Therapeutic Bispecific Antibodies Research Articles

Overcoming the inconsistent cell culture performance caused by single-use bioreactors during scaling-up the manufacturing process for a therapeutic bispecific antibody

Single use technology has been widely used in modern biopharmaceutical process development and manufacturing. However, there are concerns about the adverse effects of disposable materials on bioprocess, product quality and patient safety. Recently, we observed slow cell growth, increased lactate accumulation, and decreased production titer during scaling up a bispecific antibody (BsAb) upstream process from 3L to 500L bioreactor. To investigate the phenomenon, we conducted medium incubation and leachable spike-in experiments. We found that cell culture medium incubated in CX5-14 bag caused poor cell culture performance, which was correlated with the concentration of bis(2,4-di-tert-butylphenyl)-phosphate (bDtBPP), a breakdown product of the antioxidant Irgafos®168 in polyethylene film. In addition, when the media were spiked with 0.15μg/ml or a greater concentration of bDtBPP, slow cell growth and reduced BsAb expression level were observed. In contrast, Aegis5-14 film produced bDtBPP at a level of below detection during medium incubation. Thus, the bispecific-expressing CHO cell line restores normal cell culture performance and production titer when Aegis5-14 single-use bioreactor was employed in large-scale manufacturing. Our results reveal the importance of evaluating leachable profiles from disposable materials during process development of bispecifics and other novel therapeutic modalities.

Novel therapeutic bispecific antibodies for B-cell lymphoma targeting IgM and other antigens on the B-cell surface

Novel therapeutic bispecific antibodies for B-cell lymphoma targeting IgM and other antigens on the B-cell surface

Abstract 6712: LBL-043, a novel LILRB4xCD3 T cell engager, for the treatment of LILRB4 positive leukemia

Abstract Background: Acute myeloid leukemia (AML) is a malignant clonal disorder linked to a broad spectrum of molecular alterations. It is still an incurable cancer, the prognosis is even worse in elderly patients, where overall survival (OS) at 1 year is approximately 10-15%. Now, several CD3 bispecific immunotherapies that harness T cells against AML are at various stages of preclinical and clinical development, but most of them are hampered by the lack of specific targets to distinguish tumor cells from normal hematopoietic stem cells (HSC). Leukocyte immunoglobulin-like receptor B4 (LILRB4) is an immune checkpoint inhibitory receptor, which is highly expressed on FAB M4 and M5 AML cells with a lack of expression on normal HSC and progenitor cells. LILRB4 expression level inversely correlates with OS of patients with M4 and M5 AML. All of these indicate that LILRB4 represents a promising target for developing a T cell redirecting bispecific antibody to treat patients with AML, especially for more aggressive M4 and M5 subtype. Herein a novel bispecific antibody targeting LILRB4 and CD3, LBL-043, is developed to specifically kill LILRB4 positive tumor cells by engaging T cells. Methods: LBL-043 was constructed using our proprietary LeadsBodyTM T cell engager platform, which is a 2:1 format bispecific antibody with two VHH arms targeting LILRB4 with high affinity and one scFv arm targeting CD3 with fine-tuned low affinity. The binding affinity of LBL-043 to LILRB4 and CD3 was determined with Fortebio, while the activity was measured using several cell based assays including reporter gene and TDCC assays. The anti-tumor activity of LBL-043 was investigated in B-NDG/hu-PBMC reconstructed mice implanted with MOLM-13 (human AML-M5a) tumor model. Results: The binding affinity of LBL-043 to LILRB4 and CD3 protein was 0.724 nM and 35.7 nM, respectively. In CD3 reporter gene assays, LBL-043 could activate the NFAT reporter signaling through LILRB4 binding dependent CD3 cross-linking with an EC50 value of 2.459 nM. LBL-043 was shown a potent TDCC activity in different LILRB4 expression level MOLM-13 cells and HL-60 cells with T cell activation and cytokine release. In B-NDG/hu-PBMC reconstructed mice implanted with MOLM-13 tumor cells, LBL-043 was shown significant anti-tumor activity. Simultaneously, LBL-043 can effectively improve the survival rate and maintain relatively stable body weight in the study compared to the vehicle group. Conclusion: LBL-043, a novel bispecific antibody targeting CD3 and LILRB4 with affinity differentiation, induces T cell killing of AML cells by simultaneously binding LILRB4 expressed on tumor cells and CD3 on T cells, redirecting T cells to efficiently killing of AML cells. It is also shown a great anti-tumor efficacy in CDX mouse models. These data support LBL-043 as a novel therapeutic bispecific antibody for the treatment of LILRB4 positive AML and CMML leukemia patients. Citation Format: Yujia Dang, Xiao Huang, Yurong Qin, Xiaoya Liu, Min Chen, Duqing Jiang, Guojin Wu, Mi Ye, Jianming Sun, Baohui Wang, Jing Guan, Tingting Li, Jordan Zhu, Shoupeng Lai, Xiaoqiang Kang, Hong Ling. LBL-043, a novel LILRB4xCD3 T cell engager, for the treatment of LILRB4 positive leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6712.

Abstract 2732: NB203: A Novel therapeutic bispecific antibody targeting CEACAM1/VEGF for tumor immunity and angiogenesis modulation in the tumor microenvironment

Abstract The carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is detected on leukocytes, epithelia and endothelia, and involved in cell adhesion through homophilic or heterophilic binding. In various gastrointestinal cancers, NSCLC (non-small cell lung cancer), and melanoma, CEACAM1 is highly expressed on tumor cells and tumor stroma, with its levels increasing as the disease progresses. Notably, CEACAM1 has been found to dampen T and NK cell functions while promoting tumor angiogenesis and metastasis. In this study, we present NB203, a novel CEACAM1/VEGF bispecific antibody developed using Neologics CEACAM-plus, a bifunctional molecule engineering platform designed to target and modulate the tumor microenvironment (TME). NB203 effectively accumulated in the TME and hindered the homophilic or heterophilic interactions of CEACAM1. It demonstrated a significant enhancement in T cell activities and NK cell-mediated killing of CEACAM1-expressing tumor cells, and the inhibition of tumor cell migration and polyploidy through macrophages. More importantly, NB203 was specifically designed to impede tumor angiogenesis. It exhibited an increased binding capacity to both CEACAM1 and VEGF in pro-angiogenic TME, and undermined intratumoral vessel maturation by blocking VEGF and negatively regulating myeloid cell-dependent tumor angiogenesis. In summary, our preclinical data indicate that NB203 exhibits effective anti-tumor activity by mediating NK and cytotoxic T cell activities, improving the immune-suppressive TME, and suppressing tumor angiogenesis. The use of NB203 holds promise in overcoming resistance to anti-VEGF therapy and substantially improving the safety of therapeutic targeting of angiogenesis in cancer treatment. Citation Format: Jinyu Dong, Binbin Wang, Dong Wang, Jie Ni, Tingting Bu, Haojie Wang, Bishnu Nayak, Baiyang Wang, Xin Dong, Yu Zhang. NB203: A Novel therapeutic bispecific antibody targeting CEACAM1/VEGF for tumor immunity and angiogenesis modulation in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2732.

Development of a platform method for rapid detection and characterization of domain-specific post-translational modifications in bispecific antibodies

Charge heterogeneity is inherent to all therapeutic antibodies and arises from post-translational modifications (PTMs) and/or protein degradation events that may occur during manufacturing. Among therapeutic antibodies, the bispecific antibody (bsAb) containing two unique Fab arms directed against two different targets presents an additional layer of complexity to the charge profile. In the context of a bsAb, a single domain-specific PTM within one of the Fab domains may be sufficient to compromise target binding and could potentially impact the stability, safety, potency, and efficacy of the drug product. Therefore, characterization and routine monitoring of domain-specific modifications is critical to ensure the quality of therapeutic bispecific antibody products. We developed a Digestion-assisted imaged Capillary isoElectric focusing (DiCE) method to detect and quantitate domain-specific charge variants of therapeutic bispecific antibodies (bsAbs). The method involves enzymatic digestion using immunoglobulin G (IgG)-degrading enzyme of S. pyogenes (IdeS) to generate F(ab)2 and Fc fragments, followed by imaged capillary isoelectric focusing (icIEF) under reduced, denaturing conditions to separate the light chains (LCs) from the Fd domains. Our results suggest that DiCE is a highly sensitive method that is capable of quantitating domain-specific PTMs of a bsAb. In one case study, DiCE was used to quantitate unprocessed C-terminal lysine and site-specific glycation of Lys98 in the complementarity-determining region (CDR) of a bsAb that could not be accurately quantitated using conventional, platform-based charge variant analysis, such as intact icIEF. Quantitation of these PTMs by DiCE was comparable to results from peptide mapping, demonstrating that DiCE is a valuable orthogonal method for ensuring product quality. This method may also have potential applications for characterizing fusion proteins, antibody-drug conjugates, and co-formulated antibody cocktails.

A human lymphoma organoid model for evaluating and targeting the follicular lymphoma tumor immune microenvironment

Heterogeneity in the tumor microenvironment (TME) of follicular lymphomas (FLs) can affect clinical outcomes. Current immunotherapeutic strategies, including antibody- and cell-based therapies, variably overcome pro-tumorigenic mechanisms for sustained disease control. Modeling the intact FL TME, with its native, syngeneic tumor-infiltrating leukocytes, is a major challenge. Here, we describe an organoid culture method for cultivating patient-derived lymphoma organoids (PDLOs), which include cells from the native FL TME. We define the robustness of this method by successfully culturing cryopreserved FL specimens from diverse patients and demonstrate the stability of TME cellular composition, tumor somatic mutations, gene expression profiles, and B/T cell receptor dynamics over 3weeks. PDLOs treated with CD3:CD19 and CD3:CD20 therapeutic bispecific antibodies showed B cell killing and Tcell activation. This stable system offers a robust platform for advancing precision medicine efforts in FL through patient-specific modeling, high-throughput screening, TME signature identification, and treatment response evaluation.

Structure-function relationship study for sulfated protein therapeutics using hydrophobic interaction chromatography and mass spectrometry

Protein tyrosine sulfation is a post-translational modification (PTM) that is rarely reported in recombinant therapeutic proteins. However, when sulfation does occur, the additional negative charge from the modification can influence intermolecular interactions and antigen-binding activity, making it a critical quality attribute that necessitates stringent control. In this study, we developed a unique hydrophobic interaction chromatography (HIC) method for the separation and quantification of a therapeutic bispecific antibody with varying degrees of sulfation. Despite the increased surface hydrophilicity of sulfated species, the HIC method provides enhanced retention. Baseline resolution was attained based on the degree of sulfation, independent of other PTMs such as C-terminal amidation and forced deamidation. Further structure-function relationship studies of enriched sulfated bispecific antibody species were conducted using mass spectrometry and fluorescence-linked immunosorbent assay (FLISA). These studies revealed that the tyrosine sulfation modification, which occurs in the complementarity-determining region (CDR), is a critical quality attribute and can adversely impact the antibody’s binding to its cognate antigen. The evaluation of sulfation assay using HIC method confirmed it is an effective means for controlling this critical quality attribute.

NXT007-mediated hemostatic potential is suppressed by activated protein C-catalyzed inactivation of activated factor V

BackgroundActivated protein C (APC) inactivates activated factor (F) V (FVa) and FVIIIa. NXT007, an emicizumab-based engineered therapeutic bispecific antibody, enhances the coagulation potential of FVIII-deficient plasma (FVIIIdef-plasma) to near normal levels. However, little is known about the effect of APC-induced inactivation in NXT007-mediated hemostatic function. ObjectivesTo investigate the contribution of APC-mediated reactions to NXT007-driven hemostasis. MethodsIn pooled normal plasma (PNP) or FVIIIdef-plasma spiked with NXT007 (10 μg/mL), effects of APC (0-16 nM) were measured using a thrombin generation assay (TGA). The direct effects of APC on cofactor activity of NXT007 or FVIIIa in a FXa generation assay were also measured. The FVdef-plasma and FV Leiden plasma (FVLeiden plasma) were preincubated with 2 anti-FVIII monoclonal antibodies (termed FVIII-depleted), and the APC effect in the presence of NXT007 in FVIII-depleted FVdef-plasma with the addition of exogenous FV (7.5-30 nM) or FVIII-depleted FVLeiden plasma was investigated. ResultsThe APC dose-dependent suppression effect in TGA of FVIIIdef-plasma spiked with NXT007 was similar to that of PNP. FXa generation with NXT007 was not impaired by the addition of APC, suggesting that the APC-induced reaction in TGA with NXT007 was attributed to the direct inactivation of FVa. The addition of APC to FVIII-depleted FVdef-plasma, along with NXT007 and various FV concentrations, showed a similar attenuation to PNP. The NXT007-driven thrombin generation in FVIII-depleted FVLeiden plasma was suppressed by APC, similar to the reaction in native FVLeiden plasma. ConclusionNXT007 did not impair APC-mediated downregulation of FVa in FVIIIdef-plasmas, regardless of the presence of FV mutation with APC resistance.

Cancer therapeutic trispecific antibodies recruiting both T and natural killer cells to cancer cells.

T cells and natural killer (NK) cells are major effector cells recruited by cancer therapeutic bispecific antibodies; however, differences in the populations of these cells in individual tumors limit the general use of these antibodies. In the present study, trispecific antibodies were created, namely T cell and NK cell engagers (TaKEs), that recruit both T cells and NK cells. Notably, three Fc‑fused TaKEs were designed, TaKE1‑Fc, TaKE2‑Fc and TaKE3‑Fc, using variable fragments targeting the epidermal growth factor receptor on tumor cells, CD3 on T cells, and CD16 on NK cells. Among them, TaKE1‑Fc was predicted to form a circular tetrabody‑like configuration and exhibited the highest production and greatest cancer growth inhibitory effects. TaKE1 was prepared from TaKE1‑Fc by digesting the Fc region for further functional evaluation. The resulting TaKE1 exhibited trispecificity via its ability to bind cancer cells, T cells and NK cells, as well as comparable or greater cancer growth inhibitory effects to those of two bispecific antibodies that recruit T cells and NK cells, respectively. A functional trispecific antibody with the potential to exert strong therapeutic effects independent of Tcell and NK cell populations was developed.

Clinical Application of Therapeutic Bispecific Antibodies

Clinical Application of Therapeutic Bispecific Antibodies

Abstract 2965: LBL-033, a novel bispecific antibody targeting MUC16 and CD3, for the treatment of tumors over-expressing MUC16

Abstract Background: MUC16 is a large glycoprotein overexpressed on cell membrane of solid tumors, especially in almost 80% of ovarian cancer. The extracellular part of MUC16 could be cleaved as CA125, which is well known as a diagnosis biomarker for ovarian cancer. Despite recent advances in therapies such as PARP inhibitors, advanced ovarian cancer remains a disease of high unmet need. Herein a novel bispecific antibody targeting MUC16 and CD3, LBL-033, is developed to specifically kill MUC16 positive tumor cells by engaging T cells. Methods: LBL-033 was constructed using our unique T cell engager platform, which is a 2:1 format bispecific antibody with two arms targeting MUC16 membrane-proximal domain with high affinity and one arm targeting CD3 with fine-tuned affinity. The binding affinity of LBL-033 to MUC16 and CD3 was determined with Fortebio, while the activity was measured using several cell based assays including reporter gene and TDCC assays. The anti-tumor activity of LBL-033 was investigated in C57BL/6-huCD3e mice implanted with MC38-huMUC16 cells. GLP-compliant repeat-dose toxicologic study was evaluated in cynomolgus monkeys. Results: The binding affinity of LBL-033 to MUC16 and CD3 protein was 2.67 nM and 29.6 nM, respectively. The epitope that anti-MUC16 binds was identified at the C-terminal of MUC16 extracellular domain; and the activity was not affected in the presence of soluble CA-125. In CD3 reporter gene assay, LBL-033 could activate the NFAT reporter element through MUC16 antigen-dependent CD3 cross-linking with an EC50 value of 1.068 nM. LBL-033 was shown a potent TDCC activity in OVCAR3 cells with T cell activation. In C57BL/6-huCD3e transgenic mice implanted with MC38-huMUC16 tumor cells, LBL-033 was shown a dose dependent anti-tumor activity. In addition, LBL-033 had a significant synergistic effect, when combined with anti-PD-1. LBL-033 was shown favorable pharmacokinetic properties with a good safety profile in cynomolgus monkeys. Conclusion: LBL-033, a novel bispecific antibody targeting CD3 and MUC16 with a unique epitope, which is located at the MUC16 membrane-proximal domain, with affinity differentiation between anti-MUC16 and anti-CD3. It is shown a great anti-tumor efficacy in animal models, with a good safety profile in monkeys. These data support LBL-033 as a novel therapeutic bispecific antibody for ovarian cancer and other MUC16 positive tumors. A dose escalation study in humans will begin in Q1 2023. Citation Format: Jianming Sun, Xiao Huang, Yurong Qin, XingXing Fang, Min Chen, Huan Lin, Jing Guan, Jing Huang, Tongjun Liu, Baohui Wang, Guojin Wu, Shoupeng Lai, Xiaoqiang Kang, Hong Ling. LBL-033, a novel bispecific antibody targeting MUC16 and CD3, for the treatment of tumors over-expressing MUC16 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2965.

Comprehensive Multidimensional Liquid Chromatography-Mass Spectrometry for the Characterization of Charge Variants of a Bispecific Antibody.

Identification and further characterization of antibody charge variants is a crucial step during biopharmaceutical drug development, particularly with regard to the increasing complexity of novel antibody formats. As a standard analytical approach, manual offline fractionation of charge variants by cation-exchange chromatography followed by comprehensive analytical testing is applied. These conventional workflows are time-consuming and labor-intensive and overall reach their limits in terms of chromatographic separation of enhanced structural heterogeneities raised from new antibody formats. For these reasons, we aimed to develop an alternative online characterization strategy for charge variant characterization of a therapeutic bispecific antibody by online mD-LC-MS at middle-up (2D-LC-MS) and bottom-up (4D-LC-MS) level. Using the implemented online mD-LC-MS approach, all medium- and even low-abundant product variants previously identified by offline fraction experiments and liquid chromatography mass spectrometry could be monitored. The herein reported automated online mD-LC-MS methodology therefore represents a complementary and in part alternative approach for analytical method validation including multiattribute monitoring (MAM) strategies by mass spectrometry, offering various benefits including increased throughput and reduced sample handling and combined protein information at intact protein and peptide level.

Activated Protein C-Catalyzed Inactivation Pathway Down-Regulates NXT007-Mediated Hemostatic Function

Activated protein C (APC) together with protein S (PS) inactivates factor (F)Va and FVIIIa and efficient FVIIIa inactivation requires not only PS but also cofactor function of FV. Any impairment in these inactivation pathways is one of the most common risks of venous thrombosis. NXT007, emicizumab (Emi)-based engineered therapeutic bispecific antibody (Ab), is currently undergoing the phase 1 clinical trial. NXT007 increased coagulation potential of FVIII-deficient plasma up to the similar level to healthy controls in tissue factor (TF)-triggered thrombin generation (TG; Yamaguchi, ASH 2020). Additionally, concomitant use of NXT007 and bypassing agents could be useful for improving coagulation potentials in FVIII-deficient samples (Ogiwara, ASH 2021). However, the role of APC-dependent inactivation in NXT007-mediated hemostasis reactions remains to be investigated. Here, we examined the regulatory effects by APC in NXT007-driven hemostasis using a purified FXa generation assay and TF/ellagic acid-triggered thrombin generation assay (TGA). We firstly investigated coagulation potentials in FVIII-deficient plasma (ΔFVIII) spiked with NXT007 (3-100 μg/mL) or Emi (50 μg/mL). Peak thrombin (PeakTh) in ΔFVIII spiked with NXT007 was increased in a NXT007 dose-dependent manner. ΔFVIII spiked with NXT007 (3 μg/mL) showed higher PeakTh (355±74 nM) than emicizumab (50 μg/mL; 274±48 nM). PeakTh in ΔFVIII after the addition of NXT007 (10, 30, and 100 μg/mL) were 419±12, 454±13, and 484±10 nM, respectively. The coagulant effects of NXT007 (30 and 100 μg/mL) were comparable to pooled normal plasma (PNP). To evaluate the impact of APC-mediated down regulation on the hemostatic enhancement by NXT007 in ΔFVIII, TGA in ΔFVIII reacted with NXT007 (10 μg/mL) in the presence of APC (0-16 nM) was examined. PeakTh was decreased in an APC-dose dependent manner. Comparing with PeakTh without and with APC (16 nM), PeakTh was reduced from 457±11 nM to 331±1.4 nM and the %inhibition was 28%. We next evaluated the effects of APC on TG in PNP with or without an anti-FVIII polyclonal Ab (FVIIIAb) (10 BU/mL). The %inhibition of PeakTh in PNP with APC (16 nM) was 30%, indicating that the effect of APC-mediated inactivation was not disturbed by the presence of NXT007. To clarify the association of NXT007 alone with APC-catalyzed inactivation, we examined a purified FXa generation with NXT007 (10 μg/mL) or with FVIIIa (2 nM) after the addition of APC (0-2 nM) and PS (5 nM). FXa generation with FVIIIa was reduced by ~60% at maximum level in an APC dose-dependent manner, whereas that with NXT007 was not impaired, indicating that NXT007 itself was not affected by APC-catalyzed inactivation. To elucidate the contribution of APC-catalyzed inactivation of FV(a) in NXT007-catalyzed hemostatic regulation, we evaluated the effect of APC on the TG with NXT007 (10 μg/mL) and FV (7.5-30 nM; corresponded to 25-100% of physiological concentration) in the FV-deficient plasma with FVIIIAb (10 BU/mL). The PeakTh in the presence of NXT007 and FV (7.5-30 nM) without APC was 310-340 nM and the %inhibition of PeakTh by the addition of APC (16 nM) was ~35%, showing that NXT007 showed sufficient coagulation potential even at low concentration of FV and that the inhibition of TG by APC-catalyzed FV(a) inactivation was not disturbed by the presence of NXT007 even at FV (7.5 nM). In addition, the influence on TG with APC in the FV-Leiden (FV-L) plasma without or with FVIIIAb and NXT007 (10 μg/mL) were investigated. The %inhibition of PeakTh by APC (16 nM) in FV-L plasma was ~20%. While, the %inhibition of PeakTh by APC (16 nM) in FV-L plasma with FVIIIAb and NXT007 was similar to FV-L plasma alone (~20%), indicating that APC could regulate NXT007-catalyzed hemostatic reaction by inactivation of FV(a) under the FV-L condition. We conclude that APC-catalyzed inactivation mechanisms could play a significant role on the down-regulation of coagulation in NXT007-mediated hemostasis.

Abstract 1645: Advancing the development of CD16A-based bispecific antibodies in humanized CD16A mouse models

Abstract Antibody-dependent cell cytotoxicity (ADCC) is an important mechanism in immunotherapy. CD16A (FcγRIIIA), a low-affinity receptor for the IgG Fc domain, facilitates ADCC and is responsible for triggering the lysis of target cells by natural killer (NK) cells. Human CD16A is closely related to mouse Fcgr4 and is expressed in NK cells and macrophages, but is absent in granulocytes. Alternatively, mouse Fcgr4 is expressed in granulocytes and macrophages and is absent in NK cells. CD16A-based bispecific antibodies targeting NK cells have been studied for cancer therapy. To evaluate the anti-tumor efficacy of CD16A-based bispecific antibody in mouse tumor models, we have successfully generated a novel humanized CD16A knock-in mouse strain (B-hCD16A Mice). Considering the differential expression patterns between human CD16A and mouse Fcgr4, the regulatory region and whole genome sequence of mouse Fcgr4 were replaced by the human CD16A gene. Human CD16A and mouse Fcgr4 protein expression was confirmed in heterozygous B-hCD16A mice by FACS and RT-PCR. Human CD16A protein was detected in NK cells and macrophages of B-hCD16A mice, but not granulocytes. When compared with wild type mice, there were no abnormalities in the populations of immune cells in B-hCD16A mice. CD16A-based bispecific antibody usually binds to CD16A on NK cells, whereas the other arm of the antibody binds to tumor antigen on cancer cells. Initial assessment of the tumorigenicity of MC38 cells were evaluated in B-hCD16A mice, and results indicated that these cancer cells were able to establish tumors in vivo. These data suggest that the B-hCD16A mouse model is an effective tool for future in vivo efficacy evaluation of therapeutic CD16A-based bispecific antibodies prior to clinical development. Citation Format: Jiahui Chang, Tao Li, Limei Ma, Xiaoxia Sun, Yuelei Shen. Advancing the development of CD16A-based bispecific antibodies in humanized CD16A mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1645.

Autophagy and intracellular product degradation genes identified by systems biology analysis reduce aggregation of bispecific antibody in CHO cells

Aggregation of therapeutic bispecific antibodies negatively affects the yield, shelf-life, efficacy and safety of these products. Pairs of stable Chinese hamster ovary (CHO) cell lines produced two difficult-to-express bispecific antibodies with different levels of aggregated product (10–75% aggregate) in a miniaturised bioreactor system. Here, transcriptome analysis was used to interpret the biological causes for the aggregation and to identify strategies to improve product yield and quality. Differential expression- and gene set analysis revealed upregulated proteasomal degradation, unfolded protein response and autophagy processes to be correlated with reduced protein aggregation. Fourteen candidate genes with the potential to reduce aggregation were co-expressed in the stable clones for validation. Of these, HSP90B1, DDIT3, AKT1S1, and ATG16L1, were found to significantly lower aggregation in the stable producers and two (HSP90B1 and DNAJC3) increased titres of the anti-HER2 monoclonal antibody trastuzumab by 50% during transient expression. It is suggested that this approach could be of general use for defining aggregation bottlenecks in CHO cells.

Effects of Bypassing Hemostatic Agents in the Co-Presence of NXT007, Emicizumab-Based Engineered Bispecific Antibody to Fixa/Fx, Using in Vitro Global Assays: Rough Indicators for Their Concomitant Use

Background:NXT007, emicizumab (Emi)-based engineered therapeutic bispecific antibody which increases tissue factor (TF)-triggered thrombin generation (TG) potential of factor(F) VIII-deficient plasma to non-hemophiliac ranges at around 5-30 μg/mL (Yamaguchi, ASH 2020), is currently in a phase 1/2 clinical study. In the clinical settings under NXT007-prophylaxis, bypassing hemostatic agents (BPAs), such as activated prothrombin complex concentrates (aPCC) and recombinant(r) FVIIa, may be concomitantly administered. Under Emi-prophylaxis, repeated doses of aPCC impose a thrombotic risk. Against the risk, NHF's Medical and Scientific Advisory Council (MASAC) recommends ≤50 U/kg and ≤100 U/kg of aPCC as initial dose and one day dosage, respectively. In case of NXT007-prophylaxis, concomitant-use with BPAs should be also carefully managed and thus basic non-clinical combination data are needed.Objectives:To examine in vitro effects of BPAs in the co-presence of NXT007 for providing rough indicators for determining their safe doses.Methods:First, TF-triggered TG assay was performed using commercial FVIII-deficient plasma. rFVIII, rFVIIa or aPCC was spiked in the co-presence of NXT007 or Emi (0.1-50 μg/mL). Second, the TG assay and whole blood clot viscosity test (ROTEM) using Ca 2+-trigger was performed using healthy volunteer's blood incubated with anti-FVIII antibodies (HA model), where aPCC or rFVIIa was spiked in the co-presence of NXT007. Third, ROTEM was performed as above using whole blood from persons with hemophilia A (PwHA) under Emi- or FVIII-prophylaxis.Results:Peak height of TF-triggered TG assay using FVIII-deficient plasma was increased by spiking each of the agents (rFVIII, rFVIIa or aPCC) in the co-presence of NXT007 (0.1-50 μg/mL). Peak height increase by rFVIII under NXT007 was roughly additive. Peak height increase by each BPA under NXT007 was synergistic. Synergistic effect by aPCC was more intensive than that by rFVIIa. The combination effect of 0.1 U/mL aPCC and 0.1-50 μg/mL NXT007 on peak height did not exceed that of 0.5 U/mL aPCC and 50 μg/mL Emi. Peak height at 2 μg/mL NXT007 alone was comparable to that at 50 μg/mL Emi alone. When adding BPAs to these two settings, similar synergistic effects were observed. It suggested that NXT007's combination actions with BPAs were qualitatively similar to Emi's.In the TG assay using the HA model plasma (n=2), the combined effect of aPCC and NXT007 was similarly confirmed. In ROTEM using the same HA model whole blood (n=2), clotting time (ROTEM-CT, 7380±605 sec) was shortened by spiking 5 μg/mL NXT007 (791±148 sec) to yield normal levels (1521±110 sec). Spiked aPCC (0.13, 0.65 U/mL in whole blood, equivalent to ~10, ~50 U/kg infusion) shortened ROTEM-CT (1228±549, 435±54 sec) and further shortened it in the co-presence of 5 or 15 μg/mL NXT007 (171±11, 113±1* or 143±4, 90±5* sec [* significantly ( p<0.05) shorter than that of the Emi-treated PwHA blood spiking 0.65 U/mL aPCC, 162±21 sec)].In PwHA without Emi-prophylaxis (n=2), ROTEM-CT (5148±1290 sec) was shortened by spiking 5 or 15 μg/mL NXT007 (1598±482 or 1116±14 sec) to non-hemophiliac levels (20 IU/dL rFVIII, 1569±222 sec). Spiked aPCC (0.13, 0.65 U/mL) shortened ROTEM-CT (1383±322, 680±84 sec) and further shortened it in the co-presence of 5 or 15 μg/mL NXT007 (230±35, 144±4 or 193±11, 121±0* sec). Spiked rFVIIa also shortened ROTEM-CT in the co-presence of NXT007, but the intensity was less than spiked aPCC.ROTEM using blood from PwHA under Emi-prophylaxis (n=3) were also performed and demonstrated that the effects by co-spiking BPAs and NXT007 were roughly consistent with those using PwHA blood without Emi-prophylaxis. These ROTEM data indicated that the combined effect of 0.13 U/mL aPCC and 5-15 μg/mL NXT007 was less intensive than that of 0.65 U/mL aPCC spiked to the Emi-treated PwHA blood, while that of 0.65 U/mL aPCC and 5-15 μg/mL NXT007 had more intensive effect (Table).Conclusion:In considering concomitant use of BPA under NXT007-prophylaxis, dose of aPCC should be more carefully determined than that of rFVIIa. In this non-clinical study, the combined effect of ~0.13 U/mL aPCC (equivalent to ~10 U/kg infusion) and ~15 μg/mL NXT007 did not exceed that of 0.65 U/mL aPCC (50 U/kg infusion) under Emi-prophylaxis situation corresponding to the upper limit of initial concomitant dose recommended by MASAC, which might be rough indicators in future clinical settings. [Display omitted] DisclosuresOgiwara: Chugai Pharmaceutical Co., Ltd.: Consultancy, Research Funding. Furukawa: Chugai Pharmaceutical Co., Ltd.: Research Funding. Sasai: Chugai Pharmaceutical Co., Ltd.: Research Funding. Inaba: Chugai Pharmaceutical Co., Ltd.: Current Employment. Kitazawa: Chugai Pharmaceutical Co., Ltd.: Current Employment, Current equity holder in publicly-traded company. Nogami: Chugai Pharmaceutical Co., Ltd.: Consultancy, Research Funding.

Increasing of Ternary Complex Formation By Predominant Accumulation of Factor (F)X Accompanied By Increase of Fixa Contributes to Enhancement of Clotting Function By Apcc in Combination with Emicizumab

Background: Emicizumab is a novel therapeutic bispecific antibody for treatment of patients with hemophilia A (PWHA) irrespective of FVIII inhibitors. In the phase 3 clinical study of emicizumab focusing on PWHA with inhibitor (-inh) over 12 years old (NEJM2017), several thromboembolic events were reported among participants after multiple infusions of activated prothrombin complex concentrate (APCC) for breakthrough bleeding. We recently reported that the global hemostatic function evaluated by ROTEM was additively enhanced by infusion of APCC under treatment of emicizumab in the Japanese phase 1/2 study (ISTH2017). There exist three forms of emicizumab in the circulating plasma such as i) monomer, ii) binary complex with either FIX(a) or FX, and iii) ternary complex with FIX(a) and FX. According to in vitro experiments, we recently demonstrated that the percent of the ternary complex would be predicted by KD -based simulation based on concentration of emicizumab, FIX(a) and FX (Thromb Haemost. 2017). The in vivo pharmacodynamics of FIX(a) and FX after infusion of APCC in the presence of emicizumab are to be elucidated for confirmation of safety.Aim: To clarify the relationship between pharmacodynamics of FIX(a) and FX after infusion(s) of APCC and hemostatic potential in the presence of emicizumab.Methods: We first examined the hemostatic function by addition of emicizumab (0, 10, 50, 100μg/ml) in the citrated blood samples obtained from seven PWHAs-inh (who do not participate in the clinical study) received APCC (80-100U/kg) by thrombin generation assay (TGA) using tissue factor (0.5pM) and ellagic acid (0.3µM) as trigger reagents. The plasma samples were classified by APCC-dosing at collection as summarized in Table. Class A was defined as single or prophylactic administration while class B was defined as treatment with multiple administrations. For patient 7, intra-individual evaluation was examined before (as class A) and after initiation of treatment by APCC (as class B). We further confirmed the in vivo additional effect by APCC to emicizumab in the samples obtained from one PWHA-inh (patient 8) enrolled in the Japanese phase 1/2 study. This patient received four multiple infusions of APCC (~50U/kg, every 12hr) for a major bleeding.Results: Spike of emicizumab in vitro exhibited a dose-dependent modest increase of PeakTh maximally by ~1.6-fold of that in the absence of emicizumab (212 ± 22.8 nM) in class A. On the other hand in class B, PeakTh augmented dose-dependently on emicizumab (max. to 1,538 ± 38 nM by ~12.5-fold of that in its absence) far beyond normal control (382 nM). As for the samples from patient 8, PeakTh increased from 80.6 nM to 240 nM after 4th infusion of APCC. This increase of PeakTh was less than that in class B (from 122 ± 9.73 nM to 793 ± 29.3 nM) after spike of emicizumab (10µg/ml) which is equivalent to the target concentration in patient 8. To identify the determinant of these different hemostatic enhancing effects, antigen (Ag) levels of FX and FIX(a) in the plasmas were measured by ELISAs. The median values of FX:Ag and FIX(a):Ag were 424nM (~3.1-fold of normal control) and 124nM (~1.4-fold of normal) in class A, and those in class B were 2,000nM (~15-fold of normal) and 278nM (~3-fold of normal), respectively. In the samples of patient 8 from the Japanese phase 1/2 study, FX:Ag increased up to 3.7-fold of normal after 1st administration of APCC and further increased to 7-fold of normal after 4th administration, while FIX(a):Ag was little affected by administrations of APCC different from class B, which would be attributed to that the lower dose of APCC were infused for patient 8 than that for patients in class B. Taken together, dose and/or frequency of APCC infusion(s) seemed to affect the pharmacodynamics of FIX(a) and FX in the plasma. The elevation of both of them, especially FX, resulting in the increase of ternary complex in the presence of emicizumab, contribute to hemostatic enhancement.Conclusion: In conclusion, high-dose and frequent infusions of APCC contribute to the predominant accumulation of FX accompanied by the increase of FIX(a) in the plasma enhancing the hemostatic effect in the presence of emicizumab, in addition to the bypassing reactions. [Display omitted] DisclosuresYada:Chugai Pharmaceutical Co.: Research Funding. Nogami:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Chugai Pharmaceutical Co.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Matsumoto:Chugai Pharmaceutical Co.: Research Funding. Kitazawa:Chugai Pharmaceutical Co.: Employment, Equity Ownership, Patents & Royalties. Shima:Chugai Pharmaceutical Co.: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Generation of a safe and efficacious llama single-domain antibody fragment (vHH) targeting the membrane-proximal region of 4-1BB for engineering therapeutic bispecific antibodies for cancer

BackgroundThe discovery of checkpoint inhibitors towards cytotoxic T-lymphocyte protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) has been revolutionary for the treatment of cancers. These therapies have only...

Characterization and Monitoring of a Novel Light-heavy-light Chain Mispair in a Therapeutic Bispecific Antibody.

Site-specific cysteine engineering, along with other genetic mutations, is broadly implemented in bispecific antibodies (bsAb). Thus far, homodimer, half hole antibody, one-light chain mispaired and light chain swapped variants have been reported as chain-pairing variants for the asymmetric IgG-like bispecific antibodies. Here we report a novel mispair in which the CH3 engineered cysteine on the hole heavy chain (HC) of a knob-into-hole (KiH) bsAb is linked to the engineered cysteine in CL through a disulfide bond, forming a LHL species in a bsAb construct. Due to its impact on bioactivity, it is critical to implement an analytical strategy to monitor this CQA and mitigate risk for the future products. A set of orthogonal physicochemical assays that include hydrophobic interaction chromatography (HIC), capillary electrophoresis sodium dodecyl sulfate (CE-SDS), reverse phase liquid chromatography ultra-performance chromatography mass spectrometry (RP-UPLC MS) and disulfide bond mapping have been utilized to monitor and characterize this chain-pairing impurity for manufacturing process control and product release. Our data shows the LHL mispair in condition medium (CM) is approximately 1.3 - 1.9%. LambdaFabSelect affinity chromatography removes two major chain-pairing variants in CM - i.e. the hole-hole homodimer and hole half-antibody, while retaining the LHL species. Process improvement in Capto Q (anion exchange) and HS50 (cation exchange) chromatography steps removes LHL to as low as 0.2% in the final product. We have demonstrated an orthogonal analytical methodology that is capable of characterizing and monitoring bsAb mispairing, suitable for use in manufacturing process control and product release, and can be potentially implemented for similar bsAb constructs with engineered disulfide bonds.

Augmented antibody-based anticancer therapeutics boost neutrophil cytotoxicity.

Most clinically used anticancer mAbs are of the IgG isotype, which can eliminate tumor cells through NK cell-mediated antibody-dependent cellular cytotoxicity and macrophage-mediated antibody-dependent phagocytosis. IgG, however, ineffectively recruits neutrophils as effector cells. IgA mAbs induce migration and activation of neutrophils through the IgA Fc receptor (FcαRI) but are unable to activate NK cells and have poorer half-life. Here, we combined the agonistic activity of IgG mAbs and FcαRI targeting in a therapeutic bispecific antibody format. The resulting TrisomAb molecules recruited NK cells, macrophages, and neutrophils as effector cells for eradication of tumor cells in vitro and in vivo. Moreover, TrisomAb had long in vivo half-life and strongly decreased B16F10gp75 tumor outgrowth in mice. Importantly, neutrophils of colorectal cancer patients effectively eliminated tumor cells in the presence of anti-EGFR TrisomAb but were less efficient in mediating killing in the presence of IgG anti-EGFR mAb (cetuximab). The clinical application of TrisomAb may provide potential alternatives for cancer patients who do not benefit from current IgG mAb therapy.