Targeting immune checkpoint B7-H3 antibody-chlorin e6 bioconjugates for spectroscopic photoacoustic imaging and photodynamic therapy.

In this study, we constructed bioconjugates of targeting immune checkpoint B7-H3 antibody and chlorin e6 to treat non-small cell lung cancer under the guidance of spectroscopic photoacoustic and fluorescence imaging. The B7-H3-Ce6 conjugates could display effective tumor diagnosis and therapy and provide a novel approach for immunotherapy.

Photodynamic therapy-generated vaccines: relevance of tumour cell death expression.

Recent investigations have established that tumour cells treated in vitro by photodynamic therapy (PDT) can be used for generating potent vaccines against cancers of the same origin. In the present study, cancer vaccines were prepared by treating mouse SCCVII squamous cell carcinoma cells with photosensitiser chlorin e6-based PDT and used against poorly immunogenic SCCVII tumours growing in syngeneic immunocompetent mice. The vaccine potency increased when cells were post-incubated in culture after PDT treatment for 16 h before they were injected into tumour-bearing mice. Interfering with surface expression of phosphatidylserine (annexin V treatment) and apoptosis (caspase inhibitor treatment) demonstrated that this post-incubation effect is affiliated with the expression of changes associated with vaccine cell death. The cured mice acquired resistance to re-challenge with the same tumour, while the engagement of cytotoxic T lymphocytes was demonstrated by detection of high numbers of degranulating CD8+ cells in vaccinated tumours. The vaccines prepared from ex vivo PDT-treated SCCVII tumour tissue were also highly effective, implying that surgically removed tumour tissue can be directly used for PDT vaccines. This opens attractive prospects for employing PDT vaccines tailored for individual patients targeting specific antigens of the patient's tumour.

A Phase II safety and effect on time to tumor progression study of intratumoral light infusion technology using talaporfin sodium in patients with metastatic colorectal cancer.

BACKGROUND AND OBJECTIVES: Twenty-seven patients with refractory liver metastases from colorectal cancer took part in a Phase II study of the light infusion technology (Litx) light-activated drug/device system to assess safety and evaluate time to tumor progression (TTP). METHODS: Litx consists of the light-activated drug, talaporfin sodium (LS11), activated intratumorally by a catheter-like array of light-emitting diodes (LEDs). After placement of the array via ultrasound or computed tomography (CT) guidance, LS11 was administered intravenously, followed 15-60 min later by light infusion for 2.8 hr. Patients were assessed for adverse events and tumor response using physical examination, laboratory values, and CT scan evaluation over a period of 60 days. RESULTS: The observed occurrence of Litx treatment-related adverse events was minimal and cumulative toxicity did not occur when combined with chemotherapy. Assessment of TTP and tumor response rate, although statistically non-robust, suggest potential improvement. CONCLUSIONS: The Litx system was shown to be safe for treating liver metastases from colorectal cancer and there was no cumulative toxicity when combined with standard systemic therapy. Preliminary assessments of TTP and tumor response rate justify further evaluation in a Phase III follow-up study.

Development and identification of monoclonal antibodies against meso-Tetra (alpha,alpha,alpha,alpha,-O-phenylacetamide benzene) porphyrin.

The small molecule meso-Tetra (alpha,alpha,alpha,alpha-o-phenylacetamide benzene) porphyrin was synthesized through the condensation of o-nitrobenzaldehyde and pyrrole followed by reduction of the meso-tetra (o-nitrophenyl) porphyrin. The small molecule, without carrier, was used as complete antigen to immunize BALB/ C mice. Spleen cells producing high titer antibody were removed and fused with myeloma cells of SP2/0 origin. Using a conventional immunization protocol, stable murine monoclonal antibodies (MAbs) producing cell lines to meso-Tetra (alpha,alpha,alpha,alpha-o-phenylacetamide benzene) porphyrin 1F2 were obtained. Subclass determination showed that the clones produce IgG2a types of MAbs. The analytical results of HPLC and MALDI/TOFMS suggest that the purity of MAb 1F2 is 100%, and MAb 1F2 has a relative molecular weight of 156678.8 Da. Our results demonstrated that small molecule meso-Tetra (alpha,alpha,alpha,alpha-o-phenylacetamide benzene) porphyrin, as semiantigen without carrier, can elicit the formation of MAbs.

Synthesis and in vitro investigation of cationic 5,15-diphenyl porphyrin-monoclonal antibody conjugates as targeted photodynamic sensitisers.

The synthesis of three cationic 5,15-diphenyl porphyrins, bearing an isothiocyanate group for conjugation to proteins is described. The potential of these compounds as targeted photosensitisers for use in photodynamic therapy (PDT) is demonstrated by their efficient conjugation to monoclonal antibodies and the ability of the bioconjugates to bind and photodynamically inactivate cancer cells that express the corresponding antigen.

The development and characterisation of porphyrin isothiocyanate-monoclonal antibody conjugates for photoimmunotherapy.

A promising approach to increase the specificity of photosensitizers used in photodynamic therapy has been through conjugation to monoclonal antibodies (MAb) directed against tumour-associated antigens. Many of the conjugations performed to date have relied on the activated ester method, which can lead to impure conjugate preparations and antibody crosslinking. Here, we report the development of photosensitizer-MAb conjugates utilising two porphyrin isothiocyanates. The presence of a single reactive isothiocyanate allowed facile conjugation to MAb FSP 77 and 17.1A directed against internalizing antigens, and MAb 35A7 that binds to a non-internalizing antigen. The photosensitizer-MAb conjugates substituted with 1-3 mol of photosensitizer were characterised in vitro. No appreciable loss of immunoreactivity was observed and binding specificity was comparable to that of the unconjugated MAb. Substitution with photosensitizer had a minimal effect on antibody biodistribution in vivo for the majority of the conjugates, although a decreased serum half-life was observed using a cationic photosensitizer at the higher loading ratios. Tumour-to-normal tissue ratios as high as 33.5 were observed using MAb 35A7 conjugates. The internalizing conjugate showed a higher level of phototoxicity as compared with the non-internalizing reagent, using a cell line engineered to express both target antigens. These data demonstrate the applicability of the isothiocyanate group for the development of high-quality conjugates, and the use of internalizing MAb to significantly increase the photodynamic efficiency of conjugates during photoimmunotherapy.

Studies of the reactivity of artificial peroxidase-like hemoproteins based on antibodies elicited against a specifically designed ortho-carboxy substituted tetraarylporphyrin.

The temperature and pH dependence as well as the selectivity of the peroxidase activity of a complex associating a monoclonal antibody 13G10 with its iron(III)-alpha,alpha,alpha,beta-mesotetrakis(ortho-carboxyphenyl) porphyrin (Fe(ToCPP)) hapten have been studied and compared to those of Fe(ToCPP) alone. It first appears that the peroxidase activity of the 13G10-Fe(ToCPP) complex is remarkably thermostable and remains about 5 times higher than that of Fe(ToCPP) alone until at least 80 degrees C. Secondly, this complex is able to use not only H2O2 as oxidant but also a wide range of hydroperoxides such as alkyl, aralkyl and fatty acid hydroperoxides and catalyze their reduction 2-6-fold faster than Fe(ToCPP) alone. It is also able to catalyze the oxidation by H202 of a variety of reducing cosubstrates such as 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine (TMB) and 3,3'-dimethoxybenzidine 3-8-fold faster than Fe(ToCPP) alone, the bicyclic aromatic ABTS and TMB being the best reducing cosubstrates. Finally, a pH dependence study, between pH 4.6 and 7.5, of the oxidation of ABTS by H2O2 in the presence of either 13G10-Fe(ToCPP) or Fe(ToCPP) shows that Km(H2O2) values vary very similarly for both catalysts, whereas very different variations are found for the k(cat) values. With Fe(ToCPP) as catalyst the k(cat) value remains constant around 100 min(-1) whereas with the 13G10-Fe(ToCPP) complex, it increases sharply below pH 5 to reach 540 min -1 at pH 4.6. This could be due to the participation of a carboxylic acid side chain of the antibody protein, as a general acid-base catalyst, to the heterolytic cleavage of the O-O bond of H2O2 leading to the highly reactive iron(V)-oxo intermediate in the peroxidase mechanism. Accordingly, the modification of the carboxylic acid residues of antibody 13G10 by glycinamide leads to a 50% decrease of the peroxidase activity of the 13G10-Fe(ToCPP) complex.

Hemoabzymes. Different strategies for obtaining artificial hemoproteins based on antibodies.

Besides existing models of chemical or biotechnological origin for hemoproteins like peroxidases and cytochromes P450, catalytic antibodies (Abs) with a metalloporphyrin cofactor represent a promising alternative route to catalysts tailored for selective oxidation reactions. A brief overview of the literature shows that, until now, the first strategy for obtaining such artificial hemoproteins has been to produce antiporphyrin Abs, raised against various free-base, N-substituted, Sn-, Pd-, or Fe-porphyrins. Four of them exhibited, in the presence of the corresponding Fe-porphyrin cofactor, a significant peroxidase activity, with kcat/K(m) values of 10(2) to 5 x 10(3)/M/s. This value remained low when compared to that of peroxidases, probably because neither a proximal ligand of the Fe, nor amino acid residues participating in the catalysis of the heterolytic cleavage of the O-O bond of H2O2, have been induced in those Abs. This strategy has been shown to be insufficient for the elaboration of effective models of cytochromes P450, because only one set of Abs, raised against meso-tetrakis(para-carboxyvinylphenyl)porphyrin, was reported to catalyze the nonstereoselective oxidation of styrene by iodosyl benzene using a Mn-porphyrin cofactor, and attempts to generate Abs having binding sites for both the substrate and the metalloporphyrin cofactor, using as a hapten a porphyrin covalently linked to the substrate, were not successful. A second strategy is then proposed, which involves the chemical labeling of antisubstrate Abs with a metalloporphyrin. As an example, preliminary results are presented on the covalent linkage of an Fe-porphyrin to an antiestradiol Ab, in order to obtain semisynthetic catalytic Abs able to catalyze the selective oxidation of steroids.

Structural characterization of mouse monoclonal antibody 13-1 against a porphyrin derivative: identification of a disulfide bond in CDR-H3 of Mab 13-1.

The amino acid sequence of a mouse monoclonal antibody Mab13-1, a catalytic antibody against TCPP (meso-tetrakis(4-carboxyphenyl)porphyrin), was confirmed by mass spectrometric (MS) peptide mapping. The amino-terminal sequence of the heavy chain was established by MS/MS analysis of the isolated N-terminal peptide. The presence of a unique disulfide bond between Cys93H and Cys102H was identified by MS peptide mapping and sequence analysis of an S-S containing peptide. Positions of other disulfide bonds were identified to be conserved. The non-conserved disulfide bridge was found to be resistant as other intra-chain disulfide bonds against reduction under non-denaturing condition, and to be buried inside the molecule. This extra disulfide bond is expected to support antigen-binding by restricting the flexibility of CDR-H3 loop, and it might be favorable for the recognition of a plane antigen, a porphyrin derivative.

Studies of high thermostability and peroxidase activity of recombinant antibody L chain-porphyrin Fe(III) complex.

A complex of an independent L chain from anti-mesotetrakis (4-carboxyphenyl) porphyrin (TCPP) monoclonal antibody 13-1 and TCPP Fe(III) was designated as L-zyme and shown to exhibit high peroxidase activity and high optimal reaction temperature (90 degrees C). Heat denaturation study and circular dichroism (CD) spectra analysis suggested that refolded structure of 13-1 L chain exhibited significantly reduced inactivation rate after heat treatment. The secondary structure of 13-1 L chain changed slightly by the encapsulation of TCPP Fe(III) and the complex was found to be less thermostable than the L chain alone. Furthermore, by characterization of truncated forms of the L chain, it was revealed that the hydrophobic region (115-146) and hydrophilic region (147-189) in CL are important for thermostability and activity, respectively. Tertiary structure of L-zyme was predicted by AbM. Comparison of residues of L-zyme with those in the active centre of known structure of the peroxidase from Arthromyces ramosus (ARP) indicated that His38(CDR1), His94(CDR3), Arg96(CDR3) of L-zyme are important residues for peroxidase activity. Moreover, the steric arrangements of these residues in both L-zyme and ARP are similar, respectively. Distance between proximal His and distal His in L-zyme is 9.09 A, whereas that of ARP is 7.8 A.

Singlet oxygen producing photosensitizers in photophoresis.

In this study, the immunosuppressive properties of two photosensitizers (benzoporphyrin derivative monoacid ring A (BPD) and Photofrin (HPD)), used for the photodynamic therapy of cancer, were investigated. The investigations were performed in our rat model for photophoresis. The validity of this model has been amply demonstrated. It enables a distinction to be made between antigen-specific and antigen non-specific immune suppression. With this model, the immune response which can be specifically suppressed is the contact hypersensitivity (CHS). CHS is induced by 2,4-dinitrofluorobenzene (DNFB). Both BPD and HPD are able to suppress CHS induced by DNFB. Furthermore, this generated suppression is transferable by the spleen cells of treated animals and is antigen non-specific.

Thermostable peroxidase activity with a recombinant antibody L chain-porphyrin Fe(III) complex.

In order to engineer a new type of catalytic antibody, we attempt to use a monoclonal antibody L chain as a host protein for a porphyrin. TCPP (meso-tetrakis(4-carboxyphenyl)porphyine) was chemically synthesized and Balb/c mice were immunized using TCPP as a hapten. Two hybridoma cells (03-1, 13-1), that produce monoclonal antibody against TCPP, were obtained. Genes for both H and L chains of monoclonal antibodies were cloned, sequenced and overexpressed using E. coli as a host. ELISA and fluorescence quenching method show that the independent antibody L chains from both Mab03-1 and Mab13-1 have specific interaction with TCPP. Furthermore, the recombinant antibody L chain from Mab13-1 exhibits much higher peroxidase activity than TCPP Fe(III) alone. The enzyme activity was detectable with pyrogallol and ABTS (2,2-azinobis-3-ethylbenzthiazolin-6-sulfonic acid) but not with catechol. This new catalytic antibody was extremely thermostable. Optimum temperature of the peroxidase reaction by the complex of 13-1L chain and TCPP Fe(III) was 90 degrees C, while that the TCPP Fe(III) alone was 60 degrees C.

[Nonspecific photophysiological immunization]. / O nespetsificheskoi fotofiziologicheskoi immunizatsii.

A hypothesis is suggested on the positive effect of light radiation on the formation of protective immune mechanism related to the photophysiological destruction of porphyrins. References to experimental data are given which illustrate the possibility for existence of such a mechanism.

Facile assignment of sequence ions of a peptide labelled with 18O at the carboxyl terminus.

The combination of collision-induced dissociation (CID) and linked-scan analysis was used for analysing the sequence ions from the precursor ion of a peptide, which had been labelled with 18O at its carboxyl terminus (C-terminus) using 40 atom % H2 18O. The CID and linked-scan mass spectrum of the labelled peptide gave two series of sequence-ion signals: the one, originating from the C-terminus of the labelled peptide, showed a doublet signal due to the part-incorporation of 18O into the carboxyl group at the C-terminus, while the other, originating from the amino terminus (N-terminus), has the natural isotopic ion distribution. From the distribution of the isotopic ions in a single CID spectrum, the sequence ions containing the C-terminus could be readily differentiated from those containing the N-terminus, allowing the facile assignment of sequence ions to the amino-acid sequence of a peptide by CID and linked-scan analysis. This method was successfully applied to determination of the amino-acid sequence of the light-chain of mouse anti-porphyrin monoclonal antibody.

Urinary excretion of porphyrins is increased in patients with HIV-1 infection.

Urine concentrations of total porphyrins and of porphyrin precursors, delta-aminolaevulinic acid and porphobilinogen, were analysed in the first-morning urine samples of 36 people with established HIV-1 infection. For comparison, we also analysed the urine samples of 26 healthy HIV-seronegative people. In patients with HIV-1 infection concentrations of total porphyrins were found to be significantly higher than in healthy controls. Furthermore, there was a close correlation between concentrations of total porphyrins and porphobilinogen and urine neopterin concentrations in patients. Our data indicate an association between chronic immune activation and altered porphyrin metabolism. The data may provide a rationale for the observation of acute porphyria in patients with HIV-1 infection.

[Fluorescent immunoanalysis. Synthesis, spectro-fluorescent and immunologic properties of conjugates of coproporphyrin I with antibodies]. / Fluorestsentni immunoanaliz. Sintez, spektral'no-fluorestsentnye i immunologicheskie svoistva kon''iugatov koproporfirina I s antitelami

The synthesis of protein conjugates with the new high-efficient fluorescent labile coproporphyrin-I was optimized. A number of conjugates of monoclonal antibodies with different coproporphyrin-I content were synthesized, and their spectral properties were studied in water and micellar solutions, i.e. adsorption, excitation and emission spectra, fluorescence quantum yields, fluorescence pH-dependences. The binding constants of coproporphyrin-I and its protein conjugates with serum albumin were determined. The antibodies labelled with coproporphyrin-I retain the functional activity and photochemically stable in water solutions. The sensitivity of fluorometric detection of coproporphyrin-I and its conjugates with proteins is more than 10 times greater than in case of FITC.

Factors responsible for the formation of different N-alkylated porphyrins in rat liver microsomal systems exposed to norethindrone. The role of 3 alpha-hydroxysteroid dehydrogenase.

Incubation of rat liver microsomes with norethindrone and a NADPH-generating system leads to the formation of one N-alkylated porphyrin (green pigment, GP1). Administration of this steroid to male rats in vivo results in the formation of three more-polar green pigments (GP2, 3 and 4). A cytosolic protein (green-pigment converting protein) has been purified from rat liver that, when added to liver microsomal mixtures containing norethindrone (0.03 mM) and a NADPH-generating system, results in the formation of all four green pigments (GP1, 2, 3 and 4). Field-desorption mass spectrometry of the purified green pigments gave protonated molecules, [M + H]+, at m/z 905 for GP1, m/z 909 for GP2, m/z 925 for GP3 and 4. The Mr of the purified cytosolic protein on SDS/polyacrylamide-gel electrophoresis or gel filtration was 37000. Polyacrylamide-gel isoelectric focusing gave a pI value of 5.9. Antibodies raised in rabbits against this protein, after preincubation with rat liver cytosol, subsequently prevented the formation of the more-polar norethindrone-induced green pigments (GP2, 3 and 4). The purified protein in the presence of either NADH or NADPH catalysed the reduction of delta 4-ring-reduced norethindrone, 5 alpha-oestran-17 alpha-ethynyl-17 beta-ol-3-one and, with the appropriate cofactor, the oxidation and reduction of steroids lacking the ethynyl function, e.g. androsterone or dihydrotestosterone. Indomethacin inhibited the reduction of dihydrotestosterone by this protein with an I50 (concn. causing 50% inhibition) value of 4.9 microM. From its physical and enzymic properties it is concluded that green-pigment converting protein is the same as 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50).

Serum antibodies against porphyric hepatocytes in patients with porphyria cutanea tarda and liver disease.

The existence of serum antibodies against porphyric or normal rat hepatocytes was investigated in patients with porphyria cutanea tarda and in other forms of chronic liver disease. Ten patients with porphyria cutanea tarda, 7 of them with chronic active hepatitis or cirrhosis (group 1a) and 3 without significant liver damage (group 1b), 8 patients with nonporphyric chronic active hepatitis (group 2), and 8 alcoholic cirrhotics, 3 of them with superimposed severe alcoholic hepatitis (group 3), were studied. In an antibody-dependent cell-mediated cytotoxicity test using isolated hepatocytes from normal and hexachlorobenzene-treated (porphyric) rats as targets, it was found that sera from group 1a produced high cytotoxicity against porphyric hepatocytes and low or zero cytotoxicity against normal hepatocytes (p less than 0.001). The opposite cytotoxic pattern was observed when sera from group 2 was tested. Sera from groups 1b and 3 failed to produce cytotoxicity against both targets. The cytotoxic effect on porphyric hepatocytes was significantly reduced by preincubation of serum with free uroporphyrin or by serum absorption with a sepharose-uroporphyrin immunosorbent. Immunofluorescence studies confirmed the existence of antiporphyric hepatocyte antibodies in group 1a. In conclusion, our results show that antiporphyric hepatocytes antibodies are present in some patients with porphyria cutanea tarda and indicate that hepatocellular porphyrin might be partially responsible for the antigenicity of the liver cells. The role of these antibodies in the pathogenesis of the liver lesion remains to be elucidated.