Natural Human Protein Research Articles

Immunogenic Treatment of Metastatic Breast Cancer Using Targeted Carbon Nanotube Mediated Photothermal Therapy in Combination with Anti-Programmed Cell Death Protein-1.

The high prevalence of breast cancer is a global health concern, compounded by the lack of safe or effective treatments for its advanced stages. These facts urge the development of novel treatment strategies. Annexin A5 (ANXA5) is a natural human protein that binds with high specificity to phosphatidylserine, a phospholipid tightly maintained in the inner leaflet of the cell membrane on most healthy cells but externalized in tumor cells and the tumor vasculature. Here, we have developed a targeted photosensitizer for photothermal therapy (PTT) of solid tumors through the functionalization of single-walled carbon nanotubes (SWCNTs) to ANXA5-the SWCNT-ANXA5 conjugate. The ablation of tumors through the SWCNT-ANXA5-mediated PTT synergizes with checkpoint inhibition, creating a systemic anticancer immune response. In vitro ablation of cells incubated with the conjugate promoted cell death in a dose-dependent and targeted manner. This treatment strategy was tested in vivo with the orthotopic EMT6 breast tumor model in female balb/cJ mice. Enhanced therapeutic effects were achieved by using intratumoral injection of the conjugate and treating tumors at a lower PTT temperature (45°C). Intratumoral injection prevented the accumulation of the SWCNTs in major clearance organs. When combined with checkpoint inhibition of anti-programmed cell death protein-1, SWCNT-ANXA5-mediated PTT increased survival and 80% of the mice survived for 100 days. Evidence of immune system activation by flow cytometry of splenic cells strengthens the hypothesis of an abscopal effect as a mechanism of prolonged survival. SIGNIFICANCE STATEMENT: This study demonstrated a relatively high survival rate (80% at 100 days) of mice with aggressive breast cancer when treated with photothermal therapy using the SWCNT-ANXA5 conjugate injected intratumorally and combined with immune stimulation using the anti-programmed cell death protein-1 checkpoint inhibitor. Photothermal therapy was accomplished by maintaining the tumor temperature at a relatively low level of 45°C and avoiding accumulation of the nanotubes in the clearance organs by using intratumoral administration.

Saposin D acting on macrophage bacteriostatic function in experimental tuberculosis infection

The protection against tuberculosis infection is largely determined by the ability of host tissue macrophages to limit the growth and spread of mycobacteria. Able to multiply within the host macrophages, mycobacteria have developed a number of protective mechanisms preventing phagosome-lysosome fusion, thereby evading damaging effects of lysosomal enzymes. Saposins are small, acidic, thermostable, non-enzymatic glycoproteins that participate as co-fac-tors in degradation of short oligosaccharide head group glycosphingolipids. Saposins A, B, C and D are formed in acidic endosomes due to cleavage of initial prosaposin molecule. The effect of saposins on human immune response is mediated by their involvement in presenting mycobacterial antigens on CD1 molecules. Preliminary studies with electron microscopy allowed to uncover saposin D-bound damaging effect on Mycobacterium tuberculosis in acidic environment. These data allowed us to suggest that saposin D is an important protective component fighting against TB infection. The aim of the study was to explore how saposin D deficiency might affect formation of anti-tuberculosis immune response and ability of macrophages to inhibit M. tuberculosis growth. Materials and methods. Interstitial pulmonary macrophages and peritoneal macrophages were isolated from wild type C57BL/6 strain and saposin D deficient C57BL/6-SapD-/- mouse strains. Results. It was found that as compared to macrophages from mice, macrophages from wild type strain significantly better controlled mycobacteria growth in vitro. To study an opportunity of compensating for deficient saposin D in peritoneal macrophages from C57BL/6-SapD-/- mice, a saposin D gene-bearing lentiviral vector was created. Transfection of SAPD-deficient peritoneal macrophages with expression vector compensated for saposin D deficiency in such cells and restored bactericidal function. The mechanisms of action for current anti-TB drugs are mediated by various metabolic pathways in mycobacteria (inhibited biosynthesis of fatty acids, arabinogalactan, peptidoglycan and protein; inhibition of DNA-dependent processes, proton pumps and cytochrome P450-dependent monooxygenases). Conclusion. It was shown that saposin D deficiency affects activation of macrophage bactericidal function in vitro. Our study data may be a prerequisite for biologically substantiated potential of using a vector construct bearing natural human protein gene such as saposin D, as a new anti-tuberculosis drug.

Ingénierie de charpentes protéiques comme alternative aux anticorps

Recent advances in combinatorial protein engineering have made it possible to develop non-Ig protein scaffolds that can potentially substitute for most whole antibody-associated properties. These protein scaffolds display most of the binding properties associated with the variable domain of antibodies. In theory, many different natural human protein backbones are suitable to be used as recombinant templates for engineering ; in practice however, only a few have yielded the necessary properties to be translated into << druggable biologicals >>. Amongst these properties, potential broad specificities towards any kind of target, ease of production, small size, good tolerability and low immunogenicity are essential. Intellectual property is another key issue. In this review, a particular emphasis will be given to the most validated non-Ig scaffolds that have reached the clinical development phase.

Development of Novel Protein Scaffolds as Alternatives to Whole Antibodies for Imaging and Therapy: Status on Discovery Research and Clinical Validation

Recent advances in combinatorial protein engineering have made it possible to develop antibody-based and non-Ig protein scaffolds that can potentially substitute for most whole antibody-associated properties. In theory, many different natural human protein backbones are suitable to be used as recombinant templates for engineering : antibody-derived scaffolds, carrier proteins that display a single binding interface, backbones that provide a rigid core structure suitable for grafting loops or protein scaffolds allowing the incorporation of variable loops in a favorable 3D configuration. In practice however, only a few have yielded the necessary properties to be translated into 'druggable Biologicals'. Amongst these properties, potential broad specificities towards any kind of target, ease of production, small size, good tolerability and low immunogenicity are essential and will be discussed in this review. Intellectual property is another key issue for the development of these protein scaffolds; although circumventing antibody-associated patents is often a major if not primary goal, clear advantages compared to whole antibodies must be presented to translate scaffold discovery into successful therapeutic drug candidates. In this review, a particular emphasis will be given to the most validated scaffolds that have reached the clinical development phase. Although the question of their immunogenicity is still open, preliminary clinical data do not point to any particular adverse immunogenic reactions although these are highly dependent on dosage, administration route and therapeutic indication. Finally, some of the emerging Biotechs developing protein scaffolds have been associated during the last two years with successful acquisitions by Big Pharmas and we will speak on the perspective positions of these proteins within the global Biologicals market.

IMP321 and weekly paclitaxel as first-line chemoimmunotherapy for metastatic breast cancer (MBC)

1045 Background: IMP321 (hLAG-3Ig), a novel immunomodulator:derived from the natural human protein LAG-3 (CD223), a ligand for MHC class II molecules, in the form of a soluble fusion protein; acts indirectly on T cell responses by MHC class II+APC activation. Two intended clinical settings: low dose (250 μg) as T-cell adjuvant for therapeutic vaccines and high dose (>1 mg) as monotherapy (i.e. without antigen) or combined with standard chemotherapy. Pharmacokinetics study on patient's plasma shows systemic exposure to IMP321 for at least 24 hr following s.c. injection of doses above 1 mg. Chemoimmunotherapy protocol rationale: boosting the DC network when it is loaded with tumour antigens for the several days following chemotherapy (secondary to tumour cell apoptosis) with repeated s.c. injection of the APC-specific activator IMP321 may increase cytotoxic CD8 T cell responses. Direct and prolonged activation of APC such as monocytes may also occur. Methods: This is an open label fixed dose-escalation phase I study, performed in ambulatory setting in patients receiving as a first-line chemotherapy for MBC the standard 6 cycles of weekly paclitaxel (80 mg/m² at D1, D8 and D15 of a 4-week cycle. Two IMP321 doses, 0.25 and 1.25 mg given s.c., are being tested, given at D2 and D16 of this 4-week cycle, for 6 courses. Results: IMP321 was very well tolerated. Twelve pts have been treated to date with no grade III or IV adverse events related to the product. A three-fold increase in MHC class II+ monocyte blood counts was observed two weeks after the last injection. In addition, a two-fold increase in activated CD8+ circulating T cells was observed in these pts that mimics in vivo the in vitro effect of IMP321 on human PBMCs. Conclusions: IMP321 in MBC patients treated with first-line chemotherapy was well tolerated when given s.c. over 6 months and this low IMP321 dose (i.e. for systemic exposure) was already efficacious at enhancing significantly the number of effector cytotoxic cells in vivo. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Immutep Immutep

Oxidative Stress in Critical Conditions and its Correction

Severe postoperative complications and critical conditions caused by profuse hemorrhage, pulmonary thromboembolism, anaphylactic shock in response to drugs, as well as circulatory arrest, pyoseptic processes are accompanied by significant activation of free radical lipid oxidation along with progressive depletion of the antioxidative defense system, i. e. the development of oxidative stress underlying the development of acute multiple organ dysfunctions (MOD) of posthypoxic or toxemic genesis, and they require special treatment policy. The active antioxidants Ceruloplasmin and Laprot based on natural human protein antioxidants are the drugs of choice in correcting oxidative stress. Their use as part of traditional intensive care restores imbalance of oxidative-antioxidative processes in the body and significantly enhances the efficiency of therapy for critical conditions and it should be regarded as an essential pathogenetic component of complex intensive care. The developed tactics of effective antioxidant therapy promotes optimization of the course of a rehabilitative period in patients after prior critical conditions and reduces the need for invasive hardware methods of correcting endotoxemia and MOD, which determines its economic expediency in addition to therapeutic one. Key words: critical conditions, oxidative stress, the antioxidants Ceruloplasmin and Laprot.

Comparison of mRNA-Display-Based Selections Using Synthetic Peptide and Natural Protein Libraries

mRNA display is a genotype-phenotype conjugation method that allows the amplification-based, iterative rounds of in vitro selection to be applied to peptides and proteins. Compared to prior protein selection techniques, mRNA display can be used to select functional sequences from both long natural protein and short combinatorial peptide libraries with much higher complexities. To investigate the basic features and problems of using mRNA display in studying conditional protein-protein interactions, we compared the target-binding selections against calmodulin (CaM) using both a natural protein library and a combinatorial peptide library. The selections were efficient in both cases and required only two rounds to isolate numerous Ca2+/CaM-binding natural proteins and synthetic peptides with a wide range of affinities. Many known and novel CaM-binding proteins were identified from the natural human protein library. More than 2000 CaM-binding peptides were selected from the combinatorial peptide library. Unlike sequences from prior CaM-binding selections that correlated poorly with naturally occurring proteins, synthetic peptides homologous to the Ca2+/CaM-binding motifs in natural proteins were isolated. Interestingly, a large number of synthetic peptides that lack the conventional CaM-binding secondary structures bound to CaM tightly and specifically, suggesting the presence of other interaction modes between CaM and its downstream binding targets. Our results indicate that mRNA display is an ideal approach to the identification of Ca2+-dependent protein-protein interactions, which are important in the regulation of numerous signaling pathways.

Unique recombinant human ribonuclease and inhibition of Kaposi's sarcoma cell growth.

Preparations of human chorionic gonadotropin (hCG) have been shown to exhibit anti-Kaposi's sarcoma (KS) activity, but the identity of the responsible agent(s) remains controversial. One candidate agent is an eosinophil-derived neurotoxin (EDN)-like polypeptide that contaminates preparations of hCG. We have genetically engineered a unique form of hEDN, which is a ribonuclease, and have evaluated the cytotoxic effects of the recombinant protein on KS Y-1 cells and on cells of other cancer types. The amino-terminus of hEDN was extended by four amino acid residues, corresponding to the proximal part of the hEDN signal peptide (serine, leucine, histidine, and valine; positions -4 to -1, respectively), by use of the polymerase chain reaction and an hEDN complementary DNA. The recombinant protein was isolated from bacterial inclusion bodies. The cytotoxic activity of this hEDN variant, (-4)rhEDN, was tested on KS Y-1 cells and human glioma, melanoma, breast carcinoma, and renal carcinoma cells. Approximately half of the anti-KS activity in a crude commercial preparation of hCG was associated with a polypeptide that reacted with anti-recombinant-hEDN (rhEDN) polyclonal antibodies. Although rhEDN protein displayed little cytotoxicity against KS Y-1 cells (IC50 [50% inhibition concentration] = >100 microg/mL), (-4)rhEDN markedly inhibited cell viability (IC50 = 6 microg/mL). Neither version of rhEDN inhibited the viability of other tested human cancer cell types. A four amino acid extension of the amino-terminus of rhEDN confers cytotoxicity against KS Y-1 cells in vitro. Design of the (-4)rhEDN variant was based on the sequence of a natural human protein associated with hCG. Our results suggest that (-4)rhEDN is one of the agents in hCG responsible for anti-KS activity. A purified molecule is thus available for in vitro and in vivo mechanistic and, possibly, future clinical studies.

Thermal Stability and Domain-Domain Interactions in Natural and Recombinant Protein C

Scanning microcalorimetry and spectrofluorimetry were applied to a study of the thermal stability and interaction of the modules within natural human protein C (PC) and recombinant protein C (rPC), a potential therapeutic anticoagulant expressed in transgenic pigs. Upon heating in the presence of 2 mM EDTA, pH 8.5, each protein exhibited a similar heat absorption peak with a Tm of approximately 62 degrees C corresponding to the melting of the serine protease (SP) module. Deconvolution of this peak indicated that the SP module consists of two domains that unfold independently. At pH below 3.8, a second peak appeared at extremely high temperature corresponding to the unfolding of the two interacting epidermal growth factor-like (EGF) domains. This second peak occurred at a temperature about 20 degrees C lower in rPC than in PC indicating that the EGF domains in the recombinant protein are less stable. The isolated 6-kDa gamma-carboxyglutamic acid-rich (Gla) fragment as well as a 25-kDa Gla-(EGF)2 fragment both exhibited a sigmoidal fluorescence-detected denaturation transition in the same temperature region as the SP domains, but only in the presence of Ca2+. In 2 mM Ca2+, the first heat absorption peak in both intact proteins became biphasic, indicating Ca(2+)-induced structural changes. By contrast, Ca2+ had very little effect on the melting of Gla-domain-less protein C. This indicates that not Ca2+ itself but the Ca(2+)-loaded Gla domain is responsible for conformational changes in the SP domain of the parent protein. Detailed analysis of the shape of the endotherms obtained in Ca2+ and EDTA suggests that Ca2+ induces compact structure in the Gla domain which appears to interact strongly with the SP domain(s) of protein C.

Correct processing and secretion of a human prostatic secretory protein (PSP 94) in Escherichia coli

The cDNA for PSP 94, a cysteine-rich protein secreted by the human prostate, was unidirectionally digested with exonuclease III to generate deletion mutants with varying 5' ends. These were placed under the control of the lac promoter of the Bluescribe plasmid (pbs) to encode hybrid proteins containing the N terminus of β-galactosidase (βGal) and various fragments of PSP 94. Escherichia coli clones transformed by these constructs and expressing PSP 94 epitopes were identified by radioimmunoassay of cellular and periplasmic extracts. One such clone (I-25) secreted most of its immunoreactive material into the periplasmic space. Nucleotide sequencing showed that a new consensus ribosome-binding site had been generated fortuitously, allowing expression of pre-PSP 94 free of any βSGal sequence. Periplasmic PSP 94 is indistinguishable from the natural human protein, indicating correct processing and folding of this cysteine-rich protein in bacteria.