancreatic beta-cells controls insulin secretion.

Evidence is presented showing that a neuronal isoform of nitric oxide synthase (NOS) is expressed in rat pancreatic islets and INS-1 cells. Sequencing of the coding region indicated a 99.8% homology with rat neuronal NOS (nNOS) with four mutations, three of them resulting in modifications of the amino acid sequence. Double-immunofluorescence studies demonstrated the presence of nNOS in insulin-secreting beta-cells. Electron microscopy studies showed that nNOS was mainly localized in insulin secretory granules and to a lesser extent in the mitochondria and the nucleus. We also studied the mechanism involved in the dysfunction of the beta-cell response to arginine and glucose after nNOS blockade with N(G)-nitro-L-arginine methyl ester. Our data show that miconazole, an inhibitor of nNOS cytochrome c reductase activity, either alone for the experiments with arginine or combined with sodium nitroprusside for glucose, is able to restore normal secretory patterns in response to the two secretagogues. Furthermore, these results were corroborated by the demonstration of a direct enzyme-substrate interaction between nNOS and cytochrome c, which is strongly reinforced in the presence of the NOS inhibitor. Thus, we provide immunochemical and pharmacological evidence that beta-cell nNOS exerts, like brain nNOS, two catalytic activities: a nitric oxide production and an NOS nonoxidating reductase activity, both of which are essential for normal beta-cell function. In conclusion, we suggest that an imbalance between these activities might be implicated in beta-cell dysregulation involved in certain pathological hyperinsulinic states.

Pertinence of kappa and lambda recombinant antibodies directed against thyroid peroxidase in thyroid autoimmune disease.

Forty-one single-chain variable region fragments (scFvs) directed against thyroid peroxidase (TPO) were obtained by phage display libraries constructed from thyroid-infiltrating B cells of Graves' disease patients. Among these scFvs, 24.4% used a Vkappa light chain whereas 75.6% shows a light chain of Vlamda origin. Study of light chain gene usage in the TPO antibody repertoire demonstrated a dominance of the Vkappa 1-39 and Vlambda 1-51 genes. Thyroid peroxidase probing of overlapping peptides covering the amino acid sequences of anti-TPO T2/kappa and T13/lambda variable regions demonstrated a more restricted antigen recognition on T13/lambda than on T2/kappa. These two recombinant antibodies, expressed as whole IgG1 in the baculovirus/insect cell system, inhibited the binding to TPO of serum TPO autoantibodies whatever the light chain. Our study indicates that lambda as well as kappa light chain usage are found in the TPO antibody repertoire of thyroid-infiltrating B cells and are pertinent in the pathogenesis of autoimmune thyroid disease.

Efficient amplification and direct sequencing of mouse variable regions from any immunoglobulin gene family.

We have designed two original sets of oligonucleotide primers hybridizing the relatively conserved motifs within the immunoglobulin signal sequences of each of the 15 heavy chain and 18 kappa light chain gene families. Comparison of these 5' primers with the immunoglobulin signal sequences referenced in the Kabat database suggests that these oligonucleotide primers should hybridize with 89.4% of the 428 mouse heavy chain signal sequences and with 91.8% of the 320 kappa light chain signal sequences with no mismatch. Following PCR amplification using the designed primers and direct sequencing of the amplified products, we obtained full-length variable sequences belonging to major (V(H)1, V(H)2, V(H)3, Vkappa1 and Vkappa21) but also small-sized (V(H)9, V(H)14, Vkappa2, Vkappa9A/9B, Vkappa12/13, Vkappa23 and Vkappa33/34) gene families, from nine murine monoclonal antibodies. This strategy could be a powerful tool for antibody sequence assessment whatever the V gene family before humanization of mouse monoclonal antibody or identification of paratope-derived peptides.

Synthetic peptides derived from the variable regions of an anti-CD4 monoclonal antibody bind to CD4 and inhibit HIV-1 promoter activation in virus-infected cells.

The monoclonal antibody (mAb) ST40, specific for the immunoglobulin complementarity-determining region (CDR) 3-like loop in domain 1 of the CD4 molecule, inhibits human immunodeficiency virus type 1 (HIV-1) promoter activity and viral transcription in HIV-infected cells. To design synthetic peptides from the ST40 paratope that could mimic these biological properties, a set of 220 overlapping 12-mer peptides frameshifted by one residue, corresponding to the deduced ST40 amino acid sequence, was synthesized by the Spot method and tested for binding to recombinant soluble CD4 antigen. Several peptides that included in their sequences amino acids from the CDRs of the antibody and framework residues flanking the CDRs were found to bind soluble CD4. Eleven paratope-derived peptides (termed CM1-CM11) were synthesized in a cyclic and soluble form. All the synthetic peptides showed CD4 binding capacity with affinities ranging from 1.6 to 86.4 nM. Moreover, peptides CM2, CM6, CM7, CM9, and CM11 were able to bind a cyclic peptide corresponding to the CDR3-like loop in domain 1 of CD4 (amino acids 81-92 of CD4). Peptide CM9 from the light chain variable region of mAb ST40 and, to a lesser extent, peptides CM2 and CM11 were able to inhibit HIV-1 promoter long terminal repeat-driven beta-galactosidase gene expression in the HeLa P4 HIV-1 long terminal repeat beta-galactosidase indicator cell line infected with HIV-1. The binding of mAb ST40 to CD4 was also efficiently displaced by peptides CM2, CM9, and CM11. Our results indicate that the information gained from a systematic exploration of the antigen binding capacity of synthetic peptides from immunoglobulin variable sequences can lead to the identification of bioactive paratope-derived peptides of potential pharmacological interest.

Mapping of B epitopes in GRA4, a dense granule antigen of Toxoplasma gondii and protection studies using recombinant proteins administered by the oral route.

GRA4, a dense granule protein of Toxoplasma gondii elicits both mucosal and systemic immune responses following oral infection of mice with cysts. We studied the antigenicity and immunogenicity of truncated and soluble forms of GRA4 expressed as glutathione S-transferase fusion proteins in Escherichia coli. Protein C (amino-acids 297-345) was particularly well recognized by serum IgG antibodies, milk IgA antibodies and intestinal IgA antibodies from T. gondii infected mice and by serum IgG antibodies from T. gondii infected humans and T. gondii infected sheep. One major B epitope was localized within the last 11 C-terminal residues of GRA4. A second epitope, recognized with lower frequency, was mapped within the region 318-334. In contrast, the N domain of GRA4 (amino acids 25-276) was poorly recognized. Oral immunization of C57BL/6 mice with N, C or NC (amino acids 25-276 fused to 297-345) in association with cholera toxin induced a significant production of serum anti-GRA4 IgG antibodies but a weak and inconsistent intestinal anti-GRA4 IgG antibody response and afforded partial resistance to oral infection with T. gondii. These results provide new molecular and immunological understanding of GRA4 and indicate that it is a potential candidate for oral vaccination against T. gondii.

Anti-digoxin scFv fragments expressed in bacteria and in insect cells have different antigen binding properties.

A gene encoding a single-chain antibody fragment directed against digoxin (named 1C10 scFv) was cloned in two expression systems. For this purpose, a new baculovirus transfer cassette fully compatible with the procaryotic pHEN vector was constructed. Baculovirus production led to higher yield than did Escherichia coli expression. The procaryotic fragment showed variations in the fine specificity profile but an affinity constant nearly identical to that of the 1C10 Fab, whereas the eucaryotic scFv fragment had a lower affinity with a specificity profile identical to original mAb. The half-lives of the digoxin:scFv complexes and the global specificity are compatible with therapeutic use of this antibody fragment.

Peritoneal macrophages from C57BL/6 mice orally immunized with Toxoplasma gondii antigens in association with cholera toxin possess an enhanced ability to inhibit parasite multiplication.

Gamma-interferon (IFN-gamma) has been reported to be a major mediator of resistance to toxoplasma infection, mainly through macrophage activation. Cholera toxin used as oral adjuvant induces enhanced protection. Following oral immunization of C57BL/6 mice with a Toxoplasma gondii sonicate (TSo), in association with either cholera toxin (CT) or its B subunit (CTB), the ability of primed sensitized peritoneal macrophages (PM phi) to prevent T. gondii intracellular proliferation in vitro was examined both with and without rIFN-gamma activation. Under these conditions, the inhibition of T. gondii multiplication was greatly enhanced in PM phi from mice immunized with a TSo and CT as an oral adjuvant. In contrast, PM phi from mice immunized with a TSo in association with CTB showed a decrease in their microbiostatic activity towards T. gondii. This negative effect on IFN-gamma-treated PM phi was cancelled out by the addition of a small amount of CT in association with TSo and CTB in the immunization regimen. These data suggest that CT could act as an oral adjuvant in vaccination against toxoplasmosis by increasing the microbiostatic activity of M phi activated with IFN-gamma. Further studies, using intestinal effector cells such as enterocytes, are needed to confirm the value of CT for enhancing this major mechanism of protection against T. gondii infection.

Toxoplasma gondii oral infection induces specific cytotoxic CD8 alpha/beta+ Thy-1+ gut intraepithelial lymphocytes, lytic for parasite-infected enterocytes.

The first line of defense of the host after primary infection by Toxoplasma gondii is the intestinal mucosal surface, which consists of epithelial cells, intraepithelial lymphocytes (IEL) (mainly of CD8 phenotype), mucus, and secretory Igs. T. gondii cysts were administered orally to CBA/J mice to determine whether parasite-specific cytotoxic IEL can be elicited within the intestine. We found that oral infection led to an increase in the relative percentages of the CD8 beta + and Thy-1+ IEL populations between day 9 and day 13 after infection. At these times, T. gondii-primed, but not control mice, generated parasite-specific cytotoxic effector IEL for toxoplasma-infected macrophages. This cytotoxic activity was genetically restricted. By using magnetically activated cell sorting, the effector IEL were shown to be CD8 alpha/beta + Thy-1+ T lymphocytes and FACScan analysis revealed that they mainly express TCR-alpha beta. A significant level of cytotoxicity was also observed against T. gondii-infected epithelial cells from the MODE-K cell line, suggesting that parasite-specific IEL may ensure epithelial integrity by rapidly killing infected enterocytes. Finally, toxoplasma stimulation in vitro led to IFN-gamma production by T. gondii-primed IEL. Taken together, these data suggest that antigen-specific IEL, bearing the CD8 alpha/beta + Thy-1+, TCR-alpha beta + phenotype, can act directly as effector cells through a specific CTL activity at the intestinal level and may indirectly activate other mucosal effector mechanisms through IFN-gamma secretion.

Epitopic analysis of the Toxoplasma gondii major surface antigen SAG1.

T and B cell epitopes of the major Toxoplasma gondii surface antigen SAG1 were studied following CNBr fragmentation. Three fragments, F1, F2 and F3, were obtained, of 19, 16.5 and 14 kDa, respectively. The positions of F1 F2 and F3 within the SAG1 protein were identified by N-terminal sequence determination. The F1 fragment located on residues 125-269 contains the C-terminus, and the fragment F2 (residues 1-124) is located at the N-terminal region. F3 is a C-terminal peptide about 40 amino acids shorter than the F1 fragment (residues 165-269). Polyclonal antibodies obtained from infected animals or humans and a monoclonal anti-SAG1 antibody did not recognize either the reduced protein or the reduced fragments on immunoblotting. The monoclonal antibody 1E5 did not recognize fragment F1. Mouse IgA and IgG antibodies from infected mouse sera and intestinal secretions, as well as human IgG antibodies, only recognized the whole protein and the F1 fragment. These results suggest that the fragment F1 encompasses all B cell epitopes recognized on the SAG1 protein after infection with the parasite and that the sequence 125-165 is essential for the structural integrity of these B cell epitopes. Murine anti-SAG1 T cell proliferation was observed in SAG1 immunized CBA/J mice (H-2k) and BALB/c mice (H-2d), but not in C57BL/6 mice (H-2b). The three fragments F1, F2 and F3 were able to induce specific proliferation of anti-SAG1 T cells from CBA/J mice, while only the F1 and F2 fragments induced specific blastogenesis of anti-SAG1 T cells from BALB/c mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Oral immunization with Toxoplasma gondii antigens in association with cholera toxin induces enhanced protective and cell-mediated immunity in C57BL/6 mice.

Following oral immunization of C57BL/6 mice with a Toxoplasma gondii sonicate (TSo) in association with either cholera toxin (CT) or CT B subunit, the T. gondii-specific in vitro proliferation of splenic T lymphocytes was determined. Cytokines produced by these T cells were then characterized. After oral challenge with T. gondii 76K cysts, the percentage of cumulative survival was assessed, as was the number of brain cysts in the mice which survived. The TSo-specific proliferation of splenic T lymphocytes was greatly enhanced by the use of CT, whereas CT B subunit alone did not lead to amplification of splenic T-cell proliferation. The use of CT was associated with an increase of interleukin-2 (IL-2) and gamma interferon synthesis by TSo-stimulated splenic T cells, whereas no enhancement of IL-5 and IL-6 production was observed. IL-4 was not detected. A significant protection of mice immunized orally with TSo plus CT was observed in comparison with those immunized with TSo alone. This protection was associated with a large decrease in the number of brain cysts compared with the number found in naive mice infected orally with a sublethal dose of T. gondii 76K cysts. Further studies, using well-defined T. gondii proteins which are known to induce both mucosal and systemic immune responses, are needed to confirm the value of CT in the enhancement of protection against oral toxoplasmosis.

Mucosal and systemic cellular immune responses induced by Toxoplasma gondii antigens in cyst orally infected mice.

This study was performed to determine the T-cellular immune responses following Toxoplasma gondii oral infection and to assess further toxoplasma antigens on their ability to stimulate in vitro mucosal and systemic T-cell immunity. Parasite-specific cellular immune responses in Peyer's patches (PP), in mesenteric lymph nodes (MLN) and in spleen (SPL) were investigated using a lymphoblastic transformation test following oral infection of mice with strain 76K cysts of T. gondii. An early toxoplasma sonicate-induced mucosal T-cell proliferation occurred in MLN and PP with a peak responsiveness on day 6 post-infection (PI) and rapidly reached background levels on day 7 PI in PP and on day 8 PI in mesenteric lymph nodes. A later splenic cellular blastogenesis was observed from day 28 PI and persisted throughout the experiment (day 91). At the time of T-cell proliferation, FACS analyses revealed a decrease in the relative percentages of CD4+ and CD8+ T cells with a predominance of CD8+ lymphocytes which leads to an inversion of the CD4/CD8 ratios. We found that CBA/J is a high responder mouse strain in the induction of mesenteric and splenic T-lymphocyte blastogenesis compared to the intermediate responder BALB/c and low responder C57BL/6. Toxoplasma gondii antigens SAG1 (30,000 MW) and GRA4 (40,000-41,000 MW), which are known to induce locally IgA antibodies, are shown to stimulate primed mucosal T lymphocytes from CBA/J and BALB/c mice whereas no proliferation was demonstrated with C57BL/6 T cells. 229-242 peptide, derived from the deduced amino acid sequence of GRA4, only induces detectable proliferation of primed-CBA/J T lymphocytes. Following oral experimental infection, the in vitro mesenteric response to a toxoplasma sonicate is dominated by a Th2-type cytokine pattern whereas a predominant Th1 cytokine response is observed in the spleen. Finally, in vitro stimulation of mesenteric T cells with the three defined toxoplasma antigens resulted in secretion of interleukin-5 (IL-5) and IL-6 (except for SAG1) and interferon-gamma (IFN-gamma) whereas no detectable IL-2 or IL-4 was observed.

Molecular cloning of GRA4, a Toxoplasma gondii dense granule protein, recognized by mucosal IgA antibodies.

Clones which were selected from a Toxoplasma gondii expression library with the immune serum from a T. gondii-infected rabbit, were further screened using milk and intestinal secretions from mice which had been orally infected with T. gondii cysts. The gene products of several clones reacted strongly with milk IgA and weakly with intestinal IgA. Three of these clones (5.1, 36.1, 37.4) were shown to encode a dense granule protein of 40 kDa (GRA4). The GRA4 protein co-migrates with one of the T. gondii antigens recognized by mucosal IgA. The complete nucleotide sequence of GRA4 has been obtained by cloning genomic T. gondii BamHI fragments containing the 37.4 DNA insert. The coding sequence contains no intron. The deduced amino acid sequence indicates a proline rich (12%) product with an internal hydrophobic region of 19 amino acids and a potential site of N-glycosylation. The primary translation product with a theoretical size of 36,260 Da contains a putative N-terminal signal sequence of 20 amino acids but no apparent glycolipid anchor sequence. Quantitation of the GRA4 gene and Southern blot analysis suggested that the GRA4 gene is single copy. GRA4 gene is translated in tachyzoites to yield a single mRNA species of about 1900 bases.

Immunological characterization of a 17-kDa antigen from Cryptosporidium parvum recognized early by mucosal IgA antibodies.

Cryptosporidium parvum antigens were characterized by immunoblot analysis of sera and intestinal secretions of BALB/c mice orally infected with 10(5) oocysts. A major band at 17 kDa under non-reduced conditions and at 18 kDa under reduced conditions was recognized by anti-C. parvum IgA and IgG in serum and intestinal secretions from day 15 post-infection. This recognition persisted throughout the experiment (day 30). Mouse-serum antibodies raised against the 17-kDa purified antigen (P17) showed no cross-reactivity with other C. parvum antigens. Immunofluorescence study revealed that this antigen is located on the sporozoite. It is suggested that this antigen could be a good candidate for studies of mucosal immune response to C. parvum and for vaccination.