Quantitative ELISA for SERPINA4/kallistatin

Background: SERPINA4/kallistatin is a multifunctional protein expressed from the liver; its concentration in blood circulation reflects the degree of liver dysfunction and may serve as a diagnostic/prognostic biomarker for chronic liver diseases (CLD). Materials & methods: Antibodies specific for SERPINA4/kallistatin were used for the development of an enzyme-linked immunosorbent assay (ELISA). For correlative studies, blood samples from patients with cirrhotic liver and healthy patients were collected from R.L. Jalappa Hospital & Research Centre, Kolar. Results: Interference of other SERPINs was ruled out using western blot analysis. Quantitative ELISA was developed using monospecific antibodies as capture antibodies. Conclusion: The accuracy of the developed ELISA was determined by inter- and intra-assay precision. Linearity was defined using a spiked sample with serial dilutions. Reduced levels of SERPINA4/kallistatin were observed in patients with CLD compared with healthy controls.

Hydrolysis of a second Asp-Pro site at the N-terminus of NOTCH3 in inherited vascular dementia

Cerebrovascular pathology at the biochemical level has been informed by the study of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a vascular disorder caused by NOTCH3 mutations. Previous work in CADASIL described N-terminal proteolysis of NOTCH3 generated by specific non-enzymatic cleavage of the first Asp-Pro sequence of the protein. Here, we investigated whether the second Asp-Pro peptide bond (residues 121–122) of NOTCH3 is cleaved in CADASIL. Monospecific antibodies were generated that recognize the neo-epitope predicted to be generated by cleavage after Asp121. These antibodies were used to localize cleavage events at Asp121 in post-mortem CADASIL and control brain tissue and to investigate factors that regulate cleavage at Asp121. We report that cleavage at Asp121 occurs at a high level in the arterial media of CADASIL cerebral arteries. Leptomeningeal arteries demonstrated substantially more cleavage product than penetrating arteries in the white matter, and control vessels harbored only a small amount of cleaved NOTCH3. Proteolysis at Asp121 occurred in purified preparations of NOTCH3 ectodomain, was increased by acidic pH and reductive conditions, and required native protein conformation for cleavage. Increasing the concentration of NOTCH3 EGF-like domain protein elevated the level of proteolysis. On the other hand, several polyanionic chemicals potently blocked cleavage at Asp121. These studies demonstrate that the NOTCH3 protein in CADASIL is cleaved in multiple locations at labile Asp-Pro peptide bonds. As such, chronic brain vascular disease, like other neurodegenerative conditions, features proteolysis of pathological proteins at multiple sites which may generate small pathological peptides.

Bioassay Development for Bispecific Antibodies—Challenges and Opportunities

Antibody therapeutics are expanding with promising clinical outcomes, and diverse formats of antibodies are further developed and available for patients of the most challenging disease areas. Bispecific antibodies (BsAbs) have several significant advantages over monospecific antibodies by engaging two antigen targets. Due to the complicated mechanism of action, diverse structural variations, and dual-target binding, developing bioassays and other types of assays to characterize BsAbs is challenging. Developing bioassays for BsAbs requires a good understanding of the mechanism of action of the molecule, principles and applications of different bioanalytical methods, and phase-appropriate considerations per regulatory guidelines. Here, we review recent advances and case studies to provide strategies and insights for bioassay development for different types of bispecific molecules.

Cirrhosis of liver: Comparative cross reactivity for quantification of SERPINA4/Kallistatin

Cirrhosis of liver is an end stage of chronic liver insults from varied etiologies which leads to impaired liver functions. Proteins expressed from liver and enters into circulation reflects degree of liver dysfunction. Serpins (Serine protease inhibitors) are class of plasma proteins expressed from liver; SERPINA4/Kallistatin is a multifunctional serpin clade A protein expressed from liver and concentration in serum is the reflection of extent of liver dysfunction. The present study aimed to compare cross reactivity of serpins for polyclonal and monospecific antibodies in both cirrhotic liver and healthy subjects. Blood samples were collected from 20 subjects (10 cirrhotic liver, 10 healthy) from R. L. Jalappa Hospital and Research Centre, Kolar, Karnataka, India. Separation of proteins was carried out by SDS-PAGE. Cross reactivity study was analyzed using western blot. Proteins present in cirrhotic liver and healthy subject’s serum were separated by SDS PAGE. There was no band detection on both (cirrhotic liver and healthy) PVDF (polyvinylidene diflouride) membranes with polyclonal antibodies. However, a significant band was observed with protein of interest in healthy PVDF membrane with monospecific antibodies. There was no band in cirrhotic liver PVDF membrane even with monospecific antibodies. Comparative cross reactivity analysis of serpins in quantification of SERPINA4/Kallistatin in the present study demonstrated that there will not be any immunological cross reactivity between serpins and SERPINA4/Kallistatin due to the absence of identical epitope in cirrhotic liver and healthy subjects.

Development of monospecific polyclonal antibodies against hypervirulent Klebsiella pneumoniae

Darniati, Setiyaningsih S, Agungpriyono DR, Handharyani E. 2021. Development of monospecific polyclonal antibodies against hypervirulent Klebsiella pneumoniae. Biodiversitas 22: 99-105. The aim of the research was to produce monospecific antibodies against Klebsiella pneumoniae serotype K1 and K2. Klebsiella pneumonia isolates were recovered from pneumonic lungs of Aceh cattle. The bacteria were identified by rpoB (RNA polymerase β subunit) PCR amplification specific for K. pneumoniae. The presence of capsule cluster gene magA and k2A was used to characterize capsular serotype K1 and K2, respectively. Antisera were produced using serial immunization of New Zealand White rabbits with K. pneumoniae serotypes K1 and K2. The presence of antibodies was determined by using immunodiffusion test and purification was performed by immunoaffinity purification method. K. pneumoniae antibodies began to appear on the 14th day after the first injection and progressively intensified after the 1st and 2nd booster. The cross-reactivity between the antigen was eliminated by absorbing the antisera with the opposite antigen and several serotypes of non-K1/K2. After purification, serum protein concentrations were substantially decreased from 58.48 µg/µL and 53.99 µg/µL to 2.38 µg / µL and 3.72 µg / µL for K1 and K2, respectively). The SDS-PAGE analysis showed two bands with molecular weight at 54 kDa and 25 kDa representing the heavy and light chains of immunoglobulin G, respectively. Purified polyclonal antiseras against K. pneumoniae serotypes K1 and K2 showed high affinity and specificity for homologous antigens in immunodiffusion and direct agglutination tests, and immunohistochemical staining.

858 A bispecific antibody targeting CD40 and EpCAM induces superior anti-tumor effects compared to the combination of the monospecific antibodies

BackgroundAlligator has developed a new concept, Neo-X’, to enable antigen presenting cells to efficiently enhance priming of neoantigen-specific T cells, which may be the missing aspect in tumors that lack T cell infiltration. We hypothesize that binding of the CD40 x EpCAM bsAb (4224) to CD40 on DCs and EpCAM on tumor exosomes or tumor debris leads to i) activation of the DC, ii) uptake of the tumor material, iii) cross-presentation of tumor-derived neoantigen (present in exosomes or debris) and iiii) priming of tumor neoantigen-specific T cells, resulting in an increased quantity and/or quality of the tumor-targeting T cell pool. CD40 crosslinking by engagement with a tumor antigen on a tumor cell is required to achieve a functional agonistic effect, and subsequent DC activation will therefore only be achieved in the presence of tumor antigens.Methods4224 evaluated in vitro using human monocyte-derived DC, co-cultured with cells expressing EpCAM. In addition the functional effects were evaluated using tumor cell lines and B-cell lines expressing CD40. In vivo, the anti-tumor efficacy of the CD40 x EpCAM bsAb was determined in human CD40 transgenic mice bearing MB49 bladder carcinoma tumors transfected with human EpCAM or controls.ResultsIn vitro, we have demonstrated that the CD40 x EpCAM bsAb induces tumor target dependent activation of dendritic cells, as analyzed by flow cytometry measuring HLA-DR and CD86 expression on the DC and by measuring IL-12p40 levels in the supernatant. Further, the ability of bsAbs within the Neo-X’ concept to mediate co-localization of tumor debris and CD40 expressing antigen presenting cells depends on the receptor density of the tumor target. In vivo, 4224 displayed a potent, EpCAM-dependent anti-tumor effect with significantly reduced tumor growth and improved survival compared to an equivalent dose of the combination of the monospecific CD40 Ab and EpCAM targeting antibody. The tumor-localizing property of 4224 also shows potential for improved safety compared to CD40 monospecific antibodies. A biodistribution analysis demonstrated that the bispecific 4224 in the RUBY-format displayed similar half-life as the monospecific CD40 mAb in mice.ConclusionsIn conclusion, the Neo-X’ concept, by targeting CD40 and a tumor specific antigen, has the potential to mediate an expansion of the tumor-specific T cell repertoire, resulting in increased T cell infiltration and potent anti-tumor effects.

Greatest Hits—Innovative Technologies for High Throughput Identification of Bispecific Antibodies

Recent years have shown a tremendous increase and diversification in antibody-based therapeutics with advances in production techniques and formats. The plethora of currently investigated bi- to multi-specific antibody architectures can be harnessed to elicit a broad variety of specific modes of actions in oncology and immunology, spanning from enhanced selectivity to effector cell recruitment, all of which cannot be addressed by monospecific antibodies. Despite continuously growing efforts and methodologies, the identification of an optimal bispecific antibody as the best possible combination of two parental monospecific binders, however, remains challenging, due to tedious cloning and production, often resulting in undesired extended development times and increased expenses. Although automated high throughput screening approaches have matured for pharmaceutical small molecule development, it was only recently that protein bioconjugation technologies have been developed for the facile generation of bispecific antibodies in a ‘plug and play’ manner. In this review, we provide an overview of the most relevant methodologies for bispecific screening purposes—the DuoBody concept, paired light chain single cell production approaches, Sortase A and Transglutaminase, the SpyTag/SpyCatcher system, and inteins—and elaborate on the benefits as well as drawbacks of the different technologies.

Bispecific antibodies in cancer immunotherapy

Following the clinical success of immune checkpoint antibodies targeting CTLA-4, PD-1 or PD-L1 in cancer treatment, bispecific antibodies are now emerging as a growing class of immunotherapies with potential to further improve clinical efficacy and safety. We describe three classes of immunotherapeutic bispecific antibodies: (a) cytotoxic effector cell redirectors; (b) tumor-targeted immunomodulators; and (c) dual immunomodulators. Cytotoxic effector cell redirectors are dominated by T-cell redirecting compounds, bispecific compounds engaging a tumor-associated antigen and the T-cell receptor/CD3 complex, thereby redirecting T-cell cytotoxicity to malignant cells. This is the most established class of bispecific immunotherapies, with two compounds having reached the market and numerous compounds in clinical development. Tumor-targeted immunomodulators are bispecific compounds binding to a tumor-associated antigen and an immunomodulating receptor, such as CD40 or 4-1BB. Such compounds are usually designed to be inactive until binding the tumor antigen, thereby localizing immune stimulation to the tumor environment, while minimizing immune activation elsewhere. This is expected to induce powerful activation of tumor-specific T cells with reduced risk of immune-related adverse events. Finally, dual immunomodulators are bispecific compounds that bind two distinct immunomodulating targets, often combining targeting of PD-1 or PD-L1 with that of LAG-3 or TIM-3. The rationale is to induce superior tumor immunity compared to monospecific antibodies to the same targets. In this review, we describe each of these classes of bispecific antibodies, and present examples of compounds in development.