Development of tibulizumab,a tetravalent bispecific antibody targeting BAFF and IL-17A for the treatment of autoimmune disease

ABSTRACT

We describe a bispecific dual-antagonist antibody against human B cell activating factor (BAFF) and interleukin 17A (IL-17). An anti-IL-17 single-chain variable fragment (scFv) derived from ixekizumab (Taltz®) was fused via a glycine-rich linker to anti-BAFF tabalumab. The IgG-scFv bound both BAFF and IL-17 simultaneously with identical stoichiometry as the parental mAbs. Stability studies of the initial IgG-scFv revealed chemical degradation and aggregation not observed in either parental antibody. The anti-IL-17 scFv showed a high melting temperature (Tm) by differential scanning calorimetry (73.1°C), but also concentration-dependent, initially reversible, protein self-association. To engineer scFv stability, three parallel approaches were taken: labile complementary-determining region (CDR) residues were replaced by stable, affinity-neutral amino acids, CDR charge distribution was balanced, and a H44-L100 interface disulfide bond was introduced. The Tm of the disulfide-stabilized scFv was largely unperturbed, yet it remained monodispersed at high protein concentration. Fluorescent dye binding titrations indicated reduced solvent exposure of hydrophobic residues and decreased proteolytic susceptibility was observed, both indicative of enhanced conformational stability. Superimposition of the H44-L100 scFv (PDB id: 6NOU) and ixekizumab antigen-binding fragment (PDB id: 6NOV) crystal structures revealed nearly identical orientation of the frameworks and CDR loops. The stabilized bispecific molecule LY3090106 (tibulizumab) potently antagonized both BAFF and IL-17 in cell-based and in vivo mouse models. In cynomolgus monkey, it suppressed B cell development and survival and remained functionally intact in circulation, with a prolonged half-life. In summary, we engineered a potent bispecific antibody targeting two key cytokines involved in human autoimmunity amenable to clinical development.     

Simultaneous targeting of TNF and Ang2 with a novel bispecific antibody enhances efficacy in an in vivo model of arthritis

Despite the clinical success of anti-tumor necrosis factor (TNF) therapies in the treatment of inflammatory conditions such as rheumatoid arthritis, Crohn disease and psoriasis, full control of the diseases only occurs in a subset of patients and there is a need for new therapeutics with improved efficacy against broader patient populations. One possible approach is to combine biological therapeutics, but both the cost of the therapeutics and the potential for additional toxicities needs to be considered. In addition to the various mediators of immune and inflammatory pathways, angiogenesis is reported to contribute substantially to the overall pathogenesis of inflammatory diseases. The combination of an anti-angiogenic agent with anti-TNF into one molecule could be more efficacious without the risk of severe immunosuppression. To evaluate this approach with our Zybody technology, we generated bispecific antibodies that contain an Ang2 targeting peptide genetically fused to the anti-TNF antibody adalimumab (Humira®). The bispecific molecules retain the binding and functional characteristics of the anti-TNF antibody, but with additional activity that neutralizes Ang2. In a TNF transgenic mouse model of arthritis, the bispecific anti-TNF-Ang2 molecules showed a dose-dependent reduction in both clinical symptoms and histological scores that were significantly better than that achieved by adalimumab alone.     

Simultaneous targeting of TNF and Ang2 with a novel bispecific antibody enhances efficacy in an in vivo model of arthritis

Despite the clinical success of anti-tumor necrosis factor (TNF) therapies in the treatment of inflammatory conditions such as rheumatoid arthritis, Crohn disease and psoriasis, full control of the diseases only occurs in a subset of patients and there is a need for new therapeutics with improved efficacy against broader patient populations. One possible approach is to combine biological therapeutics, but both the cost of the therapeutics and the potential for additional toxicities needs to be considered. In addition to the various mediators of immune and inflammatory pathways, angiogenesis is reported to contribute substantially to the overall pathogenesis of inflammatory diseases. The combination of an anti-angiogenic agent with anti-TNF into one molecule could be more efficacious without the risk of severe immunosuppression. To evaluate this approach with our Zybody technology, we generated bispecific antibodies that contain an Ang2 targeting peptide genetically fused to the anti-TNF antibody adalimumab (Humira®). The bispecific molecules retain the binding and functional characteristics of the anti-TNF antibody, but with additional activity that neutralizes Ang2. In a TNF transgenic mouse model of arthritis, the bispecific anti-TNF-Ang2 molecules showed a dose-dependent reduction in both clinical symptoms and histological scores that were significantly better than that achieved by adalimumab alone.     

A bispecific antibody targeting HER2 and PD-L1 inhibits tumor growth with superior efficacy

Activation of the programmed cell death protein 1 and programmed cell death ligand 1 (PD-1/PD-L1) signaling axis plays important roles in intrinsic or acquired resistance to human epidermal growth factor receptor 2 (HER2)-directed therapies in the clinic. Therefore, therapies simultaneously targeting both HER2 and PD-1/PD-L1 signaling pathways are of great significance. Here, aiming to direct the anti-PD-L1 responses toward HER2-expressing tumor cells, we constructed a humanized bispecific IgG1 subclass antibody targeting both HER2 and PD-L1 (HER2/PD-L1; BsAb), which displayed satisfactory purity, thermostability, and serum stability. We found that BsAb showed enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity in vitro. In the late phase of peripheral blood mononuclear cell (PBMC)-humanized HER2⁺ tumor xenograft models, BsAb showed superior therapeutic efficacies as compared with monoclonal antibodies (mAbs) or combination treatment strategies. In cynomolgus monkeys, BsAb showed favorable pharmacokinetics and toxicity profiles when administered at a 10 mg/kg dosage. Thus, HER2/PD-L1 BsAb was demonstrated as a potentially effective option for managing HER2⁺ and trastuzumab-resistant tumors in the clinic. We propose that the enhanced antitumor activities of BsAb in vivo may be due to direct inhibition of HER2 signaling or activation of T cells.     

Single chain Fv antibody against angiopoietin-2 inhibits VEGF-induced endothelial cell proliferation and migration in vitro

Angiopoietin-2 (Ang2) promotes tumor growth and metastasis by specifically priming endothelial cells for angiogenesis. Multiple angiogenic factors up-regulate expression of Ang2, suggesting that Ang2 may be the common pathway in growth factor initiated-angiogenesis. Using phage display technology, we generated single chain Fv molecule against human Ang2 (scFv-Ang2) with high affinity (K(d)=0.01 microM) from a mouse phage antibody library. Compared with control scFv, the mouse scFv-Ang2 completely inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) treated with vascular endothelial growth factor (VEGF, 10 ng/ml), but not that of the cells treated with either basic fibroblast growth factor, or angiotensin II, or Ang2. Chemotaxis assay showed that scFv-Ang2 could block completely Ang2-induced (100%) and partially VEGF-induced (49%) migration of HUVECs. The results indicate that Ang2 takes part in the VEGF-induced angiogenesis and scFv-Ang2 might be a promising compound in blocking both VEGF and Ang2 induced angiogenesis.     

Enhanced expression of angiopoietin-2 and the Tie2 receptor but not angiopoietin-1 or the Tie1 receptor in a rat model of myocardial infarction

Modulation of Tie2 receptor activity by angiopoietin ligands is crucial for angiogenesis, blood vessel maturation, and vascular endothelium integrity. The role of the angiopoietin (Ang) and Tie system in myocardial infarction is not well understood. To investigate the participation of the Ang/Tie in myocardial infarction, adult Sprague-Dawley rats with ligation of the left anterior descending coronary artery to induce myocardial infarction were studied. Ang1, Ang2, Tie1, and Tie2 were measured immediately after ligation of the coronary artery, and at 6 h, 1 and 3 days, and 1, 2, 3 and 4 weeks after ligation by Northern blotting, Western blotting, and immunohistochemical staining. Ang2 mRNA significantly increased from 2 weeks (2.1-fold) to 4 weeks (2.9-fold) after the infarction in the left ventricular free wall. Tie2 mRNA increased significantly from 1 week (2.1-fold) to 4 weeks (3.8-fold) after the infarction. Ang2 protein also significantly increased from 3 days (1.9-fold) to 4 weeks (3-fold) after the infarction in the left ventricular free wall. Tie2 protein increased 2.4-fold at 3 weeks and 2.8-fold at 4 weeks after the infarction. Neither Ang1 nor Tie1 mRNA or protein showed any significant change at any time point after the infarction. The ratio of Ang2/Ang1 mRNA and protein in the study group was higher than that in the control group. Ang2 and Tie2 expression in nonischemic myocardium showed no significant change. Immunohistochemical study also showed increased immunoreactivity of Ang2 and Tie2 at the infarct border. In conclusion, Ang2 and Tie2 expressions significantly increased both spatial and temporal patterns after myocardial infarction in the rat ventricular myocardium, while Ang1 and Tie1 receptor expression did not.     

Broad neutralization of SARS-CoV-2 variants by an inhalable bispecific single-domain antibody

1. Download : Download high-res image (193KB)
2. Download : Download full-size image

     

CODV-Ig,a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications

Bispecific immunoglobulins (Igs) typically contain at least two distinct variable domains (Fv) that bind to two different target proteins. They are conceived to facilitate clinical development of biotherapeutic agents for diseases where improved clinical outcome is obtained or expected by combination therapy compared to treatment by single agents. Almost all existing formats are linear in their concept and differ widely in drug-like and manufacture-related properties. To overcome their major limitations, we designed cross-over dual variable Ig-like proteins (CODV-Ig). Their design is akin to the design of circularly closed repeat architectures. Indeed, initial results showed that the traditional approach of utilizing (G4S)_x linkers for biotherapeutics design does not identify functional CODV-Igs. Therefore, we applied an unprecedented molecular modeling strategy for linker design that consistently results in CODV-Igs with excellent biochemical and biophysical properties. CODV architecture results in a circular self-contained structure functioning as a self-supporting truss that maintains the parental antibody affinities for both antigens without positional effects. The format is universally suitable for therapeutic applications targeting both circulating and membrane-localized proteins. Due to the full functionality of the Fc domains, serum half-life extension as well as antibody- or complement-dependent cytotoxicity may support biological efficiency of CODV-Igs. We show that judicious choice in combination of epitopes and paratope orientations of bispecific biotherapeutics is anticipated to be critical for clinical outcome. Uniting the major advantages of alternative bispecific biotherapeutics, CODV-Igs are applicable in a wide range of disease areas for fast-track multi-parametric drug optimization.     

Angiogenic activity of endothelial progenitor cells through angiopoietin-1 and angiopoietin-2

Angiogenesis is a regulated process involving the proliferation, migration, and remodeling of different cell types particularly mature endothelial cells and recently discovered progenitor cells, named as endothelial progenitor cells (EPCs). Up to now, many attempts have been made to understand the dynamic balance of pro- and anti-angiogenic factors on EPCs on different milieu. It has been accepted that Ang-1, -2 and Tie-1, -2 signaling play a key role on angiogenesis pathways in endothelial lineage cells. In the current experiment, the angiogenic/angio-modulatory potency of Ang-1 and -2 was investigated on isolated EPCs. Freshly isolated EPCs were exposed to different concentrations of Ang-1 and -2 (25 and 50?ng/ml) over a course of 7 and 14 days. Corroborating to our results, a superior effect of Ang-1 on angiogenic properties, including an increased concentration of vascular endothelial growth factor, in vitro tubulogenesis, EPC migratory, Tie-2 expression and clonogenicity, was determined. A large amount of positive mature endothelium markers was achieved in EPCs being-exposed to Ang-1 peptide. Nonetheless, the number of CD133 positive cells increased in the presence of Ang-2. Collectively, we conclude that Ang-1 potentially induces functional and mature vascular-like behavior in EPCs more than Ang-2.     

A novel bispecific diabody targeting both vascular endothelial growth factor receptor 2 and epidermal growth factor receptor for enhanced antitumor activity

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) are receptor tyrosine kinases known to play critical roles in the development and progression of tumors. Based on the cross‐talk between EGFR and VEGFR2 signal pathways, we designed and produced a bispecific diabody (bDAb) targeting both EGFR and VEGFR2 simultaneously. The bispecific molecule (EK‐02) demonstrated that it could bind to HUVEC (VEGFR2 high‐expressing) and A431 (EGFR overexpressing) cells. Additionally, similar to the parental antibodies, it was able to inhibit proliferation and migration, and induced apoptosis in these cells (HUVECs and A431), demonstrating that it had retained the functional properties of its parental antibodies. Furthermore, the efficacy of EK‐02 was evaluated using the human colon adenocarcinoma cell line HT29 (VEGFR2 and EGFR coexpressing). In vitro assay showed that EK‐02 could bind to HT29 cells, restrain cell growth and migration, and induce apoptosis with enhanced efficacy compared to both parental antibodies. Further, it inhibited the neovascularization and tumor formation on an HT29 cell bearing chicken chorioallantoic membrane (CAM) tumor model in vivo. In conclusion, these data suggest that the novel bDAb (EK‐02) has antiangiogenesis and antitumor capacity both in vitro and in vivo, and can possibly be used as cotargeted therapy for the treatment of EGFR and VEGFR2 overexpressing tumors. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:294–302, 2016     

Angiopoietin 1 (Ang-1), angiopoietin 2 (Ang-2) and Tie-2 (a receptor tyrosine kinase) concentrations in peripheral blood of patients with thyroid cancers

There are limited and conflicting studies investigating the role of the angiopoietin family in human thyroid cancer development and progression. We have investigated cytokines angiopoietin-1, -2 and their receptor (Tie-2), known to be involved in angiogenesis, in the serum of 52 thyroid cancer patients (21 cases of papillary cancers, PTC; 8 follicular cancers, FTC; 12 medullary cancers, MTC and 11 anaplastic cancers, ATC), using ELISA assays. The control consisted of 27 healthy volunteers. Statistically significant lower concentrations of Ang-1 were found in patients with thyroid cancers as compared with the control (p<0.003). The levels of Ang-2 and Tie-2 did not differ significantly between thyroid cancer patients and control. We have also compared the results of Ang-1, Ang-2, and Tie-2 determinations obtained in different histopathological subgroups of cancer patients. These results revealed lower Ang-1 concentrations in ATC (p<0.05), MTC (p<0.02), FTC (p<0.01) and in PTC patients (p<0.05) than control. We have also observed lower Ang-2 concentration in PTC patients (p<0.03) and Tie-2 in FTC patients (p<0.02 ) in comparison to controls. In conclusion, the Angs/Tie-2 system dysfunction may play an important role in thyroid cancerogenesis and decreased concentration of Ang-1 in serum can be a useful additional biomarker for the presence of thyroid cancers.     

miR-29a suppresses growth and invasion of gastric cancer cells in vitro by targeting VEGF-A

Increasing data shows miR-29a is a key regulator of oncogenic processes. It is significantly down-regulated in some kind of human tumors and possibly functionally linked to cellular proliferation, survival and migration. However, the mechanism remains unclear. In this study, we report miR-29a is significantly under-expressed in gastric cancer compared to the healthy donor. The microvessel density is negatively related to miR-29a expression in gastric cancer tissues. The ectopic expression of miR-29a significantly inhibits proliferation and invasion of gastric cancer cells. Furthermore, western blot combined with the luciferase reporter assays demonstrate that vascular endothelial growth factor A (VEGF-A) is direct target of miR-29a. This is the first time miR-29a was found to suppress the tumor microvessel density in gastric cancer by targeting VEGF-A. Taken together, these results suggest that miR-29a is a tumor suppressor in gastric cancer. Restoration of miR-29a in gastric cancer may be a promising therapeutic approach. [BMB Reports 2014; 47(1):39-44]     

Construction,expression and characterization of the engineered antibody against tumor surface antigen,p185^c—erbB—2

The c-erbB-2 proto-oncogene encodes a 185kD protein p185,which belongs to epidermal growth factor receptor family.Amplification of this gene has been shown to correlate with poor clinical prognosis for certain cancer patients.The monoclonal antibody A21 which directed against p185 specifically inhibits proliferation of tumor cells overexpressing p185,hence allows it to be a candidate for targeted therapy.In order to overcome several drawbacks of murine MAb,we cloned its VH and VL genes and constructed the single-chain FV(scFv)through a peptide linker.The recombinant scFv A21 was expressed in Escherichia coli and purified by the affinity column.Subsequently it was characterized by ELISA,Western blot,cell immunohistochemistry and FACS.All these assays showed the binding activity to extracellular domain(ECD)of p185.Based on those properties of scFvA21,we further constructed the scFv-Fc fusion molecule with a homodimer form and the recombinant product was expressed in mammalian cells.In a series of subsequent analysis this fusion protein showed identical antigen binding site and activity with the parent antibody.These anti-p185 engineered antibodies have promised to be further modified as a tumor targeting drugs,with a view of application in the diagnosis and treatment of human breast cancer.     

Tumor-specific targeting of T helper type 1 (Th1) cells by anti-CD3 × anti-c-ErbB-2 bispecific antibody

T helper type1 (Th1) or type2 (Th2) cells were induced from naive Th cells obtained from ovalbumin-specific T cell receptor (TCR) transgenic mice. Th1 cells producing interferon γ (IFNγ) exhibited stronger antigen-specific cytotoxicity against ovalbumin-(323–339)-peptide-pulsed A20 tumor cells than did Th2 cells. To develop a general method for applying antigen-nonspecific Th1 cells to tumor immunotherapy, we examined the targeting of Th1 cells to tumor cells using a bispecific antibody (bsAb) consisting of anti-(mouse CD3) mAb and anti-(human c-ErbB-2) mAb. When ovalbumin-specific Th1 or Th2 cells were cocultured with c-erbB-2-positive transfectants (CMS7HE), neither type of cell showed significant cytotoxicity or cytokine production in response to tumor cells. However, addition of bsAb resulted in the triggering of both Th1 and Th2 cells. Th1 cells showed higher levels of bsAb-dependent cytotoxicity against CMS7HE tumor cells than did Th2 cells. The targeting of Th1 cells to CMS7HE tumor cells by bsAb also triggered the production of cytokines such as IFNγ, interleukin-2 and tumor necrosis factor α (TNFα). The released TNFα was demonstrated to be a critical cytolytic factor in bsAb-mediated cytotoxicity by Th1 cells. Finally, Th1 cells were demonstrated to show antitumor activity in vivo against human c-erbB-2-positive tumor cells implanted in nude mice. These results suggest that Th1 cells are useful effector cells for the application to adoptive tumor immunotherapy in conjunction with bsAb. Received: 22 April 1999 / Accepted: 2 July 1999     

Potentiation of long-term-cultured lymphokine-activated killer cell cytotoxicity against small-cell lung carcinoma by anti-CD3 x anti-(tumor-associated antigen) bispecific antibody

Lymphokine-activated killer (LAK) cells exhibit a potent cytotoxicity to malignant cells in vitro. However, a satisfactory effect has not been obtained in many clinical studies except for a few cases. One of the most important reasons why cytolytic activity could not be exhibited in vivo is that LAK cells do not accumulate in the tumor tissue because of a lack of specificity. In the present study, we show the effect of a bispecific antibody (bsAb) on the accumulation of LAK cells around the small-cell lung carcinoma (SCLC) cell and the subsequent enhancement of LAK cell cytotoxicity against SCLC. When short-term (4 days)-cultured LAK cells were used, OKT3xLU246 bsAb, which direct CD3+ T-LAK cells to the target cell, induced a similar level of cytotoxicity to that induced by 3G8xLU246 bsAb, which directs CD16+ LAK cells. Longterm (21 days)-cultured LAK cells exhibited a reduced spontaneous cytotoxicity but retained high cytotoxic activity, which could be directed by OKT3xLU246 or 3G8xLU246 bsAb. The inhibitory effect of LAK cells on tumor cell clonogenicity in soft agar was also enhanced by both bsAb. These results indicate that application of the therapy with LAK cells and OKT3xLU246 bsAb to SCLC patients might be a promising new method of adoptive immunotherapy.     

LC-MS characterization and purity assessment of a prototype bispecific antibody

Bispecific IgG asymmetric (heterodimeric) antibodies offer enhanced therapeutic efficacy, but present unique challenges for drug development. These challenges are related to the proper assembly of heavy and light chains. Impurities such as symmetric (homodimeric) antibodies can arise with improper assembly. A new method to assess heterodimer purity of such bispecific antibody products is needed because traditional separation-based purity assays are unable to separate or quantify homodimer impurities. This paper presents a liquid chromatography-mass spectrometry (LC-MS)-based method for evaluating heterodimeric purity of a prototype asymmetric antibody containing two different heavy chains and two identical light chains. The heterodimer and independently expressed homodimeric standards were characterized by two complementary LC-MS techniques: Intact protein mass measurement of deglycosylated antibody and peptide map analyses. Intact protein mass analysis was used to check molecular integrity and composition. LC-MS^E peptide mapping of Lys-C digests was used to verify protein sequences and characterize post-translational modifications, including C-terminal truncation species. Guided by the characterization results, a heterodimer purity assay was demonstrated by intact protein mass analysis of pure deglycosylated heterodimer spiked with each deglycosylated homodimeric standard. The assay was capable of detecting low levels (2%) of spiked homodimers in conjunction with co-eluting half antibodies and multiple mass species present in the homodimer standards and providing relative purity differences between samples. Detection of minor homodimer and half-antibody C-terminal truncation species at levels as low as 0.6% demonstrates the sensitivity of the method. This method is suitable for purity assessment of heterodimer samples during process and purification development of bispecific antibodies, e.g., clone selection.     

Changes and significance of serum angiopoietin-2 levels in patients with coronary heart disease

Coronary heart disease (CHD) is characterized by inflammatory process and endothelial dysfunction. To investigate angiopoietin-2 (Ang-2) profiles, we evaluated serum Ang-2 levels in different types of CHD in 166 subjects. Ang-2 was measured by enzyme-linked immunosorbent assay. Serum Ang-2 levels were significantly elevated in patients with CHD and gradually increased with advance of CHD. Ang-2 was positively correlated with Gensini scores and hs-CRP. Ang-2 might have potential implication in detecting and monitoring the progression of CHD.     

Design,selection and optimization of an anti-TRAIL-R2/anti-CD3 bispecific antibody able to educate T cells to recognize and destroy cancer cells

Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or TRAIL-receptor agonistic monoclonal antibodies promote apoptosis in most cancer cells, and the differential expression of TRAIL-R2 between tumor and normal tissues allows its exploitation as a tumor-associated antigen. The use of these antibodies as anticancer agents has been extensively studied, but the results of clinical trials were disappointing. The observed lack of anticancer activity could be attributed to intrinsic or acquired resistance of tumor cells to this type of treatment. A possible strategy to circumvent drug resistance would be to strike tumor cells with a second modality based on a different mechanism of action. We therefore set out to generate and optimize a bispecific antibody targeting TRAIL-R2 and CD3. After the construction of different bispecific antibodies in tandem-scFv or single-chain diabody formats to reduce possible immunogenicity, we selected a humanized bispecific antibody with very low aggregates and long-term high stability and functionality. This antibody triggered TRAIL-R2 in an agonistic manner and its anticancer activity proved dramatically potentiated by the redirection of cytotoxic T cells against both sensitive and resistant melanoma cells. The results of our study show that combining the TRAIL-based antitumor strategy with an immunotherapeutic approach in a single molecule could be an effective addition to the anticancer armamentarium.