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Colorectal cancer (CRC) remains a significant global health burden, emphasizing the need for innovative treatment strategies. 95% of the CRC population are microsatellite stable (MSS), insensitive to classical immunotherapies such as anti-PD-1; on the other hand, responders can become resistant and relapse. Recently, the use of cancer vaccines enhanced the immune response against tumor cells. In this context, we developed a therapeutic vaccine based on Stimulated Tumor Cells (STC) platform technology. This vaccine is composed of selected tumor cell lines stressed and haptenated in vitro to generate a factory of immunogenic cancer-related antigens validated by a proteomic cross analysis with patient’s biopsies. This technology allows a multi-specific education of the immune system to target tumor cells harboring resistant clones. Here, we report safety and antitumor efficacy of the murine version of the STC vaccine on CT26 BALB/c CRC syngeneic murine models. We showed that one cell line (1CL)-based STC vaccine suppressed tumor growth and extended survival. In addition, three cell lines (3CL)-based STC vaccine significantly improves these parameters by presenting additional tumor-related antigens inducing a multi-specific anti-tumor immune response. Furthermore, proteomic analyses validated that the 3CL-based STC vaccine represents a wider quality range of tumor-related proteins than the 1CL-based STC vaccine covering key categories of tumor antigens related to tumor plasticity and treatment resistance. We also evaluated the efficacy of STC vaccine in an MC38 anti-PD-1 resistant syngeneic murine model. Vaccination with the 3CL-based STC vaccine significantly improved survival and showed a confirmed complete response with an antitumor activity carried by the increase of CD8+ lymphocyte T cells and M1 macrophage infiltration. These results demonstrate the potential of this technology to produce human vaccines for the treatment of patients with CRC. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) remains a significant global health burden, emphasizing the need for innovative treatment strategies. 95% of the CRC population are microsatellite stable (MSS), insensitive to classical immunotherapies such as anti-PD-1; on the other hand, responders can become resistant and relapse. Recently, the use of cancer vaccines enhanced the immune response against tumor cells. In this context, we developed a therapeutic vaccine based on Stimulated Tumor Cells (STC) platform technology. This vaccine is composed of selected tumor cell lines stressed and haptenated in vitro to generate a factory of immunogenic cancer-related antigens validated by a proteomic cross analysis with patient’s biopsies. This technology allows a multi-specific education of the immune system to target tumor cells harboring resistant clones. Here, we report safety and antitumor efficacy of the murine version of the STC vaccine on CT26 BALB/c CRC syngeneic murine models. We showed that one cell line (1CL)-based STC vaccine suppressed tumor growth and extended survival. In addition, three cell lines (3CL)-based STC vaccine significantly improves these parameters by presenting additional tumor-related antigens inducing a multi-specific anti-tumor immune response. Furthermore, proteomic analyses validated that the 3CL-based STC vaccine represents a wider quality range of tumor-related proteins than the 1CL-based STC vaccine covering key categories of tumor antigens related to tumor plasticity and treatment resistance. We also evaluated the efficacy of STC vaccine in an MC38 anti-PD-1 resistant syngeneic murine model. Vaccination with the 3CL-based STC vaccine significantly improved survival and showed a confirmed complete response with an antitumor activity carried by the increase of CD8+ lymphocyte T cells and M1 macrophage infiltration. These results demonstrate the potential of this technology to produce human vaccines for the treatment of patients with CRC.

Clavulanic acid (CLV) and amoxicillin, frequently administered in combination, can be independently involved in allergic reactions. Protein haptenation with β-lactams is considered necessary to activate the immune system. The aim of this study was to assess the suitability of biotinylated analogues of CLV as probes to study protein haptenation by this β-lactam. Two synthetic approaches afforded the labeling of CLV through esterification of its carboxylic group with a biotin moiety, via either direct binding (CLV-B) or tetraethylenglycol linker (CLV-TEG-B). The second analogue offered advantages as solubility in aqueous solution and potential lower steric hindrance for both intended interactions, with the protein and with avidin. NMR reactivity studies showed that both CLV and CLV-TEG-B reacts through β-lactam ring opening by aliphatic amino nitrogen, however with different stability of resulting conjugates. Unlike CLV conjugates, that promoted the decomposition of clavulanate fragment, the conjugates obtained with the CLV-TEG-B remained linked, as a whole structure including biotin, to nucleophile and showed a better stability. This was a desired key feature to allow CLV-TEG-B conjugated protein detection at great sensitivity. We have used biotin detection and mass spectrometry (MS) to detect the haptenation of human serum albumin (HSA) and human serum proteins. MS of conjugates showed that HSA could be modified by CLV-TEG-B. Remarkably, HSA preincubation with CLV excess only reduced moderately the incorporation of CLV-TEG-B, which could be attributed to different protein interferences. The CLV-TEG-B fragment with opened β-lactam was detected bound to the 404–430HSA peptide of the treated protein. Incubation of human serum with CLV-TEG-B resulted in the haptenation of several proteins that were identified by 2D-electrophoresis and peptide mass fingerprinting as HSA, haptoglobin, and heavy and light chains of immunoglobulins. Taken together, our results show that tagged-CLV keeps some of the CLV features. Moreover, although we observe a different behavior in the conjugate stability and in the site of protein modification, the similar reactivity indicates that it could constitute a valuable tool to identify protein targets for haptenation by CLV with high sensitivity to get insights into the activation of the immune system by CLV and mechanisms involved in β-lactams allergy.

Background Allergic reactions to β-lactams are among the most frequent causes of drug allergy and constitute an important clinical problem. Drug covalent binding to endogenous proteins (haptenation) is thought to be required for activation of the immune system. Nevertheless, neither the nature nor the role of the drug protein targets involved in this process is fully understood. Here, we aim to identify novel intracellular targets for haptenation by amoxicillin ( AX) and their cellular fate. Methods We have treated B lymphocytes with either AX or a biotinylated analog ( AX-B). The identification of protein targets for haptenation by AX has been approached by mass spectrometry and immunoaffinity techniques. In addition, intercellular communication mediated by the delivery of vesicles loaded with AX-B-protein adducts has been explored by microscopy techniques. Results We have observed a complex pattern of AX-haptenated proteins. Several novel targets for haptenation by AX in B lymphocytes have been identified. AX-haptenated proteins were detected in cell lysates and extracellularly, either as soluble proteins or in lymphocyte-derived extracellular vesicles. Interestingly, exosomes from AX-B-treated cells showed a positive biotin signal in electron microscopy. Moreover, they were internalized by endothelial cells, thus supporting their involvement in intercellular transfer of haptenated proteins. Conclusions These results represent the first identification of AX-mediated haptenation of intracellular proteins. Moreover, they show that exosomes can constitute a novel vehicle for haptenated proteins, and raise the hypothesis that they could provide antigens for activation of the immune system during the allergic response. [ABSTRACT FROM AUTHOR]

Diisocyanates (dNCOs) are low molecular weight chemical sensitizers that react with autologous proteins to produce neoantigens. dNCO-haptenated proteins have been used as immunogens for generation of dNCO-specific antibodies and as antigens to screen for dNCO-specific antibodies in exposed individuals. Detection of dNCO-specific antibodies in exposed individuals for diagnosis of dNCO asthma has been hampered by poor sensitivities of the assay methods in that specific IgE can only be detected in approximately 25% of the dNCO asthmatics. Apart from characterization of the conjugates used for these immunoassays, the choice of the carrier protein and the dNCO used are important parameters that can influence the detection of dNCO-specific antibodies. Human serum albumin (HSA) is the most common carrier protein used for detection of dNCO specific-IgE and -IgG but the immunogenicity and/or antigenicity of other proteins that may be modified by dNCO in vivo is not well documented. In the current study, 2,4-toluene diisocyanate (TDI) and 1,6-hexamethylene diisocyanate (HDI) were reacted with HSA and human hemoglobin (Hb) and the resultant adducts were characterized by (i) HPLC quantification of the diamine produced from acid hydrolysis of the adducts, (ii) 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay to assess extent of cross-linking, (iii) electrophoretic migration in polyacrylamide gels to analyze intra- and inter-molecular cross-linking, and (iv) evaluation of antigenicity using a monoclonal antibody developed previously to TDI conjugated to Keyhole limpet hemocyanin (KLH). Concentration-dependent increases in the amount of dNCO bound to HDI and TDI, cross-linking, migration in gels, and antibody-binding were observed. TDI reactivity with both HSA and Hb was significantly higher than HDI. Hb–TDI antigenicity was approximately 30% that of HSA–TDI. In conclusion, this data suggests that both, the extent of haptenation as well as the degree of cross-linking differs between the two diisocyanate species studied, which may influence their relative immunogenicity and/or antigenicity. [ABSTRACT FROM AUTHOR]

The schematic model of ACC modified SPCE immobilized with different potency of skin sensitizers (extreme/strong (red), moderate (green) and weak/non sensitizers (blue)). [Display omitted] • To explore the haptenation of skin sensitizer with a modified screen printed carbon electrode and a self–assembled monolayer of gold nanoparticles and cysteine using the impedance technique. • The interaction of extreme/strong skin sensitizer was discovered with a high value of changes of charge transfer resistance of skin sensitizer ( Δ R C T s k i n s e n s i t i z e r ). • A total of 9 min was reported to be the optimum contact time with skin sensitizer. • The adsorption isotherm studies showed Langmuir isotherm adsorption and spontaneous mechanism. Haptenation of skin sensitizer is indicated because of the covalent attachment of a skin sensitizer to a skin protein. The idea of this research is to explore the haptenation of skin sensitizer with a modified screen printed carbon electrode (SPCE) and a self–assembled monolayer of gold nanoparticles (AuNPs) and cysteine (designated as ACC modified SPCE) using the impedance technique. The ACC modified SPCE was characterized using Fourier–Transform infrared spectroscopy–attenuated total reflectance (FTIR–ATR), atomic force microscopy (AFM), and Energy Dispersive X–Ray Spectroscopy (EDX). The presence of AuNPs and cysteine on the working electrode of the ACC modified electrode was detected by FTIR–ATR and EDX. AuNPs, possessing a homogeneous dot shape, and cysteine was randomly distributed on the carbon surface of SPCE shown by FESEM images. The AFM images indicated the possibility of a monolayer of adsorption of skin sensitizer onto ACC modified SPCE. The ACC modified SPCE has good reproducibility and stability with an RSD of 8.43 %. In addition, the interaction of extreme/strong skin sensitizer with the ACC modified SPCE was discovered with a high value of changes of charge transfer resistance of skin sensitizer ( Δ R C T s k i n s e n s i t i z e r ) when compared to moderate and weak/non skin sensitizers. A total of 9 min was reported to be the optimum contact time of the skin sensitizer onto ACC modified SPCE. The adsorption isotherm studies of skin sensitizers with ACC modified SPCE showed Langmuir isotherm adsorption and spontaneous mechanism. Maleic anhydride (as a model of extreme/strong skin sensitizers) showed a fast–binding rate (18.2149 M−1) with the addition of a slow dissociation rate of 0.0549 M, while glycerol (as a model of weak/non skin sensitizer) showed a slow binding rate of 4.5977 M−1 as a result of a rapid dissociation rate of 0.2175 M. The change in Gibbs free energy (ΔG°) was calculated for ACC modified SPCE with glycerol and maleic anhydride to be at –3.78 × 10+03 kJ/mol and –7.19 × 10+03 kJ/mol, respectively. This study proved that the ACC modified SPCE could be used for routine screening during the early product development stage in qualitative skin sensitization measurements. [ABSTRACT FROM AUTHOR]

• The interaction of skin sensitizers with the cysteine modified screen printed carbon electrode (SPCE) was analyzed using EIS. • The extreme/strong skin sensitizer resulted in high Δ R C T s k i n s e n s i t i z e r values compared to moderate and weak/non skin sensitizer. • Analysis conducted using AFM and FTIR-ATR supported the data obtained using EIS. • The adsorption kinetic studies for ETSC modified SPCE with skin sensitizer based on the different classes of skin sensitizer followed the Langmuir isotherm adsorption. • ETSC modified SPCE has the potential to be used for routine screening purposes during early cosmetics and personal care products development. Skin sensitization is defined as an allergic response to a skin sensitizer upon contact with the skin. Skin sensitization is induced through covalent binding of a skin sensitizer to skin proteins (haptenation process). In this research work, the working electrodes of screen printed carbon electrodes (SPCEs) were modified by electrodepositing gold nanoparticles (AuNPs), addition of thiourea and then followed by self–assembly of AuNPs and cysteine (designated as ETSC). The main purpose of this research was to study the interaction of skin sensitizers with the cysteine and gold nanoparticles modified screen printed carbon electrode (SPCE) analyzed using impedance technique. The interaction of maleic anhydride (as extreme/strong skin sensitizer) with the cysteine modified SPCE was found to result in high changes in the value of charge transfer resistance of skin sensitizer ( Δ R C T s k i n s e n s i t i z e r ) compared to moderate sensitizer (isoeugenol) and weak/non skin sensitizer (glycerol). The ETSC modified SPCE with skin sensitizers were characterised using Fourier–Transform infrared spectroscopy–attenuated total reflectance (FTIR–ATR), atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). The presence of skin sensitizers on the working electrode of ETSC modified electrode was detected by FTIR–ATR. AFM images indicated different patterns with different surface roughness for extreme/strong, moderate and weak/non skin sensitizers. Using EIS, the estimation for fractional surface coverage (θIS) of ETSC modified SPCE when contacted with maleic anhydride was higher than isoeugenol and glycerol. Other than that, the active site radius (r a), and the distance between two adjacent sites (2r b) for ETSC modified SPCE when contacted with maleic anhydride were shorter than the r a and 2rb obtained for isoeugenol and glycerol, indicating higher surface average roughness for ETSC modified SPCE that was contacted with maleic anhydride. Data obtained from EIS agreed with images obtained using AFM. The adsorption kinetic studies for ETSC modified SPCE with skin sensitizers showed that the skin sensitizers displayed Langmuir isotherm adsorption. This work showed that the ETSC modified SPCE has the potential to be employed for the screening of potential skin sensitizers during the early stage of cosmetic and personal care products development. [ABSTRACT FROM AUTHOR]

Haptenation of model nucleophiles, representing the key MIE in skin sensitisation, is routinely measured in chemico to provide data for skin allergy risk assessment. Better understanding of the dynamics of haptenation in human skin could provide the metrics required to improve determination of sensitiser potency for risk assessment of chemicals. We have previously demonstrated the applicability and sensitivity of the dual stable isotope labelling approach to detect low level haptenation in complex mixtures of proteins. In the present study, we investigated haptenation in a relevant living cell model over time at a subtoxic concentration. DNCB, an extremely potent sensitiser, caused minimal changes in overall protein differential expression in HaCaT cells and haptenated approximately 0.25 % of all available nucleophiles when applied at a subtoxic concentration (10μM) for 4 h. The data shows that the maximum level of haptenation occurs at 2 h and that DNCB, whilst being a promiscuous hapten, shows a preference for Cys residues, despite the considerably higher concentration of amine-based nucleophiles. Although a proportion of highly abundant proteins were haptenated, numerous haptenated sites were also detected on low abundant proteins. Certain proteins were modified at residues buried deep inside the protein structure which are less accessible to haptenation compared with surface exposed nucleophiles. The microenvironment of the buried residues may be a result of several factors influencing the reactivity of both the target nucleophile and the hapten. [ABSTRACT FROM AUTHOR]

Clavulanic acid (CLV) and amoxicillin, frequently administered in combination, can be independently involved in allergic reactions. Protein haptenation with β-lactams is considered necessary to activate the immune system. The aim of this study was to assess the suitability of biotinylated analogues of CLV as probes to study protein haptenation by this β-lactam. Two synthetic approaches afforded the labeling of CLV through esterification of its carboxylic group with a biotin moiety, via either direct binding (CLV-B) or tetraethylenglycol linker (CLV-TEG-B). The second analogue offered advantages as solubility in aqueous solution and potential lower steric hindrance for both intended interactions, with the protein and with avidin. NMR reactivity studies showed that both CLV and CLV-TEG-B reacts through β-lactam ring opening by aliphatic amino nitrogen, however with different stability of resulting conjugates. Unlike CLV conjugates, that promoted the decomposition of clavulanate fragment, the conjugates obtained with the CLV-TEG-B remained linked, as a whole structure including biotin, to nucleophile and showed a better stability. This was a desired key feature to allow CLV-TEG-B conjugated protein detection at great sensitivity. We have used biotin detection and mass spectrometry (MS) to detect the haptenation of human serum albumin (HSA) and human serum proteins. MS of conjugates showed that HSA could be modified by CLV-TEG-B. Remarkably, HSA preincubation with CLV excess only reduced moderately the incorporation of CLV-TEG-B, which could be attributed to different protein interferences. The CLV-TEG-B fragment with opened β-lactam was detected bound to the 404–430HSA peptide of the treated protein. Incubation of human serum with CLV-TEG-B resulted in the haptenation of several proteins that were identified by 2D-electrophoresis and peptide mass fingerprinting as HSA, haptoglobin, and heavy and light chains of immunoglobulins. Taken together, our results show that tagged-CLV keeps some of the CLV features. Moreover, although we observe a different behavior in the conjugate stability and in the site of protein modification, the similar reactivity indicates that it could constitute a valuable tool to identify protein targets for haptenation by CLV with high sensitivity to get insights into the activation of the immune system by CLV and mechanisms involved in β-lactams allergy. [ABSTRACT FROM AUTHOR]