Bispecific antibodies (Bispecifics) demonstrate exceptional clinical potential to address some of the most complex diseases. However, Bispecific production in a single cell often requires the correct pairing of multiple polypeptide chains for desired assembly. This is a considerable hurdle that hinders the development of many immunoglobulin G (IgG)-like bispecific formats. Our approach focuses on the rational engineering of charged residues to facilitate the chain pairing of distinct heavy chains (HC). Here, we deploy structure-guided protein design to engineer charge pair mutations (CPMs) placed in the CH3-CH3′ interface of the fragment crystallizable (Fc) region of an antibody (Ab) to correctly steer heavy chain pairing. When used in combination with our stable effector functionless 2 (SEFL2.2) technology, we observed high pairing efficiency without significant losses in expression yields. Furthermore, we investigate the relationship between CPMs and the sequence diversity in the parental antibodies, proposing a rational strategy to deploy these engineering technologies.
Protein-based biotherapeutics are produced in engineered cells through complex processes and may contain a wide variety of variants and post-translational modifications that must be monitored or controlled to ensure product quality. Recently, a low level (~1-5%) impurity was observed in a number of proteins derived from stably transfected Chinese hamster ovary (CHO) cells using mass spectrometry. These molecules include antibodies and Fc fusion proteins where Fc is on the C-terminus of the construct. By liquid chromatography-mass spectrometry (LC-MS), the impurity was found to be ~1177 Da larger than the expected mass. After tryptic digestion and analysis by LC-MS/MS, the impurity was localized to the C-terminus of Fc in the form of an Fc sequence extension. Targeted higher-energy collision dissociation was performed using various normalized collision energies (NCE) on two charge states of the extended peptide, resulting in nearly complete fragment ion coverage. The amino acid sequence, SLSLSPEAEAASASELFQ, obtained by the de novo sequencing effort matches a portion of the vector sequence used in the transfection of the CHO cells, specifically in the promoter region of the selection cassette downstream of the protein coding sequence. The modification was the result of an unexpected splicing event, caused by the resemblance of the commonly used GGU codon of the C-terminal glycine to a consensus splicing donor. Three alternative codons for glycine were tested to alleviate the modification, and all were found to completely eliminate the undesirable C-terminal extension, thus improving product quality.
Glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) has been identified in multiple genome-wide association studies (GWAS) as a contributor to obesity, and GIPR knockout mice are protected against diet-induced obesity (DIO). On the basis of this genetic evidence, we developed anti-GIPR antagonistic antibodies as a potential therapeutic strategy for the treatment of obesity and observed that a mouse anti-murine GIPR antibody (muGIPR-Ab) protected against body weight gain, improved multiple metabolic parameters, and was associated with reduced food intake and resting respiratory exchange ratio (RER) in DIO mice. We replicated these results in obese nonhuman primates (NHPs) using an anti-human GIPR antibody (hGIPR-Ab) and found that weight loss was more pronounced than in mice. In addition, we observed enhanced weight loss in DIO mice and NHPs when anti-GIPR antibodies were codosed with glucagon-like peptide-1 receptor (GLP-1R) agonists. Mechanistic and crystallographic studies demonstrated that hGIPR-Ab displaced GIP and bound to GIPR using the same conserved hydrophobic residues as GIP. Further, using a conditional knockout mouse model, we excluded the role of GIPR in pancreatic β-cells in the regulation of body weight and response to GIPR antagonism. In conclusion, these data provide preclinical validation of a therapeutic approach to treat obesity with anti-GIPR antibodies.
Elevated plasma low density lipoprotein cholesterol (LDL-C) level is a risk factor for the development of atherosclerosis and associated cardiovascular disorders. Studies by Brown and Goldstein have firmly demonstrated the pivotal function played by the LDL receptor (LDLR) in cholesterol metabolism. The recently discovered proprotein convertase subtilisin/kexin type 9 (PCSK9) is another important regulator of LDL-C level. The protein promotes degradation of the LDLR, thus abolishing the receptor function. Specific inhibition of PCSK9 may therefore provide another novel therapeutic approach in the treatment of hypercholesterolemia. To evaluate human PCSK9 as a therapeutic target for monoclonal antibody development, the recombinant protein has been expressed in CHO system. The mature protein consists of 2 tightly associated subunits: a pro-domain of ~14 kD containing a single cysteine residue and a C-terminal catalytic domain of ~62 kD. The latter contains a total of 25 cysteinyl residues and a single N-linked glycosylation site. To show that the recombinant protein has been properly folded, we attempted to identify the location of the free cysteinyl residue(s) in the C-terminal domain. LC-MS analysis of the intact material after incubation with iodoacetamide in the presence of denaturant showed only a single site was alkylated. The labeled protein was completely reduced and pyridyethylated prior to proteolytic degradation. Analysis by LC-MS/MS showed that Cys301 was the only residue S-carboxamidomethylated, indicating this is the single free cysteine. The data therefore confirms the prior structural information obtained by X-ray crystallography. Peptide mapping also shows Asn533 is glycosylated. The predominant glycoforms are biantennary glycans, followed by triantennary and with very low amount of tetraantennary oligosaccharides. It is interesting to note that Asn533 is located in a cysteine rich area and is proximal to a cysteine residue that is disulfide cross linked (NCS). Thus, N-glycosylation was probably complete prior to cystine bridges formation in the maturation of PCSK9.
Since the basic concept of using magnetic sensor to detect biomolecules labelled with magnetic particles (MP) was first disclosed by Baselt [1], significant development has been made on the material and structure of magnetic transducers. The tri-layer spin-valve GMR device can yield 4 times as much signal as the magnetoresistive device mentioned by Baselt. Magnetic tunnel junction (MTJ) device can produce 100 times as much signal as the magnetoresistive devices. In this work, an active biochip consisting of 12 million magnetic-tunnel-junction (MTJ) transducers was developed for high throughput and high sensitivity detection of biomolecules. This chip can detect 12 million different types of molecules simultaneously.
In recent years, there have been notable advances with the development of anticancer drugs including those targeting protein tyrosine kinases such as the c-Met receptor, which has been implicated in the development and progression of several cancers. However, despite such progress, drug resistance continues to be the single most important cause of cancer treatment failure, and understanding the mechanisms of drug resistance remains a major hurdle in treating patients with recurrent disease. PF-04217903 is a small-molecule c-Met kinase inhibitor that potently inhibits c-Met-driven processes such as cell growth (proliferation and survival), motility, invasion, and morphology of a variety of tumor cells. Resistance to PF-04217903 was observed in GTL-16, a gastric carcinoma cell line with a constitutively activated c-Met receptor. In this report, mass spectrometry (MS) based quantitative phosphoproteomic analysis was used to determine changes in signaling pathways in the parental cells in response to c-Met inhibition and to investigate the changes in protein levels and related canonical pathways in both parental and PF-04217903 resistant (R3) clones in response to c-Met inhibition. The quantitative MS workflow included phosphoprotein enrichment of cell lysates from six treatment conditions: in-solution digestion, chemical labeling of peptides with a set of 6-plex isobaric tandem mass tags (TMT), HILIC fractionation, phosphopeptide enrichment, and nano LC-MS/MS on a LTQ-Orbitrap mass spectrometer. An investigation of these quantitative datasets using Ingenuity Pathways Analysis (IPA) revealed pathway changes in the various treatments that were consistent with previously observed transcriptomic and phenotypic changes. Proteomic analysis also revealed an increase in B-Raf expression in R3 clones. Expression profiling confirmed that B-Raf gene copy number was up-regulated and also indicated the presence of a mutated form of B-Raf. Using a bottom-up MS approach, SND-1 was identified as the B-Raf fusion partner. The discovery of this novel B-Raf fusion protein presents a novel target with potential clinical implications in the treatment of patients resistant to c-Met inhibitors.
AskGene has established a proprietary cytokine prodrug platform (Smartkine®) to achieve its overarching objective of modulating immune reactions at a disease site in a selective and controlled manner. Cytokines are potent molecules, yet their broad application as therapeutics has been hampered due to short PK, severe systemic toxicity, and narrow therapeutic window. To improve the therapeutic potential of cytokines, AskGene has developed several antibody-cytokine prodrug fusion molecules using its proprietary SmartKine® platform.
Methods
The in vitro activities of ASKG915 were evaluated using reporter cell line and PBMC-based assays. Peripheral immune activation was evaluated in a GvHD model with human PBMC-engrafted NSG mice. Anti-tumor activities were tested in a human PBMC-engrafted tumor xenograft model and a syngeneic tumor model. The PK/PD properties and safety profiles of ASKG915 were assessed in non-human primates (NHPs) following three IV injections every two weeks.
Results
ASKG915 showed minimal activity prior to protease-dependent activation and significantly enhanced activity after protease-dependent activation in vitro. Specifically, it has significantly higher activities stimulating PD-1+ immune cells, presumably through"cis activation". In in vivo efficacy studies, it showed similar potency as a reference anti-PD-1-IL-15 fusion molecule (not masked) while having a better safety profile. In addition, in a GvHD study, ASKG915 at 10 mg/kg i.p. induced lower interferon gamma levels in the periphery at Day 4 compared to the reference molecule at 1 mg/kg i.p. These results showed that, compared to the reference molecule, ASKG915 had comparable immune stimulation in the tumor while having significantly reduced immune stimulation in the periphery. In NHPs, ASKG915 demonstrated prolonged and antibody-like PK profiles. More importantly ASKG915 was well tolerated at the highest dosage tested in NHP, with no cytokine release syndrome (CRS) and minimal immune reaction at injection sites.
Conclusions
Activated ASKG915 showed selective stimulation for PD-1+ immune cells in in vitro assays with human PBMC. ASKG915 in vivo showed tumor-selective activation compared to a reference molecule. In addition, it had extended antibody-like PK in NHPs and was well tolerated at the highest dosage tested in the GLP PK/PD study. It also showed a significantly expanded therapeutic window. An IND filing is planned in the second half of 2022. To our knowledge, ASKG915 is the first anti-PD-1 antibody-IL-15 prodrug fusion molecule moving into clinical development.
Ethics Approval
The use of the animals in the studies have been approved by the ethics committees of the research contract organizations (CRO).
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)