Antibody targeting of claudin-1 as a potential colorectal cancer therapy.

BACKGROUND: Metastatic colorectal cancer (mCRC) is one of the major causes of cancer-related death. Despite the substantial progress in mCRC management, it remains important to identify new therapeutic options and biological markers for personalized medicine. Here, we investigated the expression of claudin-1 (CLDN1), a major tight junction transmembrane protein, in the different colorectal cancer (CRC) molecular subtypes and then assessed the anti-tumor effect of a new anti-CLDN1 monoclonal antibody (mAb). METHODS: Gene expression profiling and immunochemistry analysis of normal and tumor tissue samples from patients with stage IV CRC were used to determine CLDN1 gene expression. Then, the 6F6 mAb against CLDN1 extracellular part was generated. Its effect on CRC cell cycle, proliferation, survival and migration was assessed in vitro, using a 3D cell culture system, flow cytometry, clonogenic and migration assays. In vivo, 6 F6 mAb efficacy was evaluated in nude mice after subcutaneous xenografts or intrasplenic injection of CRC cells. RESULTS: Compared with normal mucosa where it was almost exclusively cytoplasmic, in CRC samples CLDN1 was overexpressed (p < 0.001) and mainly localized at the membrane. Moreover, it was differentially expressed in the various CRC molecular subtypes. The strongest expressions were found in the consensus molecular subtype CMS2 (p < 0.001), the transit-ampliflying (p < 0.001) and the C5 subtypes (p < 0.001). Lower CLDN1 expression predicted a better outcome in the molecular subtypes C3 and C5 (p = 0.012 and p = 0.004, respectively). CLDN1 targeting with the 6 F6 mAb led to reduction of survival, growth and migration of CLDN1-positive cells. In preclinical mouse models, the 6F6 mAb decreased tumor growth and liver metastasis formation. CONCLUSION: Our data indicate that CLDN1 targeting with an anti-CLDN1 mAb results in decreased growth and survival of CRC cells. This suggests that CLDN1 could be a new potential therapeutic target.

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.

Isolation and characterisation of a human anti-idiotypic scFv used as a surrogate tumour antigen to elicit an anti-HER-2/neu humoral response in mice.

HER-2/neu is a tumour antigen that is overexpressed in human breast tumours. Among the vaccine strategies developed to overcome immune tolerance to self-proteins, vaccination with anti-idiotypic (anti-Id) antibodies has been described as a promising approach for treatment of several malignant diseases. To develop an active immunotherapy for cancer patients positive for HER-2/neu, we investigated immunisation with human anti-Id single-chain fragments (scFv) mimicking the conformation of HER-2/neu protein to induce a humoral response in mice. We selected by phage display two human anti-Id scFv (Ab2beta) directed against trastuzumab F(ab')2 fragments (Ab1), a humanised anti-HER-2/neu monoclonal antibody. Using competitive ELISA and Biacore biosensor analysis, we showed that anti-Id scFv 40 and scFv 69 could inhibit HER-2/neu binding to trastuzumab. Following vaccination of BALB/c mice with the soluble or phage-displayed scFv, Ab3 polyclonal antibodies, and among them Ab1' antibodies able to bind HER-2/neu, were detected in the sera of the immunised mice. These results demonstrate that the human anti-Id scFv could act as a surrogate antigen for HER-2/neu. The present study strongly suggests that the novel 30 kDa human mini-antibody could be used as an anti-idiotype-based vaccine formulation to induce an effective humoral response in patients bearing HER-2/neu-positive tumours.

Casein interactions studied by the surface plasmon resonance technique.

Surface plasmon resonance technique was investigated for the first time to study the apparent hydrophobicity and association properties of the major bovine caseins: alpha(s)-(alpha(s1)- and alpha(s2)-caseins in a 4:1 proportion), beta-, and kappa-caseins. The apparent hydrophobicities of the caseins were evaluated by a new method based on the binding level of casein on a hydrophobic sensor chip, and kinetic and equilibrium affinity constants were determined for the following casein interactions: alpha(s)/alpha(s), alpha(s)/beta, alpha(s)/kappa, beta/beta, and beta/kappa, using a sensor chip modified with covalent immobilized caseins. The study by surface plasmon resonance technology of these casein interactions under different conditions (pH, ionic strength, calcium concentration, chemical modification) demonstrated that, at neutral pH, electrostatic repulsive forces play an important role since an increase in ionic strength of the medium resulted in a stronger interaction. When charge repulsions were reduced by either acidification, increase in ionic strength, or dephosphorylation, casein interactions were reinforced, presumably due to weak attractive forces. Moreover, in this molecular model, we showed that addition of calcium greatly increased the binding response between the most phosphorylated caseins and that the added calcium (2 mM) participated directly in the formation of bridges between the phosphate groups of the casein molecules.

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.

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.

Interaction of the octapeptide angiotensin II with a high-affinity single-chain Fv and with peptides derived from the antibody paratope.

The amino-acid sequence of the very high-affinity anti-angiotensin II monoclonal antibody 4D8 was predicted from the nucleotide sequence of the heavy and light chain variable genes. The single-chain variable fragment (scFv) was constructed and expressed in Escherichia coli as a soluble protein and at the surface of the filamentous M13 phage and was compared with the full-length antibody (Ab). The scFv showed the same specificity profile and affinity constant as the intact antibody (5.0x10(10) and 8.0x10(10) M(-1), respectively, by Scatchard analysis). Several peptides from the set of overlapping dodecapeptides covering the variable domains of 4D8 mAb were found to specifically bind biotinylated angiotensin II: peptides from the L1, L2, L3 and H1 regions had the strongest capacity to bind the antigen.

Study of hydrophobic interactions between acylated proteins and phospholipid bilayers using BIACORE.

Intracellular proteins of eukaryotic cells are frequently covalently modified by the addition of long chain fatty acids. These modifications are thought to allow otherwise soluble proteins to associate with membranes by lipid-lipid based hydrophobic interactions. The purpose of this work was to quantify the effect of acyl chain length on hydrophobic interactions between acylated proteins and phospholipid monolayers. The binding of an artificially acylated model protein to electrically neutral phospholipids was studied by surface plasmon resonance, using BIACORE. Kinetic rates for the binding of bovine pancreatic ribonuclease A (RNase A), monoacylated on its N-terminal lysine with fatty acids of 10, 12, 14, 16 or 18 carbon atoms, to phospholipids on hydrophobic sensor chips, were measured. Unlike unmodified ribonuclease, acylated RNase A bound to the phospholipids, and the association level increased with the acyl chain length to reach a maximum for C16. Reproducible kinetics were obtained which did not fit a 1:1 Langmuir model but rather a two-step binding profile.

Systematic mapping of regions of human cardiac troponin I involved in binding to cardiac troponin C: N- and C-terminal low affinity contributing regions.

The Spot method of multiple peptide synthesis was used to map in a systematic manner regions of the human cardiac troponin I sequence (hcTnI) involved in interactions with its physiological partner, troponin C (cTnC). Ninety-six 20-mer peptides describing the entire hcTnI sequence were chemically assembled; their reactivity with [125I]cTnC, in the presence of 3 mM Ca2+, enabled the assignment of six sites of interaction (residues 19-32, 45-54, 129-138, 145-164, 161-178 and 191-210). For several sites, a good correlation with literature data was obtained, thus validating this methodological approach. Synthetic peptides, each containing in their sequence an interaction site, were prepared. As assessed by BIACORE, all of them exhibited an affinity for cTnC in the range of 10(-6)-10(-7) M, except for hcTnI [39-58] which showed a nanomolar affinity. This peptide was also able to block the interaction between hcTnI and cTnC. We therefore postulate that despite the existence of multiple cTnC interaction sites on the hcTnI molecule, only that region of hcTnI allows a stabilization of the complex. Residues 19-32 from the N-terminal cardio-specific extension of hcTnI were also found to be involved in interaction with cTnC; residues 19-32 may correspond to the minimal sequence of the extension which could switch between the N- and C-terminal TnC domains, depending on its phosphorylation state. Finally, two Ca(2+)-dependent cTnC binding domains within the C-terminal part of hcTnI (residues 164-178 and 191-210) were also mapped. The latter site may be linked with the cardiac dysfunction observed in stunned myocardium.

Affinity for the cognate monoclonal antibody of synthetic peptides derived from selection by phage display. Role of sequences flanking thebinding motif.

Randomized peptide sequences displayed at the surface of filamentous phages are often used to select antibody ligands. The selected sequences are generally further used in the form of synthetic peptides; however, as such, their affinity for the selecting antibody is extremely variable and factors influencing this affinity have not been fully deciphered. We have used an f88.4 phage-displayed peptide library to identify ligands of mAb 11E12, an antibody reactive to human cardiac troponin I. A majority of the sequences thus selected showed a (T/A/I/L) EP(K/R/H) motif, homologous to the Y-TEPH motif identified by multiple peptide synthesis as the critical motif recognized by mAb 11E12 in the peptide epitope. A set of 15-mer synthetic peptides derived from the phage-selected sequences was used in BIACORE to characterize their interaction with mAb 11E12. Most peptides exhibited affinities in the 7-26 nM range. These affinities represented, however, only 1.9-7. 5% of the affinity of the 15-mer peptide epitope. In circular dichroism experiments, the peptide epitope showed a propensity to have some stabilized conformation, whereas a low-affinity peptide selected by phage-display did not. To try to decipher the molecular basis of this difference in affinity, new peptides were prepared by grafting the N- or the C-terminal sequence of the peptide epitope to the Y-TEPK motif of a low-affinity peptide selected by phage-display. These hybrid peptides showed marked increases both in affinity (as assessed using BIACORE) and in inhibitory potency (as assessed in competition ELISA), compared with the parent sequence. Thus, the sequences flanking the motif, although not containing critical residues, convey some determinants necessary for high affinity. The affinity of a given peptide strongly depends on its capacity to maintain the antigenically reactive structure it has on the phage, implying that it is impossible to predict whether high- or low-affinity peptides will be obtained from phage display.

Low molecular weight serine protease inhibitors from insects are proteins with highly conserved sequences.

A low molecular weight protease inhibitor peptide found in ovaries of the desert locust Schistocerca gregaria (SGPI-2), was purified from plasma of the same locust and sequenced. It was named SGCI. It was found active towards chymotrypsin and human leukocyte elastase. SGCI was synthesized using a solid-phase procedure and the sequence of its reactive site for chymotrypsin was determined. Compared with an inhibitor purified earlier from another locust species, the total sequence of SGCI showed 88% identity. In particular, the sequence of the reactive site of these inhibitors was identical. Our search for a closely related peptide in an insect species far removed from locusts, the lepidopteran Spodoptera littoralis, was unfruitful but a different chymotrypsin inhibitor, belonging to the Kazal family, was found whose mass is greater than that of SGCI (20 vs 3.6 kDa). Its N-terminal sequence shares 80% identity with that of a chymotrypsin inhibitor purified earlier from the haemolymph of another lepidopteran. Conservation of the amino acid sequence in the reactive site seems to be an exception among protease inhibitors.

Protein-disulfide isomerase (PDI) in FRTL5 cells. pH-dependent thyroglobulin/PDI interactions determine a novel PDI function in the post-endoplasmic reticulum of thyrocytes.

Thyroglobulin (TG) is secreted by the thyrocytes into the follicular lumen of the thyroid. After maturation and hormone formation, TG is endocytosed and delivered to lysosomes. Quality control mechanisms may occur during this bidirectional traffic since 1) several molecular chaperones are cosecreted with TG in vivo, and 2) lysosomal targeting of immature TG is thought to be prevented via the interaction, in acidic conditions, between the Ser(789)-Met(1172) TG hormonogenic domain (BD) and an unidentified membrane receptor. We investigated the secretion and cell surface expression of PDI and other chaperones in the FRTL5 thyroid cell line, and then studied the characteristics of the interaction between TG and PDI. We demonstrated that PDI, but also other chaperones such as calnexin and KDEL-containing proteins are exposed at the cell surface. We observed on living cells or membrane preparations that PDI specifically binds TG in acidic conditions, and that only BD is involved in binding. Surface plasmon resonance analysis of TG/PDI interactions indicated: 1) that PDI bound TG but only in acidic conditions, and that it preferentially recognized immature molecules, and 2) BD is involved in binding even if cysteine-rich modules are deleted. The notion that PDI acts as an "escort" for immature TG in acidic post-endoplasmic reticulum compartments is discussed.

Human anti-thyroid peroxidase single-chain fragment variable of Ig isolated from a combinatorial library assembled in-cell: insights into the in vivo situation.

In an attempt to explore the natural variable heavy and light chain (VH/VL) pairing of autoantibodies involved in Graves' disease, we constructed a phage-displayed Ab library obtained by in-cell PCR of thyroid-infiltrating cells. We report here the molecular cloning and characterization of human single-chain fragment variable regions (scFv) specific for thyroid peroxidase (TPO) generated from this library. On the basis of the nucleotide sequences, three different scFvs were obtained (ICA1, ICB7, and ICA5). All were encoded by genes derived from the VH1 and Vlambda1 gene families. Using BIACORE for epitope mapping and kinetic analysis, we showed that these scFvs exhibited high affinity (Kd = 1 nM) for TPO and recognized three different epitopes. The biological relevance of these scFvs as compared with serum anti-TPO autoantibodies was assessed by competition studies. Sera from all the 29 Graves' disease patients tested were able to strongly inhibit (60-100%) the binding of the 3 scFvs to TPO. These data demonstrate that the in-cell PCR library generated human anti-TPO scFvs that retained the VH/VL pairing found in vivo and that the different epitope specificities defined by these scFvs overlapped with those found in the sera of patients with autoimmune thyroid disease.

Interleukin-6 receptor signaling. II. Bio-availability of interleukin-6 in serum.

Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. With a view to studying the physiological role of these soluble receptors, both proteins were purified from human plasma. Surface plasmon resonance was used to measure the kinetic constants of equilibria between IL-6 and natural sIL-6R, and between the IL-6/sIL-6R complex and soluble gp130. Kd values were found to be 0. 9 and 2.3 nM respectively. Soluble natural IL-6R and gp130 were also found to interact with a Kd of 2.8 nM in the absence of IL-6. By using these Kd values, a mathematical simulation predicted that 1) within a large range of IL-6, sIL-6R and sgp130 concentrations, free IL-6 represents 30% of the total circulating cytokine, 2) sIL-6R overconcentrations lead to dramatic changes of the concentration of free IL-6, 3) increased concentrations of sgp130 should produce an efficient buffering effect on the IL-6/sIL-6R complex without incidence on the level of free IL-6. According to this model, the IL-6/sIL-6R complex appears to be an important support of IL-6 signaling in the most commonly encountered in vivo situations. The concentration of this complex is directly under the control of the concentration of sIL-6R; its bio-availability should be efficiently buffered by increased sgp130 concentrations.

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.

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.

One-step conversion of amino acids into N-menthyloxycarbonyl alkyl ester derivatives for chiral gas chromatography.

Amino acids dissolved in aqueous methanol (or ethanol) and treated with optically active menthyl chloroformate were converted into N-menthyloxycarbonyl methyl (or ethyl) ester derivatives within a few minutes at room temperature. The obtained diastereomeric derivatives, with the exception of arginine and histidine, had suitable gas chromatographic properties allowing enantiomeric analysis of a large number of proteinogenic and synthetic amino acids.

Racemization of alpha-amino acid esters by aliphatic ketones in the presence of carboxylic acids.

Amino acid esters are racemized by dissolution in a mixture of aliphatic ketones and carboxylic acids. The racemization rate mainly depends on the structure of the amino acid and on the kind of ketone and carboxylic acid used, the best racemizing medium being acetone containing 15% acetic acid. The mechanism of the racemization and the practical consequences of this study in the optical resolution field are discussed.

Immobilization of enzymes on alumina by means of pyridoxal 5'-phosphate.

A number of proteases have been immobilized on alumina in a two-step procedure: the first step converted them into semisynthetic phosphoproteins which, in the second step, spontaneously bonded to alumina through their phosphate function. The immobilized enzymes thus obtained showed the physical properties typical of the inorganic carrier and a high activity on low molecular weight substrates.

Peptide and ester synthesis in organic solvents catalyzed by seryl proteases linked to alumina.

Trypsin and alpha-chymotrypsin were immobilized to alumina-phosphocolamine complex, activated by glutaraldehyde. The immobilized enzymes show a great stability toward organic solvents miscible or immiscible with water. In the presence of a low concentration of water, the immobilized enzymes catalyzed transesterification reactions as well as peptide synthesis. The synthesized peptides were stable toward the immobilized enzymes.