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Altered protein phosphorylation in cancer cells often leads to surface presentation of phosphopeptide neoantigens. However, their role in cancer immunogenicity remains unclear. Here we describe a mechanism by which an HLA-B*0702-specific acute myeloid leukemia phosphoneoantigen, pMLL 747-755 (EPR(pS)PSHSM), is recognized by a cognate T cell receptor named TCR27, a candidate for cancer immunotherapy. We show that the replacement of phosphoserine P 4 with serine or phosphomimetics does not affect pMHC conformation or peptide-MHC affinity but abrogates TCR27-dependent T cell activation and weakens binding between TCR27 and pMHC. Here we describe the crystal structures for TCR27 and cognate pMHC, map of the interface produced by nuclear magnetic resonance, and a ternary complex generated using information-driven protein docking. Our data show that non-covalent interactions between the epitope phosphate group and TCR27 are crucial for TCR specificity. This study supports development of new treatment options for cancer patients through target expansion and TCR optimization., (© 2023. The Author(s).)

CTLA-4 and CD28 exemplify a co-inhibitory and co-stimulatory signaling axis that dynamically sculpts the interaction of antigen-specific T cells with antigen-presenting cells. Anti-CTLA-4 antibodies enhance tumor-specific immunity through a variety of mechanisms including: blockade of CD80 or CD86 binding to CTLA-4, repressing regulatory T cell function and selective elimination of intratumoral regulatory T cells via an Fcγ receptor-dependent mechanism. AGEN1884 is a novel IgG1 antibody targeting CTLA-4. It potently enhanced antigen-specific T cell responsiveness that could be potentiated in combination with other immunomodulatory antibodies. AGEN1884 was well-tolerated in non-human primates and enhanced vaccine-mediated antigen-specific immunity. AGEN1884 combined effectively with PD-1 blockade to elicit a T cell proliferative response in the periphery. Interestingly, an IgG2 variant of AGEN1884 revealed distinct functional differences that may have implications for optimal dosing regimens in patients. Taken together, the pharmacological properties of AGEN1884 support its clinical investigation as a single therapeutic and combination agent.

Alzheimer's disease is characterized by pathogenic oligomerization, aggregation, and deposition of amyloid beta peptide (Aβ), resulting in severe neuronal toxicity and associated cognitive dysfunction. In particular, increases in the absolute or relative level of the major long form of Aβ, Aβ42, are associated with increased cellular toxicity and rapidity of disease progression. As a result of this observation, screening to identify potential drugs to reduce the level of Aβ42 have been undertaken by way of modulating the proteolytic activity of the gamma secretase complex without compromising its action on other essential substrates such as Notch. In this review we summarize results from a program that sought to develop such gamma secretase modulators based on novel natural products identified in the extract of Actaea racemosa, the well-known botanical black cohosh. Following isolation of compound 1 (SPI-014), an extensive medicinal chemistry effort was undertaken to define the SAR of 1 and related semisynthetic compounds. Major metabolic and physicochemical liabilities in 1 were overcome including replacement of both the sugar and acetate moieties with more stable alternatives that improved drug-like properties and resulted in development candidate 25 (SPI-1865). Unanticipated off-target adrenal toxicity, however, precluded advancement of this series of compounds into clinical development.

A screen of a library of synthetic drugs and natural product extracts identified a botanical extract that modulates the processing of amyloid precursor protein (APP) in cultured cells to produce a lowered ratio of amyloid-beta peptide (1-42) (Aβ42) relative to Aβ40. This profile is of interest as a potential treatment for Alzheimer's disease. The extract, from the black cohosh plant (Actaea racemosa), was subjected to bioassay guided fractionation to isolate active components. Using a combination of normal-phase and reverse-phase chromatography, a novel triterpene monoglycoside, 1, was isolated. This compound was found to have an IC(50) of 100 nM for selectively reducing the production of amyloidogenic Aβ42 while having a much smaller effect on the production of Aβ40 (IC(50) 6.3 μM) in cultured cells overexpressing APP. Using IP-MS methods, this compound was found to modulate the pool of total Aβ produced by reducing the proportion of Aβ42 while increasing the relative amounts of shorter and less amyloidogenic Aβ37 and Aβ39. Concentrations of 1 sufficient to lower levels of Aβ42 substantially (up to 10 μM) did not significantly affect the processing of Notch or other aspects of APP processing. When 1 (10 μg) was administered to CD-1 normal mice intracerebroventricularly, the level of Aβ42 in brain was reduced. Assays for off-target pharmacology and the absence of overt signs of toxicity in mice dosed with compound 1 suggest a comparatively selective pharmacology for this triterpenoid. Compound 1 represents a new lead for the development of potential treatments for Alzheimer's disease via modulation of gamma-secretase.

The discovery of a new series of γ-secretase modulators is disclosed. Starting from a triterpene glycoside γ-secretase modulator that gave a very low brain-to-plasma ratio, initial SAR and optimization involved replacement of a pendant sugar with a series of morpholines. This modification led to two compounds with significantly improved central nervous system (CNS) exposure.

During the last 20 years, an expanding body of research has elucidated the central role of amyloid precursor protein (APP) processing and amyloid beta peptide (Abeta) production in the risk, onset, and progression of the neurodegenerative disorder Alzheimer's disease (AD), the most common form of dementia. Ongoing research is establishing a greater level of detail for our understanding of the normal functions of APP, its proteolysis products, and the mechanisms by which this processing occurs. The importance of this processing machinery in normal cellular function, such as Notch processing, has revealed specific concerns about targeting APP processing for therapeutic purposes. Aspects of AD that are now well studied include direct and indirect genetic and other risk factors for AD, APP processing, and Abeta production. Emerging from these studies is the particular importance of the long form of Abeta, Abeta42. Elevated Abeta42 levels, as well as particularly the elevation of the ratio of Abeta42 to the shorter major form Abeta40, has been identified as important in early events in the pathogenesis of AD. The specific pathological importance of Abeta42 has drawn attention to seeking drugs that will selectively lower the levels of this peptide through reduced production or increased clearance while allowing normal protein processing to remain substantially intact. An increasing variety of compounds that modulate APP processing to reduce Abeta levels are being identified, some with Abeta42 selectivity. Such compounds are now reaching clinical evaluation to determine how they may be of benefit in the treatment of AD.

Amyloid beta peptide (Abeta) is implicated in the pathogenesis of Alzheimer s disease (AD), particularly as oligomers or polymers that are correlated with Abeta cellular toxicity. Inhibition of the formation of toxic forms of Abeta has therefore emerged as one approach to the treatment of AD. This article reviews efforts to adapt the structure of Abeta to the design and testing of peptide-based inhibitors of Abeta polymerization of interest as potential AD therapeutics.

Cholyl-LVFFA-OH (1, PPI-368) is an organic-modified peptide based on the sequence of amyloid beta-peptide (A beta). It is a potent and selective inhibitor of A beta polymerization that blocks the formation of neurotoxic species of A beta. In a nucleation-dependent polymerization assay of 50 microM A beta(1-40), equimolar concentrations of PPI-368 block polymerization based on turbidity and electron microscopy. Monomeric A beta(1-40) and A beta(1-42) are non-toxic when incubated with neuronal cell lines, but become toxic during polymerization. PPI-368 coordinately delays the onset of polymerization and the formation of neurotoxic A beta species for both peptides. In a polymerization extension assay seeded with pre-formed A beta polymer, similar inhibition and dose-dependency phenomena are observed with PPI-368. Radiolabeled PPI-368 is incorporated into fibrils during polymerization demonstrating binding to A beta peptide within afibrillar structure. Gel-filtration studies show progressive disappearance of A beta monomer and concomitant appearance of soluble higher molecular weight oligomers. In the presence of submolar concentrations of PPI-368, monomeric A beta is still present and oligomers are not observed PPI-368 does not inhibit the polymerization of other amyloidogenic proteins such as transthyretin (TTR) or islet amyloid polypeptide (IAPP(20-29).

Polymerization of the amyloid beta-peptide (Abeta) has been identified as a major feature of the pathogenesis of Alzheimer's disease (AD). Inhibition of the formation of these toxic polymers of Abeta has thus emerged as an approach to developing therapeutics for AD. Techniques for studying Abeta polymerization include the use of fibril nucleation and extension assays in a variety of formats. Detection of polymeric forms of Abeta has been achieved using turbidity, dye binding, light scattering and toxicity among other methods. Direct and indirect methods have been described for the measurement of binding affinities for Abeta fibrils. Imaging techniques include electron microscopy, X-ray diffraction and atomic force microscopy. These techniques have been used to characterize different classes of compounds that inhibit the formation of Abeta polymers. These compounds include dyes such as Congo Red, the antibiotic rifampicin, the anthracycline 4'-iodo-4'-deoxydoxorubicin, and a large variety of Abeta-derived peptides and modified peptides, among other reported inhibitors.

Cellular toxicity resulting from nucleation-dependent polymerization of amyloid beta-peptide (Abeta) is considered to be a major and possibly the primary component of Alzheimer's disease (AD). Inhibition of Abeta polymerization has thus been identified as a target for the development of therapeutic agents for the treatment of AD. The intrinsic affinity of Abeta for itself suggested that Abeta-specific interactions could be adapted to the development of compounds that would bind to Abeta and prevent it from polymerizing. Abeta-derived peptides of fifteen residues were found to be inhibitory of Abeta polymerization. The activity of these peptides was subsequently enhanced through modification of their amino termini with specific organic reagents. Additional series of compounds prepared to probe structural requirements for activity allowed reduction of the size of the inhibitors and optimization of the Abeta-derived peptide portion to afford a lead compound, cholyl-Leu-Val-Phe-Phe-Ala-OH (PPI-368), with potent polymerization inhibitory activity but limited biochemical stability. The corresponding all-D-amino acyl analogue peptide acid (PPI-433) and amide (PPI-457) retained inhibitory activity and were both stable in monkey cerebrospinal fluid for 24 h.

The potential of therapeutic gene transfer to treat human disease has prompted a diverse and growing range of basic and applied research efforts to explore and develop gene therapy strategies (1-5). Reported approaches to gene therapy mclude the uses of retroviruses (6,7), adenovirus (8,9), receptor-mediated endocytosis (10,11), direct injection (12), and liposomes (13,14), among others. Targeted delivery of DNA via receptors has been successfully applied using protein ligands to the hepatic asialoglycoprotein receptor (ASGr) (10,15-18), and, subsequently, the transferrin receptor (11). The ASGr is a cell-surface receptor that is highly represented on hepatocytes. Thus, genes targeted to this receptor can be delivered in a highly selective manner to the liver.

An asialoglycoprotein-based DNA delivery system containing an antisense oligo DNA against the polyadenylation region and adjacent upstream sequences of woodchuck hepatitis virus (WHV) was prepared. Experimental woodchucks were inoculated neonatally with the woodchuck virus 23 weeks before initiating the study, and all animals subsequently developed hepatitis as evidenced by the presence of measurable levels of circulating viral DNA. Animals were injected intravenously (i.v.) with asialoorosomucoid (AsOR)-poly-L-lysine complexes containing 0.1 mg kg-1 antisense DNA for five consecutive days. Levels of surface antigen did not differ substantially between treated and control animals. However, intravenous administration of complexed antisense DNA significantly decreased viraemia, as shown by a five- to 10-fold decrease in circulating viral DNA 25 days post treatment. The decline lasted for at least 2 weeks, after which there was a gradual increase in DNA levels. Antisense DNA alone or a complex containing a random oligo DNA of the same size and linkage failed to have any significant effect on viral DNA levels. We conclude that antisense oligo DNA can be targeted to the liver in vivo, resulting in a substantial and prolonged decrease in viral DNA levels in WHV-infected woodchucks.

In vivo gene therapy shows promise as a treatment for both genetic and acquired disorders. The hepatic asialoglycoprotein receptor (ASGPr) binds asialoorosomucoid-polylysine-DNA (ASOR-PL-DNA) complexes and allows targeted delivery to hepatocytes. The tris(N-acetylgalactosamine aminohexyl glycoside) amide of tyrosyl(glutamyl) glutamate [YEE(GalNAcAH)3] has been previously reported to have subnanomolar affinity for the ASGPr. We have used an iodinated derivative of YEE(GalNAcAH)3 linked to polylysine and complexed to the luciferase gene (pCMV-Luc) in receptor-binding experiments to establish the feasibility of substituting ASOR with the synthetic glycopeptide for gene therapy. Scatchard analyses revealed similar Kd values for both ASOR and the glycopeptide. Binding and internalization of 125I-Suc-YEE(GalNAcAH)3 were competitively inhibited with either unlabeled ASOR or glycopeptide. The reverse was also true; 125I-ASOR binding was competed with unlabeled YEE(GalNAcAH)3 suggesting specific binding to the ASGPr by both compounds. Examination of in vivo delivery revealed that the 125I-labeled glycopeptide complex mimicked previous results observed with 125I-ASOR-PL-DNA. CPM in the liver accounted for 96% of the radioactivity recovered from the five major organs (liver, spleen, kidney, heart, and lungs). Cryoautoradiography displayed iodinated glycopeptide complex bound preferentially to hepatocytes rather than nonparenchymal cells. In vitro, as well as in vivo, transfections using the glycopeptide-polylysine-pCMV-luciferase gene complex (YG3-PL-Luc) resulted in expression of the gene product. These data demonstrate that the YEE(GalNAcAH)3 synthetic glycopeptide can be used as a ligand in targeted delivery of DNA to the liver-specific ASGPr.

Asialoorosomucoid-polylysine (ASOR-PL) conjugates have been recently developed as carriers of electrostatically bound DNA for targeted delivery to the hepatic asialoglycoprotein receptor (ASGPr) for gene therapy. Using acid-urea gel electrophoresis we have found that previously reported procedures for the fractionation of ASOR-PL conjugates do not efficiently remove noncovalently bound polylysine (PL) from ASOR-PL. DNA complexes prepared with these conjugates have low solubilities, which limits their usefulness for subsequent experimentation, particularly in vivo. For ASOR-PL made by carbodiimide-mediated crosslinking with 5-kDa PL, dialysis against 1 M guanidine hydrochloride is effective to remove the low molecular weight unbound PL. Dialysis is not feasible when using higher molecular weight PLs, but preparative elution acid-urea gel electrophoresis was used to isolate crude ASOR-PL fractions free of unbound PL. ASOR-PL freed of PL by dialysis or electrophoresis was further fractionated by cation-exchange HPLC on carboxymethyl-functionalized columns eluted with a mixed pH-salt gradient. Early-eluting ASOR-PL fractions isolated by a combination of preparative elution acid-urea gel electrophoresis and cation-exchange HPLC were found to be preferred for the formation of soluble DNA complexes.

A series of peptidylglycoside derivatives, including the tris(GalNAc-aminohexyl) glycoside of tyrosyl(glutamyl)-glutamate (1) which is known to have sub-nanomolar affinity for the asialoglycoprotein receptor (ASGPr), have been synthesized using the protected tetra-O-acetyl aminohexyl glycoside (9) of N-acetylgalactosamine. The N-succinyl derivative of 1 was also prepared, providing a derivative for bioconjugate formation via carboxyl activation of the glycopeptide. Use of the protected glycoside 9 affords synthetic intermediates with increased solubility in organic solvents that are easier to purify and use in subsequent synthetic manipulations in comparison with compounds containing unprotected glycosides. These synthetic procedures will be generally applicable to the preparation of related compounds to probe binding to the ASGPr and the use of these cluster glycosides as ligands for targeted delivery to the liver.

Procedures for the synthesis and acylation of p-nitrobenzophenone oxime polystyrene resin for the preparation of protected peptide segments have been reexamined. Friedel-Crafts acylation with p-nitrobenzoyl chloride is complete in less than one hour at room temperature. Conversion of the ketoresin to the corresponding oxime requires less than six hours at 85 degrees C. Carbodiimide-mediated coupling of tert-butyloxycarbonyl-amino acids to the oxime resin requires less than one hour. By varying the quantity of p-nitrobenzoyl chloride and aluminum chloride used for acylation, the oxime substitution level of the resulting resin can be controlled between 0.2 and 0.8 milliequivalents per gram of resin. Aminoacyl oxime resin can thus be prepared in one day.

Cell-free protein synthesis in rabbit reticulocyte lysate translation mixtures has been studied during multi-hour incubations. In an impaired lysate obtained from cells stored at 0 degrees C before lysis, and showing a low initial rate of synthesis, translation could be stimulated by a factor of 4 by including RNase inhibitor and additional ATP and GTP. In translation mixtures prepared from normal lysates, protein synthesis could be improved by approximately 50% by the addition of excess GTP. The observed increases in protein synthesis were obtained by improved maintenance of the initial rate of synthesis.

The chemical synthesis of biologically active peptides and polypeptides can be achieved by using a convergent strategy of condensing protected peptide segments to form the desired molecule. An oxime support increases the ease with which intermediate protected peptides can be synthesized and makes this approach useful for the synthesis of peptides in which secondary structural elements have been redesigned. The extension of these methods to large peptides and proteins, for which folding of secondary structures into functional tertiary structures is critical, is discussed. Models of apolipoproteins, the homeo domain from the developmental protein encoded by the Antennapedia gene of Drosophila, a part of the Cro repressor, and the enzyme ribonuclease T1 and a structural analog have been synthesized with this method.