Comprehensive Stress Stability Studies Reveal the Prominent Stability of the Liquid-Formulated Biotherapeutic Asymmetric Monovalent Bispecific IgG1 Monoclonal Antibody Format.

The developed asymmetric monovalent bispecific IgG1 or Duet monoclonal antibody (Duet mAb) has two distinct fragment antigen-binding region (Fab) subunits that target two different epitope specificities sequentially or simultaneously. The design features include unique engineered disulfide bridges, knob-into-hole mutations, and kappa and lambda chains to produce Duet mAbs. These make it structurally and functionally complex, so one expects challenging developability linked to instability, degradation of products and pathways, and limited reports available. Here, we have treated the product with different sources of extreme stress over a lengthy period, including varying heat, pH, photo stress, chemical oxidative stress, accelerated stress in physiological conditions, and forced glycation conditions. The effects of different stress conditions on the product were assessed using various analytical characterization tools to measure product-related substances, post-translational modifications (PTMs), structural integrity, higher-order disulfide linkages, and biological activity. The results revealed degradation products and pathways of Duet mAb. A moderate increase in size, charge, and hydrophobic variants, PTMs, including deamidation, oxidation, isomerization, and glycation were observed, with most conditions exhibiting biological activity. In addition, the characterization of fractionated charge variants, including deamidated species, showed satisfactory biological activity. This study demonstrated the prominent stability of the Duet mAb format comparable to most marketed mAbs.

A novel anti-LAG-3/TIGIT bispecific antibody exhibits potent anti-tumor efficacy in mouse models as monotherapy or in combination with PD-1 antibody.

We report the generation of a novel anti-LAG-3/TIGIT bispecific IgG4 antibody, ZGGS15, and evaluated its anti-tumor efficacy in mouse models as monotherapy or in combination with a PD-1 antibody. ZGGS15 exhibited strong affinities for human LAG-3 and TIGIT, with KDs of 3.05 nM and 2.65 nM, respectively. ZGGS15 has EC50s of 0.69 nM and 1.87 nM for binding to human LAG-3 and TIGIT on CHO-K1 cells, respectively. ZGGS15 competitively inhibited the binding of LAG-3 to MHC-II (IC50 = 0.77 nM) and the binding of TIGIT to CD155 (IC50 = 0.24 nM). ZGGS15 does not induce ADCC, CDC, or obvious cytokine production. In vivo results showed that ZGGS15 had better anti-tumor inhibition than single anti-LAG-3 or anti-TIGIT agents and demonstrated a synergistic effect when combined with nivolumab, with a significantly higher tumor growth inhibition of 95.80% (p = 0.001). The tumor volume inhibition rate for ZGGS15 at 2 mg/kg was 69.70%, and for ZGGS15 at 5 mg/kg plus nivolumab at 1 mg/kg, it was 94.03% (p < 0.001). Our data reveal that ZGGS15 exhibits potent anti-tumor efficacy without eliciting ADCC or CDC or causing cytokine production, therefore having a safe profile.

Identification and quantification of chain-pairing variants or mispaired species of asymmetric monovalent bispecific IgG1 monoclonal antibody format using reverse-phase polyphenyl chromatography coupled electrospray ionization mass spectrometry.

Developing a knob-into-hole asymmetric bispecific IgG1 monoclonal antibody (mAb) poses manufacturing challenges due to the expression of chain pairing variants, also called mispaired species, in the desired product. The incorrect pairing of light and heavy chains could result in heterogeneous mispaired species of homodimers, heterodimers, light chain swapping, and low molecular weight species (LMWS). Standard chromatography, capillary electrophoretic, or spectroscopic methods poorly resolve these from the main variants. Here, we report a highly sensitive reverse-phase polyphenyl ultra-high-performance liquid chromatography (RP-UHPLC) method to accurately measure mispaired species of Duet mAb format, an asymmetric IgG1 bispecific mAb, for both process development and quality control analytical tests. Coupled with electrospray ionization mass spectrometry (ESI-MS), it enabled direct online characterization of mispaired species. This single direct assay detected diverse mispaired IgG-like species and LMWS. The method resolved eight disulfide bonds dissociated LMWS and three mispaired LMWS. It also resolved three different types of IgG-like mispaired species, including two homodimers and one heterodimer. The characterization and quantification simultaneously enabled the cell line selection that produces a lesser heterogeneity and lower levels of mispaired species with the desired correctly paired product. The biological activity assessment of samples with increased levels of these species quantified by the method exhibited a linear decline in potency with increasing levels of mispaired species in the desired product. We also demonstrated the utility of the technique for testing in-process intermediate materials to determine and assess downstream purification process capability in removing diverse mispaired IgG-like species and LMWS to a certain level during the downstream purification process. Our investigation demonstrates that adopting this method was vital in developing asymmetric bispecific mAb from the initial stage of cell line development to manufacturing process development. Therefore, this tool could be used in the control strategy to monitor and control mispaired species during manufacturing, thus improving the quality control of the final product.

Blinatumomab Redirects Donor Lymphocytes against CD19+ Acute Lymphoblastic Leukemia without Relevant Bystander Alloreactivity after Haploidentical Hematopoietic Stem Cell Transplantation.

Blinatumomab alone or with donor leukocyte infusions (DLI) has been used after allogeneic hematopoietic stem cell transplantation (HSCT) as a salvage therapy in relapsing patients with CD19+ hematological malignancies. It was effective in a fraction of them, with low incidence of Graft-versus-Host Disease (GvHD). Immunosuppressive drugs used as GvHD prophylaxis hinder T cell function and reduce the efficacy of the treatment. Because T cell-depleted haploidentical HSCT with donor regulatory and conventional T cells (Treg/Tcon haploidentical HSCT) does not require post-transplant immunosuppression, it is an ideal platform for the concomitant use of blinatumomab and DLI. However, the risk of GvHD is high because the donor is haploidentical. We treated two patients with CD19+ acute lymphoblastic leukemia (ALL) who had relapsed after Treg/Tcon haploidentical HSCT with blinatumomab and DLI. Despite the mismatch for one HLA haplotype, they did not develop GvHD and achieved complete remission with negative minimal residual disease. Consistently, we found that blinatumomab did not enhance T cell alloreactivity in vitro. Eventually, the two patients relapsed again because of their high disease risk. This study suggests that treatment with blinatumomab and DLI can be feasible to treat relapse after haploidentical transplantation, and its pre-emptive use should be considered to improve efficacy.

Development of a Bispecific Antibody Targeting Clinical Isolates of Acinetobacter baumannii.

BACKGROUND: We previously reported developing 2 anticapsular monoclonal antibodies (mAbs) as a novel therapy for Acinetobacter baumannii infections. We sought to determine whether a bispecific mAb (bsAb) could improve avidity and efficacy while maximizing strain coverage in one molecule. METHODS: Humanized mAb 65 was cloned into a single-chain variable fragment and attached to humanized mAb C8, combining their paratopes into a single bsAb (C73). We tested bsAb C73's strain coverage, binding affinity, ex vivo opsonic activity, and in vivo efficacy compared to each mAb alone and combined. RESULTS: The bsAb demonstrated strain coverage, binding affinity, opsonization, and in vivo efficacy superior to either original mAb alone or combined. CONCLUSIONS: A humanized bsAb targeting distinct A. baumannii capsule moieties enabled potent and effective coverage of disparate A. baumannii clinical isolates. The bsAb enhances feasibility of development by minimizing the number of components of a promising novel therapeutic for these difficult-to-treat infections.

Anti-Langmuir elution behavior of a bispecific monoclonal antibody in cation exchange chromatography: Mechanistic modeling using a pH-dependent Self-Association Steric Mass Action isotherm.

The objective of this scientific work was to model and simulate the complex anti-Langmuir elution behavior of a bispecific monoclonal antibody (bsAb) under high loading conditions on the strong cation exchange resin POROS™ XS. The bsAb exhibited anti-Langmuirian elution behavior as a consequence of self-association expressed both in uncommon retentions and peak shapes highly atypical for antibodies. The widely applied Steric Mass Action (SMA) model was unsuitable here because it can only describe Langmuirian elution behavior and is not able to describe protein-protein interactions in the form of self-association. For this reason, a Self-Association SMA (SAS-SMA) model was applied, which was extended by two activity coefficients for the salt and protein in solution. This model is able to describe protein-protein interactions in the form of self-dimerization and thus can describe anti-Langmuir elution behavior. Linear gradient elution (LGE) experiments were carried out to obtain a broad dataset ranging from pH 4.5 to 7.3 and from 50 to 375 mmol/L Na+ for model parameter determination. High loading LGE experiments were conducted with an increasing load from 0.5 up to 75.0 mgbsAb/mLresin. Thereby, pH-dependent empirical correlations for the activity coefficient of the solute protein, for the equilibrium constant of the self-dimerization process and for the shielding factor could be set up and ultimately incorporated into the SAS-SMA model. This pH-dependent SAS-SMA model was thus able to simulate anti-Langmuir behavior over extended ranges of pH, counterion concentration, and column loading. The model was confirmed by experimental verification of simulated linear pH gradient elutions up to a load of 75.0 mgbsAb/mLresin.

Preparation of an Immunoaffinity Column Based on Bispecific Monoclonal Antibody for Aflatoxin B1 and Ochratoxin A Detection Combined with ic-ELISA.

A novel and efficient immunoaffinity column (IAC) based on bispecific monoclonal antibody (BsMAb) recognizing aflatoxin B1 (AFB1) and ochratoxin A (OTA) was prepared and applied in simultaneous extraction of AFB1 and OTA from food samples and detection of AFB1/OTA combined with ic-ELISA (indirect competitive ELISA). Two deficient cell lines, hypoxanthine guanine phosphoribosyl-transferase (HGPRT) deficient anti-AFB1 hybridoma cell line and thymidine kinase (TK) deficient anti-OTA hybridoma cell line, were fused to generate a hybrid-hybridoma producing BsMAb against AFB1 and OTA. The subtype of the BsMAb was IgG1 via mouse antibody isotyping kit test. The purity and molecular weight of BsMAb were confirmed by SDS-PAGE method. The cross-reaction rate with AFB2 was 37%, with AFG1 15%, with AFM1 48%, with AFM2 10%, and with OTB 36%. Negligible cross-reaction was observed with other tested compounds. The affinity constant (Ka) was determined by ELISA. The Ka (AFB1) and Ka (OTA) was 2.43 × 108 L/mol and 1.57 × 108 L/mol, respectively. Then the anti-AFB1/OTA BsMAb was coupled with CNBr-Sepharose, and an AFB1/OTA IAC was prepared. The coupling time and elution conditions of IAC were optimized. The coupling time was 1 h with 90% coupling rate, the eluent was methanol-water (60:40, v:v, pH 2.3) containing 1 mol/L NaCl, and the eluent volume was 4 mL. The column capacities of AFB1 and OTA were 165.0 ng and 171.3 ng, respectively. After seven times of repeated use, the preservation rates of column capacity for AFB1 and OTA were 69.3% and 68.0%, respectively. The ic-ELISA for AFB1 and OTA were applied combined with IAC. The IC50 (50% inhibiting concentration) of AFB1 was 0.027 ng/mL, the limit of detection (LOD) was 0.004 ng/mL (0.032 µg/kg), and the linear range was 0.006 ng/mL~0.119 ng/mL. The IC50 of OTA was 0.878 ng/mL, the LOD was 0.126 ng/mL (1.008 µg/kg), and the linear range was 0.259 ng/mL~6.178 ng/mL. Under optimum conditions, corn and wheat samples were pretreated with AFB1-OTA IAC. The recovery rates of AFB1 and OTA were 95.4%~105.0% with ic-ELISA, and the correlations between the detection results and LC-MS were above 0.9. The developed IAC combined with ic-ELISA is reliable and could be applied to the detection of AFB1 and OTA in grains.

Characterization of robust immune responses to a bispecific antibody, a novel class of antibody therapeutics.

Background: Anti-A/B is a bispecific monoclonal antibody that blocks activities of soluble targets A and B. Robust immune responses were observed in a multiple-dose cynomolgus monkey toxicology study, negatively impacting the toxicokinetics/pharmacodynamics profile of anti-A/B in some animals. This was unexpected as similar findings were not observed in the two previously studied parental molecules. Methodology & Results: This paper discusses our characterization strategy for evaluating the immunogenic domain(s) of anti-A/B and our mitigation plan to monitor immunogenicity in the first-in-human clinical study. The characterization results from the cynomolgus monkey and Phase I studies are discussed. Conclusion: The characterization strategy discussed informed understanding of immunogenicity results and clinical impact, which can be broadly applied to other molecules with multiple-binding domains.

Monitoring glycation levels of a bispecific monoclonal antibody at subunit level by ultrahigh-resolution MALDI FT-ICR mass spectrometry.

Bispecific monoclonal antibodies (BsAbs) are engineered proteins with multiple functionalities and properties. The "bi-specificity" of these complex biopharmaceuticals is a key characteristic for the development of novel and more effective therapeutic strategies. The high structural complexity of BsAbs poses a challenge to the analytical methods needed for their characterization. Modifications of the BsAb structure, resulting from enzymatic and non-enzymatic processes, further complicate the analysis. An important example of the latter type of modification is glycation, which can occur in the manufacturing process, during storage in the formulation or in vivo after application of the drug. Glycation affects the structure, function, and stability of monoclonal antibodies, and consequently, a detailed analysis of glycation levels is required. Mass spectrometry (MS) plays a key role in the structural characterization of monoclonal antibodies and top-down, middle-up and middle-down MS approaches are increasingly used for the analysis of modifications. Here, we apply a novel middle-up strategy, based on IdeS digestion and matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) MS, to analyze all six different BsAb subunits in a single high-resolution mass spectrum, namely two light chains, two half fragment crystallizable regions and two Fd' regions, thus avoiding upfront chromatography. This method was used to monitor glycation changes during a 168 h forced-glycation experiment. In addition, hot spot glycation sites were localized using top-down and middle-down MALDI-in-source decay FT-ICR MS, which provided complementary information compared to standard bottom-up MS.

Emicizumab for the prevention of bleeds in hemophilia A.

Introduction: The management of hemophilia A with and without inhibitors is challenging with high treatment burden of prophylactic regimens, musculoskeletal complications, poor treatment compliance, poor venous access and therapies with suboptimal levels. Areas covered: Emicizumab is a bispecific monoclonal antibody recently approved for prevention of bleeds in hemophilia A patients with and without inhibitors. This review is a synthesis of several recently completed emicizumab clinical trials with the emphasis of its mechanism of action, efficacy, safety, and pharmacokinetic profile when used for prevention of bleeds in hemophilia A patients of all age groups with and without inhibitors. We also review the role of emicizumab in the era of rapidly evolving novel therapies in hemophilia A. Expert opinion: Data from completed clinical studies indicate that emicizumab is poised to address current unmet needs in hemophilia A. It has high efficacy in the prevention of bleeds and a favorable safety profile. Subcutaneous dosing and versatile dosing regimens make emicizumab an ideal drug to address current unmet needs in hemophilia A management across all age groups. How emicizumab fits in the current rapidly evolving hemophilia A therapeutic landscape will be exciting to watch in the coming period.

Bispecific Monoclonal Antibody-Based Multianalyte ELISA for Furaltadone Metabolite, Malachite Green, and Leucomalachite Green in Aquatic Products.

A new multianalyte immunoassay was designed to screen furaltadone metabolite 5-morpholinomethyl-3-amino-2-oxazolidone (AMOZ), malachite green (MG), and leucomalachite green (LMG) in aquatic products using a bispecific monoclonal antibody (BsMAb). Gradient drug mutagenesis methods were separately used to prepare an anti-3-nitrobenzaldehyde-derivatized AMOZ (3-NPAMOZ) hybridoma cell line that was hypoxanthine-guanine-phosphoribosyltransferase (HGRPT) deficient and an anti-LMG hybridoma cell line that was thymidine kinase (TK) deficient. BsMAb recognizing 3-NPAMOZ and LMG was generated using hybrid-hybridomas of HGRPT and TK deficient cell lines. For AMOZ and LMG, respectively, the BsMAb-based indirect competitive ELSIA (ic-ELISA) values of 1.7 ng/mL and 45.3 ng/mL and detection limits of 0.2 ng/mL and 4.8 ng/mL. To establish the ic-ELISA, 3-NPAMOZ derivatized from AMOZ with 3-nitrobenzaldehyde and LMG reduced from MG by potassium borohydride was recognized by BsMAb. Recoveries of AMOZ, MG, and LMG in aquatic products were satisfactory and correlated with HPLC analysis. Thus, the multianalyte ic-ELISA is suitable for rapid quantification of AMOZ, MG, and LMG in aquatic products.

Immunological changes in the ascites of cancer patients after intraperitoneal administration of the bispecific antibody catumaxomab (anti-EpCAM×anti-CD3).

OBJECTIVE: To explore the effects of intraperitoneal (i.p.) infusion of catumaxomab, a bispecific monoclonal antibody (anti-EpCAM×anti-CD3), on T cells, NK cells and macrophages in ascites of cancer patients and to understand how ascitic immune cells can be activated despite the pervasive immunosuppressive ability of ascites microenvironment. METHODS: Six patients with malignant ascites received i.p. catumaxomab infusion. Ascitic immune cells were profiled by flow cytometry and gene expression at baseline and after i.p. catumaxomab infusion. In vitro experiments enabled investigations on the adverse effect of ascites microenvironment on catumaxomab-stimulated immune cells. RESULTS: I.p. catumaxomab infusion enhanced the expression of the CD69 and CD38 activation molecules in CD4(+) and CD8(+) T cells, NK cells and macrophages, and favoured CD8(+) T cell accumulation into the peritoneal cavity. An analogous immune cell activation as well as IFN-γ and IL-2 production were induced by catumaxomab in vitro. In vitro experiments showed that the immunosuppressive milieu of ascites abrogated all the immunostimulatory activities of catumaxomab. Adding EpCAM(+) tumour cells to the culture permitted both catumaxomab Fab regions to engage cognate antigens and restored immunostimulatory catumaxomab activity. CONCLUSIONS: This is the first demonstration in a clinical setting that i.p. catumaxomab infusion activates NK cells and macrophages in addition to T cells in ascites and favours CD8(+) T cell accumulation into the peritoneal cavity. Moreover, our findings indicate that the concomitant binding of both catumaxomab Fab regions delivers an activation signal that is strong enough to activate immune cells despite the prevailing immunosuppressive environment of malignant ascites.

Pretargeted Radioimmunotherapy of Prostate Cancer with an Anti-TROP-2×Anti-HSG Bispecific Antibody and a (177)Lu-Labeled Peptide.

UNLABELLED: TROP-2 is a pancarcinoma marker that is expressed at high levels in many epithelial cancers, including prostate cancer (PC). The trivalent bispecific antibody TF12 (anti-TROP2 × anti-HSG [histamine-succinyl-glycine]) has shown to effectively target PC. In this study, the efficacy of pretargeted radioimmunotherapy (PRIT) with multiple cycles of TF12 and (177)Lu-labeled diHSG-peptide (IMP288) in mice with s.c. PC3 tumors was investigated and compared with that of conventional RIT with (177)Lu-labeled anti-TROP-2 mAb hRS7. METHODS: The potential of one, two, and three cycles of PRIT using the TF12 pretargeted (177)Lu-IMP288 (41 MBq per cycle) was determined in mice with s.c. PC3 tumors, and compared with the efficacy and toxicity of RIT with (177)Lu-hRS7 dosed at the maximum tolerated dose (11 MBq). RESULTS: PRIT of two and three cycles showed significantly higher median survival (> 150 days) compared with PRIT of one cycle of TF12 and (177)Lu-IMP288 (111 days, p < 0.001) or the controls (76 days, p < 0.0001). All mice treated with the mAb (177)Lu-hRS7 survived at the end of the experiment (150 days), compared with 80% in the mice that were treated with three cycles of PRIT and 70% in the group that received two cycles of PRIT. Clinically significant hematologic toxicity was found only in the groups that received either three cycles of PRIT (p < 0.0009) or RIT (p < 0.0001). CONCLUSIONS: TROP-2-expressing PC can be targeted efficiently with TF12 and radiolabeled IMP288. (177)Lu-IMP288 accumulated rapidly in the tumors. PRIT of multiple cycles inhibited the growth of s.c. PC3 tumors. Clinically relevant hematological toxicity was observed in the group that received three cycles of PRIT; however, conventional RIT with the parent mAb (177)Lu-hRS7 was at least as effective with similar toxicity.

Mapping biological behaviors by application of longer-lived positron emitting radionuclides.

With the technological development of positron emission tomography (PET) and the advent of novel antibody-directed drug delivery systems, longer-lived positron-emitting radionuclides are moving to the forefront to take important roles in tracking the distribution of biotherapeutics such as antibodies, and for monitoring biological processes and responses. Longer half-life radionuclides possess advantages of convenient on-site preparation procedures for both clinical and non-clinical applications. The suitability of the long half-life radionuclides for imaging intact monoclonal antibodies (mAbs) and their respective fragments, which have inherently long biological half-lives, has attracted increased interest in recent years. In this review, we provide a survey of the recent literature as it applies to the development of nine-selected longer-lived positron emitters with half-lives of 9-140h (e.g., (124)I, (64)Cu, (86)Y and (89)Zr), and describe the biological behaviors of radionuclide-labeled mAbs with respect to distribution and targeting characteristics, potential toxicities, biological applications, and clinical translation potentials.