A patient with melanoma that became sensitized to immunotherapy after treatment with a CDK4/6 inhibitor.

Background: Despite the profound effect that checkpoint inhibitors and BRAF/MEK inhibitors have had on survival in patients with metastatic melanoma, treatment options remain limited for those who demonstrate poor response or develop resistance to these modalities. The prospect of tumor sensitization to these treatments is therefore an attractive one. Results: We describe the case of a patient who developed a sustained response to trametinib and pembrolizumab, despite prior resistance to both these therapies, after receiving treatment with a CDK4/6 inhibitor. Discussion: We further outline the preclinical data supporting a possible role for the use of CDK4/6 inhibitors in tumor sensitization to immunotherapy.

A totally synthetic lipopeptide-based self-adjuvanting vaccine induces neutralizing antibodies against heat-stable enterotoxin from enterotoxigenic Escherichia coli.

ST-based lipopeptide vaccine candidates were constructed in which ST was chemically synthesized and folded into the correct conformation prior to ligation to a module containing a T-helper cell epitope (T(H)) and the Toll-like receptor 2 (TLR2) agonist, S-[2,3-bis(palmitoyloxy)propyl]cysteine (P2C). Two different chemistries, thioether-based and oxime-based, were then used to ligate ST to the lipidated T(H) epitope. The enterotoxic activity of synthetic ST and the ST-based lipopeptide vaccines was determined in mice followed by an evaluation of immunological efficacy. The importance of the fine detail in chemical composition used in vaccine design was demonstrated by the findings that (i) the oxime-based vaccine exhibited little or no toxicity but the thioether-based vaccine, exhibited residual toxicity in suckling mice, (ii) although each of the synthetic vaccines generated specific anti-ST antibodies, it was the low titer antibodies induced by the oxime-based vaccine that demonstrated better neutralizing activity suggesting that the chemical linkage also affects the specificity of antibodies, (iii) the geometric arrangement of ST within a vaccine can profoundly affect the specificity and biological function of the antibodies that are elicited, and (iv) the lipopeptide-based ST vaccine candidate assembled using oxime chemistry induced a better neutralizing antibody response to ST when administered by the mucosal (intranasal) route.

Influence of different length linker containing DHEA-7-CMO-enzyme conjugates on sensitivity and specificity of DHEA-17-CMO-antibody.

Introduction of spacers in enzyme conjugates is known to exert an influence on the assay parameters of steroid enzyme immunoassays. We have introduced 3 to 10 atomic length linkers between enzyme and steroid moieties and studied their effects on sensitivity and specificity of dehydroepiandrosterone enzyme immunoassays. Dehydroepiandrosterone-17-carboxymethyloxime-bovine serum albumin (DHEA-17-CMO-BSA) was used as an immunogen to raise the antiserum in New Zealand white rabbits. Five enzyme conjugates were prepared using DHEA-7-CMO as carboxylic derivative of DHEA and horseradish peroxidase (HRP) as label. These were DHEA-7-CMO-HRP, DHEA-7-CMO-urea-HRP (DHEA-7-CMO-U-HRP), DHEA-7-CMO-ehylenediamine-HRP (DHEA-7-CMO-EDA-HRP), DHEA-7-CMO-carbohydrazide-HRP (DHEA-7-CMO-CH-HRP), and DHEA-7-CMO-adipic acid dihydrazide-HRP (DHEA-7-CMO-ADH-HRP). The influence of different atomic length linkers on sensitivity and specificity were studied with reference to label without linker. The results of the present investigation revealed that with incorporation of linkers, the sensitivity improves, whereas specificity only marginally improves. These differential behaviors of various linkers toward the sensitivity and specificity of assays might be due to the difference in the magnitude of overall forces of attraction between the antibody and the enzyme conjugates.

Antigen heterologous enzyme linked immunosorbent assay for the measurement of DHEA in serum.

Homologous and heterologous combinations of enzyme conjugate and antibody in steroid enzyme immunoassay (EIA) influences unlabeled steroid recognition by antibody that affects sensitivity of the assay. To develop dehydroepiandrosterone (DHEA) antigen heterologous enzyme linked immunosorbent assay (ELISA), antibodies were generated against DHEA-3-hemisuccinate-bovine serum albumin (DHEA-3-HS-BSA), DHEA-7-carboxymethyloxime-bovine serum albumin (DHEA-7-CMO-BSA), and DHEA-17-carboxymethyloxime-bovine serum albumin (DHEA-17-CMO-BSA). Five horseradish peroxidase (HRP) enzyme conjugates were prepared using five testosterone derivatives [testosterone-3-CMO (T-3-CMO), testosterone-17-HS (T-17-HS), testosterone-17-glucuronoside (T-17-G), testosterone-19-carboxymethylether (T-19-CME), and testosterone-11-HS (T-11-HS)]. Fifteen antigen heterologous combinations of antibody and enzyme conjugates were evaluated in the standard binding assay; only two combinations showed binding. The use of antigen heterologous combination (different antigen in label than the immunogen) resulted in development of a simple, direct, and convenient assay as it permits the direct addition of the serum sample into the assay and it requires only 1.5 h to complete.

Preparation for aflatoxin B(1)-cationized bovine serum albumin based on Mannich-type reaction.

Aflatoxin B(1) (AFB(1)), which is commonly found in agricultural commodities, is one of the most potent carcinogenic mycotoxins. To ensure food safety, rapid and low-cost immunological methods have been applied to detect AFB(1) worldwide. A key step in these immunological methods is coupling AFB(1) to carrier proteins; AFB(1) is usually deviated to AFB(1)-oxime and coupled to carrier proteins to form the AFB(1)-oxime-protein conjugate. In the current research, AFB(1) was directly coupled with cationized bovine serum albumin (cBSA) using a method based on Mannich-type principles. The coupling effects were investigated with different initial molar ratios of AFB(1) to cBSA. The conjugate molar ratio was 6.4:1 when the initial molar ratio was 40:1. The cationized proteins and their conjugates were identified by UV-Vis and FT-IR spectra, which showed the characteristic bands of the ethylendiamine group and AFB(1), respectively. After BALB/c mice were immunized with AFB(1)-cBSA, a quicker immunological response and a similar sensitivity of antisera against AFB(1) were observed, compared with immunization by AFB(1)-oxime-BSA. This suggests that the Mannich-type reaction might be an alternative method of preparation for AFB(1)-protein.

A totally synthetic polyoxime malaria vaccine containing Plasmodium falciparum B cell and universal T cell epitopes elicits immune responses in volunteers of diverse HLA types.

This open-labeled phase I study provides the first demonstration of the immunogenicity of a precisely defined synthetic polyoxime malaria vaccine in volunteers of diverse HLA types. The polyoxime, designated (T1BT(*))(4)-P3C, was constructed by chemoselective ligation, via oxime bonds, of a tetrabranched core with a peptide module containing B cell epitopes and a universal T cell epitope of the Plasmodium falciparum circumsporozoite protein. The triepitope polyoxime malaria vaccine was immunogenic in the absence of any exogenous adjuvant, using instead a core modified with the lipopeptide P3C as an endogenous adjuvant. This totally synthetic vaccine formulation can be characterized by mass spectroscopy, thus enabling the reproducible production of precisely defined vaccines for human use. The majority of the polyoxime-immunized volunteers (7/10) developed high levels of anti-repeat Abs that reacted with the native circumsporozoite on P. falciparum sporozoites. In addition, these seven volunteers all developed T cells specific for the universal epitope, termed T(*), which was originally defined using CD4(+) T cells from protected volunteers immunized with irradiated P. falciparum sporozoites. The excellent correlation of T(*)-specific cellular responses with high anti-repeat Ab titers suggests that the T(*) epitope functioned as a universal Th cell epitope, as predicted by previous peptide/HLA binding assays and by immunogenicity studies in mice of diverse H-2 haplotypes. The current phase I trial suggests that polyoximes may prove useful for the development of highly immunogenic, multicomponent synthetic vaccines for malaria, as well as for other pathogens.

[Preparation of conjugated antigens based on zearalenone carboxymethyloxime and their use in immunoenzyme analysis]. / Poluchenie kon''iugirovannykh antigenov na osnove karboksimetiloksima zearalenona i ikh primenenie v immunofermentnom analize.

Zearalenone-6'-carboxymethyloxime was synthesized, and its conjugates with albumins and gelatin were prepared. Polyclonal rabbit antibodies against the conjugate with bovine serum albumin were shown to be highly specific to zearalenone and to have a lower cross-reactivity toward its structural analogues (alpha-zearalenol--28%, beta-zearalenol--6%, zearalanone--12%, and alpha-zearalanol--5%). The sensitivity of enzyme immunoassay using gelatin-based immobilized conjugates for determination of zearalenone in solutions was 1 ng/ml, and this allowed us to determine this substance in feed at a threshold concentration of 200 micrograms/kg.

Totally synthetic lipid-containing polyoxime peptide constructs are potent immunogens.

A synthetic peptide corresponding to a sequence from influenza hemagglutinin was used as a model antigen to study the immunogenicity of polyoxime constructs. In the absence of any adjuvant, tetrameric forms of different polyoxime constructs did not elicit an antibody response. High and long-lasting levels of antibody were induced, however, by polyoxime constructs to which Pam3Cys (tripalmitoyl-S-glyceryl cysteine) was attached. Comparable serum antibody levels were achieved with Tetraoxime-Pam3Cys administered by the intraperitoneal or intranasal routes to those obtained when the monomeric peptide was administered by the intraperitoneal route in complete Freund's adjuvant (CFA). Mice receiving Tetraoxime-Pam3Cys and Pam3Cys-peptide intranasally developed peptide-specific antibody secreting cells (ASCs) in their lungs and mediastinal lymph nodes. At low dose, the Tetraoxime-Pam3Cys induced higher levels of antibody compared to those elicited by the monomeric Pam3Cys-peptide delivered by either route. These results show that lipo-tetraoxime constructs assembled by polyoxime chemistry can be potent inducers of systemic and mucosal immunity.

Antibody binding characteristics of geometrical isomers of testosterone 3-(O-carboxymethyl)oxime.

Geometrical isomers of testosterone 3-(O-carboxymethyl)oxime and their histamine derivatives were purified on reverse-phase high pressure liquid chromatography, and their antibody binding characteristics were studied. Using a competitive testosterone enzyme immunoassay, the unfractionated mixture of the oximes showed 75% cross-reactivity with respect to testosterone, whereas the isolated 3Z- and 3E-isomers showed 124% and 26% cross-reactivity, respectively. The cross-reactivity was increased in the histamine derivatives, but the difference in cross-reactivity of the two isomers was reduced. Suppression of the ionization of the carboxyl group by lowering the pH of the incubation mixture in the antigen-antibody binding step raised the cross-reactivity of the mixture of free oximes to 128%, at pH 4.0. Thus, the geometry and ionization state of the carboxymethyl oxime group has a profound effect on the affinity of the isomers for the antibody.

Plasmodium falciparum polyoximes: highly immunogenic synthetic vaccines constructed by chemoselective ligation of repeat B-cell epitopes and a universal T-cell epitope of CS protein.

Effective immunoprophylaxis directed against the pre-erythrocytic stages of the malaria parasite requires a vaccine that can elicit humoral and cell mediated immunity in individuals of diverse genetic background. In order for a synthetic peptide malaria vaccine to meet these requirements, problems associated with genetic restriction, peptide chemistry, adjuvant formulation and physiochemical characterization of the final synthetic vaccine product must first be overcome. To address these issues, five polyoxime vaccine candidates have been constructed by ligating purified peptide epitopes of the P. falciparum CS protein to a branched template via oxime bonds. All five constructs, including two based on templates containing the synthetic adjuvant tripalmitoyl-S-glyceryl cysteine (Pam3Cys), were of sufficient purity for characterization by mass spectrometry. The immunogenicity of the malaria polyoximes in different murine strains was compared to that of multiple antigen peptide (MAP) constructs synthesized by standard step-wise synthesis. A tri-epitope polyoxime-Pam3Cys construct, based on the repeats and a universal T-cell epitope that contains both helper and CTL epitopes of the CS protein, was shown to be a precisely-defined synthetic malaria vaccine candidate that was highly immunogenic in murine strains of diverse H-2 haplotypes.

A synthetic peptide-based polyoxime vaccine construct of high purity and activity.

An artificial protein containing four copies of a peptide comprising the C-terminal 23 residues of influenza virus hemagglutinin was constructed using oxime chemistry and compared with two tetrameric multiple antigenic peptide (MAP) constructions of the same peptide displayed either radially or linearly which were made by conventional techniques. The tetra-oxime was much more homogeneous yielding a single peak on reversed phase HLPC and the correct mass spectrum. In addition, the tetra-oxime was found to be recognized by anti-peptide antibodies, to stimulate at low concentrations a T-cell clone and also to elicit in mice high titres of antibodies which were able to recognize native virus. The modular polyoxime approach, which permits artificial proteins to be assembled rapidly, in high yield and in high purity, is expected to lead to an increase in the use of artificial proteins in vaccine technology.

Radioimmunoassay of oestriol in the urine of non-pregnant women.

A radioimmunoassay for oestriol in the urine of non-pregnant women is described. Urine is hydrolysed rapidly at room temperature with an E. coli glucuronidase preparation and extracted prior to a simple radioimmunoassay procedure. An antiserum raised against oestriol-6-(O-carboxymethyl) oxime bovine serum albumin is used with (3H)-oestriol as tracer. Free and antibody-bound fractions are separated by partition between ammonium sulphate solution and scintillant without centrifugation. The accuracy and precision of the assay are satisfactory. It gave values approximately 80% of those obtained for total oestrogens by a fluorimetric method.

Thymolysis induced by EN3638 and other drugs and its relation to immunosuppression.

The effect on the thymus of EN3638, an oxime derivative of salicylic acid, was investigated because the drug has immunosuppressive properties. It caused a moderate reduction in thymic weight of normal adult and weanling rats and it retarded the regeneration of the thymus that follows acute corticosteroid-induced involution. These effects of EN3638 were not mediated by the adrenal nor due to the salicylate part of the molecule. At high dose levels, the drug also reduced slightly the weight of spleen and lymph nodes and the rate of body growth. Thymolytic doses of EN3638 were also immunosuppressive for experimental allergic encephalomyelitis (EAE). At doses that caused equivalent degrees of thymolysis. EN3638 was somewhat more potent for suppression of EAE than cyclophophosphamide or hydrocortisone.

Direct radioimmunoassay of estradiol in serum extracts.

The feasibility of direct radioimmunoassay of unconjugated estradiol in dried serum extracts in the assessment of ovarian function in the regulation of the menstrual cycle has been investigated. Using an antiserum to an estradiol-6-conjugate, assay reliability was evaluated and the procedure standardized through a series of tests aimed at assessing accuracy, sensitivity, and precision. These included multiple titration with competing steroids, the definition of blank effects from solvent and sample, the assay both of samples to which known amounts of estradiol had been added and of serially diluted samples, and clinical validation using as a reference samples related to well-defined physiological situations. The variability of replicate estimates and the repeatability of the calibration curve were evaluated to obtain information on assay precision and sensitivity. The analytical performances proved to be perfectly adequate for the clinical purposes for which the assay was intended, in terms both of reliability of results and of methodologic practicability.

The comparative specificity of 3 oestradiol-binding proteins. Rat alpha-foetoprotein, rat liver 17beta-hydroxy steroid dehydrogenase and anti-(oestradiol-6-carboxymethyloxime-bovine serum albumin) antiserum.

1. The specificity of 3 oestradiol-binding proteins was studied. Two of these proteins are naturally occurring (rat alpha-foetoprotein and rat liver microsomal 17beta-hydroxy steroid dehydrogenase) and the third is an artificially induced model, anti-(oestradiol-6-carboxymethyloxime-bovine serum albumin) gamma-globulins. 2. A specific binding procedure for each protein model permitted a determination of its affinity for oestradiol and for 30 other steroids. 3. The results obtained have brought to light the different areas of the steroid molecule that are important for its recognition by each of the three proteins. The two naturally occurring proteins (alpha-foetoprotein and 17beta-hydroxy steroid dehydrogenase) recognize the edge of the steroid defined by C-4, C-6, C-8 and C-15. On the other hand, the gamma-globulins recognize the opposite edge, i.e. that defined by C-2, C-10, C-11 and C-17. 4. Diethylstilboestrol, whose structure is analogous to that of a steroid, is only recognized by the two naturally occurring proteins.

Radioimmunoassay of norethindrone: peripheral plasma levels after oral administration to humans and rhesus monkeys.

PIP: A radioimmunoassay for the measurement of norethindrone, utilizing an antiserum raised in sheep against norethindrone 3-(O-carboxymethyl) oximebovine serum albumin, and its application for determination of norethindrone concentrations in plasma is presented. The practical detection limit was .5 ng/ml plasma. No significant cross reaction was found for naturally occurring steroids. The cross reaction for d-norgestrel was 88% and for lynestrenol and R 2323 26%. Peripheral plasma levels of immunoreactive norethindrone were assayed at short intervals after oral administration of norethindrone in different doses to 6 human volunteers. Peak levels occurred within 1-2 hours. The plasma half-life of norethindrone was found to be about 3 hours during the first hours after ingestion and about 9 hours thereafter. A linear correlation was found between ingested dose per kg body weight and plasma peak levels. In 3 orally treated female rhesus monkeys the peak levels were lower and not as distinct as compared to that of the humans for the corresponding dose per kg body weight. The plasma half-life after the peak was about 8 1/2 hours.^ieng