Development of bispecific antibodies in China: overview and prospects
Development of bispecific antibodies in China: overview and prospects
Charge Repulsion Improved Bispecific (CRIB™) platform was developed by Alphamab Co., Ltd. (Alphamab) to manipulate the charge network among various Fc bonds (
Bispecific antibody patents from Chinese applications granted by the United States Patent and Trademark Office [
EXPRESSION, PRODUCTION, AND BIOASSAY OF BSABS
The production of an IgG-like bsAb with physical and chemical properties similar to some those of an IgG can be generated following typical bioprocesses for producing a typical monoclonal antibody. However, unlike natural mAbs, technical challenges with respect to quantity, quality, and stability of bispecific antibodies have hampered the wider clinical application and acceptance of these antibodies [
BsAbs under clinical development in China by Chinese companies as of May 2020 [
Structures of bsAbs approved for clinical trials by NMPA
The structures of 17 bsAbs among 21 projects in the clinical stage in China had been disclosed, and these can be categorized as asymmetric IgG-like bsAbs (7 of 17, 41.2%), symmetric IgG-like bsAbs (6 of 17, 35.3%), bispecific fragments (1 of 17, 5.9%), and antibody-receptor fusion bsAbs (3 of 17, 17.6%) (
MOAs of 18 bsAbs in Chinese clinical trials. (A) Among the 18 bsAbs, five of which are by T-cell redirection (5 of 18, 27.8%), six by dual-checkpoints blockade (6 of 18, 33.3%), two by dual signaling inhibitions (2 of 18, 11.1%), one by co-localized blocking (1 of 18, 5.6%), two by biparatopic bsAbs (2 of 18, 11.1%), and two by tumor-targeted immunomodulators (2 of 18, 11.1%). (B) T-cell redirection refers to bridge T cells and tumor cells by binding to both a TAA and IAA and redirecting the cytotoxic activity of effector T cells to attack specifically to the tumor cells. Represent projects in using the MOA include M802 (HER2 × CD3, by YZYBio), M701 (EPCAM×CD3, by YZYBio), A-319 (CD19 × CD3, by Generon), K193 (CD19 × CD3, by Lvzhu), and ES101 (PD-L1 × CD137, by Elpiscience). (C) Dual checkpoints blockade is by two-checkpoint blockers integrated into one antibody to inhibit two immune checkpoints simultaneously. AK104 (PD-1 × CTLA-4, by Akeso), KN046 (PD-L1 × CTLA-4, by Alphamab), IBI318 (PD-1 × PD-L1, by Innovent), HX009 (PD-1 × CD47, by HanxBio), IBI322 (PD-L1 × CD47, by Innovent), and MGD013 (PD-1 × LAG-3) are six of these examples. (D) Dual signaling inhibitions are to target two different receptors for preventing the receptors from phosphorylation and/or from the activation of both receptor-mediated signaling pathways to inhibit tumor proliferation. EMB-01 (EGFR × c-MET, by Epimab) and SI-B001 (HER3 × EGFR, by Biokin) are two of these typical bsAbs. (E) Co-localized blocking is by inhibiting two or more tumor cell intrinsic and extrinsic pathways to raise the possibility of superior antitumor activity compared with the monotherapy. SHR-1701 (PD-L1 × TGF-β, by Hengrui) is one of such bsAbs. (F) Biparatopic bsAbs are by binding to two different epitopes of the same antigen or same receptor to enhance the antigen–antibody affinity and to improve the drug efficacy. Both KN026 (HER2 × HER2, by Alphamab) and MBS301 (HER2 × HER2, by Mabworks) bind to the D2 and D4 subdomains of HER2. (G彩神网) Tumor-targeted immunomodulators are designed for binding to both one TAA (e.g. HER2, CD20) to inhibit TAA signaling pathway and one immunomodulating receptor (e.g. PD-1, CD47) to regulate the immune system to attack the tumors. IBI315 (HER2 × PD-1, by Innovent) and IMM0306 (CD20 × CD47, by ImmuneOnco) utilize such MOAs.
The bsAbs targeting both a T-cell associated antigen and a TAA can bridge T cells and tumor cells to achieve the concept that the redirecting the cytotoxic activity of the effector T cells to specifically eliminate tumor cells ( 彩神网 Identifier: NCT03631706) is currently ongoing, wherein M7824 has showed remarkably increased efficacy.
Expression and CMC
The advances in protein engineering and bioprocesses based on mammalian cells for over expression and cost-effective production of a typical bsAb have allowed many start-up companies in China to bypass the early developed quadroma technology [14,178,179] or even latterly improved chimeric quadroma technology [180,181], by which the Catumaxomab was first launched on and then off market due to the instable and low-yield production and immunogenicity in clinical consequences [182,183]. The engineered CHO cell systems are commercially available and often provide higher than 3–5 g/L of a typical IgG-like bsAb.
More efforts are also focused on searching new expression systems including co-culture methodology  and CrossMab technology [15,185] to further resolve incorrect assembly of bsAb fragments . Yet, it still remains a key goal of the field to establish idea expression system to achieve a stable bsAb production with high yield and low cost. Our opinions for how to produce a uniform bsAb with a high quality, and negligible by-products are highly dependent on the structure platform, which should be selected based on their biological and functional needs. The biology should drive the discovery of bsAbs as innovative therapeutics . Along with the discovery, multiple engineering technologies can be applied. For a full IgG-like bsAb with a symmetric or asymmetric structure, knobs-in-holes technology induced between Fc/Fc chains is an efficient way to mitigate the formations of two homodimers [29,46,186]. A common light chain [8,24,187] has been approved to be effective for eliminating the mismatch between HC/LC arms for a whole IgG type, and an scFv-IgG-like bsAb  also do so for an asymmetric structure, specifically when attenuating binding affinity to one of the antigen is required, e.g. to CD3 in T cell engaging bsAb for mitigating CRS (see Section CD3 bsAbs and CRS).
CMC technology developed with protein A-based purification for mAb production can be adapted in general for the production of IgG-like bsAb. The advent of biosimilar developments is a driving desire to achieve a lower cost of production and to globalize biologics manufacturing . High titers routinely achieved for IgG-like antibody in mammalian cell culture and high yield of an mAb production have resulted in significant evolution in process platform approaches, including the consideration of alternative expression systems [44,45], continuous biomanufacturing [188–190] and non-chromatographic separation formats [188,191]. The continuous bioprocess reduced hold steps, improved facility utilization, and reduced capital investment with less contamination risk, less deviation and high integrity. However, there are still several challenges to overcome, including high upfront investment, core technologies and experiences barrier in China, new control and validation strategies, and regulatory uncertainties . Meanwhile, CDMOs booming currently in China provide well-established technology platforms for full CMC services from DNA to bioprocess development, and to manufacturing production for national and international biologic companies. To choose CDMO for CMC development and manufacturing production can bypass upfront investments including building the team and facility, and speed up the project progress as well.
One of the challenges in developing bispecific antibodies is the immunogenicity caused by new epitopes in the artificial structures of the bsAbs. Although the specific epitope determination of the anti-drug antibodies (ADA) response is not frequently required by FDA, a more general assessment of domain specificity for a multiple-domain product, such as a bsAb, was more commonly performed . The domain specificity is generally started with ADA-positive samples confirmed using the whole molecule and further characterization may require multiple assays to measure immune responses to different domains of the molecule .
It was expected that a bsAb produced by a non-B cell might be more immunogenic than an mAb in general because its non-natural structures or intact antibody structures with additional domains could potentially provide novel epitopes leading to increased immunogenicity . The more engineering operations on a bsAb architecture, the higher risk of the molecule immunogenicity. However, this could not be verified by two marked bsAbs. Blinatumomab was constructed as a non-natural structure of scFvs from anti-CD19 and anti-CD3 sequences of variable regions of murine antibodies. However, the results from clinical trials showed that only a few cases of patients with anti-blinatumomab antibodies were detectable and the impact of the immunogenicity on its PK could not be concluded . The other marketed bsAb, Emicizumab, is an asymmetric IgG-like format with common light chains, which bridges FIXa and FX to restore the function of missing FVIIIa in people with hemophilia A. The bsAb was also shown with low immunogenicity (3.5% tested positive for ADA) in the phase III studies . Meanwhile，10 CD3 bsAbs, despite their different product attributes and structures binding to both human and monkey antigens, were shown to be ADA positive in cynomolgus monkeys for toxicology studies . Although ADA generation in the animals resulted in reduced systemic drug exposure or even loss of the exposure by the end of a one-month study, it did not interfere with overall interpretation of toxicology results in these short-term studies. There was sufficient duration of exposure (at least 2 weeks) or sufficient number of animals exposed (a subset of animals with longer than 2-week exposure) to allow for assessing the toxicities of the bsAbs. It was found that only one of the 10 bsAbs reported immunogenicity with nearly 60% of patients developing ADAs [195彩神网]. Therefore, the correlation of the immunogenicity found between animals and human patients was also not established.
It was generally accepted that higher immunogenicity comes from more artificial engineering operations: (1) a transformation of the basic domain, including Fc mutations (e.g. KiH, CRIB) and the modification of CH1 and CL (e.g. CrossMab); (2) a introduction of linkers for connecting different domains, such as a linker between VH and VL in scFv, a linker between two scFv in BiTE, etc.; (3) a deletion or addition of a sequences or a domain, which provide new surface exposed for an Ab to recognize; and (4) any change introducing aggregation of a bsAb or new post-translation modifications (e.g. O-glycosylation) on the molecule. Minimizing these structural modifications should effectively reduce the immunogenicity of the entire bsAb molecule along with humanization of the entire bsAb although the humanization of variable regions of an antibody does not always reduce the immunogenicity in humans . The specific strategies include as follows: (1) in domain modification, mutate residues facing to the interface of two domains or facing to the inside of the domain and keep the natural protein surface unchanged. The molecular structure after mutation can be predicted and confirmed by computer simulation (e.g. by discovery studio software). (2) When choosing a linker, it is recommended to use common flexible linkers, such as (GGGGS)n (n = 1 ~ 3), of which low immunogenicity have been verified in clinical trials of blinatumomab [193,197] (3). The structure of a new bsAb is currently difficult to simulate by computer modeling, and a compromise method is to compare the characteristics of the new bsAb with its parental antibodies by monitor its functional activities and stabilities. In theory, the closer in the structures of a bsAb to that of a natural mAb, the lower the risk in immunogenicity of the molecule.
How to predict the immunogenicity of a candidate molecule in earlier preclinical development is still a challenge. Three assays are currently used to assess immunogenicity: (1) T-cell epitope prediction in silico, which is a computer system from Epibase (Lonza). The protein sequence was truncated into short peptides with a length of 7–13 amino acids, which are then aligned in silico with the peptide sequences recognized by TCR in database. All the protein-derived peptides are scored according to their alignment degrees with the reference peptides to determine their immunogenicity risk . (2) In vitro assay to detect the immunogenicity of human-based mAb, which is a cell proliferation assay using flow cytometry to detect a slight increase in proliferating helper-T cells. PBMCs of multiple (20 or more) donors are treated with the candidate molecule, and a high- and a low-immunogenic antibody drugs. The degree of helper T-cell proliferation is analyzed to determine the positive threshold level. Based on the helper T-cell proliferation and threshold levels, the positive incidence of the candidate antibodies was obtained to assess their immunogenicity . (3) In vivo assay by ADA analysis in non-human primate (NHP): the immunogenicity of a bsAb is assessed through the proportion of ADA positive individuals after drug administration. Using this assay, the relative immunogenicity of 27 mAbs in NHPs and human were studied, and 59% (16) of 27 cases were detected with ADA formations, which were comparable between NHPs and human. However, NHPs and human had different types of ADAs in response to these 16 mAbs . Each of above three assays has limitations when used individually, and the integrated use of in silico and in vitro assays provides a predicted immunogenicity for selecting a candidate but not for making “go or not-go” decision during the development [200彩神网]. As ADAs could cause the failure of a bio-therapeutic development program, it is still highly required to improve and validate the evaluation assays to reduce the risk of the preclinical programs due to the immunogenicity.
Perspectives for industry of Chinese bsAbs
Prompted by the success of fast follow-up，license in/out，and collaboration of anti-PD-1/anti-PD-L1 and other IO therapeutic developments with matured pharmaceutical companies in developed countries, China is at a stage of rapid developments in biopharmaceutical innovations of therapeutic biologics, CAR-T, and regenerative medicine. BsAb is among the fastest growing class of investigational drugs. The M802 was reported with clinical potential and was approved as the first bsAb for clinical trials in China by the late 2017. Then in about two-and-half years, 21 bsAbs have been developed into clinical trials in China. Technology platforms with focus on bsAbs, such as FIT-Ig, ITab, CRIB, SMAB, Doubody, WuXiBody, and YBODY, all contribute to this new wave of innovations. Based upon the clinical results of bsAbs in China, we expect many more IND approvals from NMPA, quick indication expansion from cancers to other diseases, new combinations of bsAbs with other therapeutics or therapies, and further improved discovery platforms with defined intellectual property. Although no bsAb drugs developed in China have been approved on marketing, bsAb will maintain its rapid growth in China for the years to come.
The authors thank Dr. Shouye Wang for manuscript reviewing and Brian Chi for manuscript editing assistance.
ADA, anti-drug antibodies, ADC, antibody drug conjugation, ADCC, antibody-dependent cellular cytotoxicity, ADCP, antibody-dependent cellular phagocytosis, ALL, acute lymphoblastic leukemia, B7-H1, B7 homolog 1, BITE, bispecific T cell engager, BsAb, bispecific antibody, BpAb, biparatopic antibody, CAR-T, chimeric antigen receptor T-Cell immunotherapy, CCK-8, cell counting kit-8, CHO, Chinese hamster ovary cells, CLL-1, C-type lectin-like molecule-1, CMC, chemistry manufacturing and controls, c-MET, c-mesenchymal-epithelial transition factor, CPB, checkpoint blockade, CRIB, charge repulsion-improved bispecific, CRS, cytokine release syndrome, CTLA-4, cytotoxic T-lymphocyte-associated protein-4, CV, cell viability, Cα, constant region of α chain, Cβ, constant region of β chain, DO, dissolved oxygen, ECD, extracellular domain, EGFR, epidermal growth factor receptor, Fab, antigen-binding fragment, FAE, Fab-arm exchange, Fc, crystallizable fragment, FDA, the US Food and Drug Administration, FISH, fluorescence in situ hybridization, FIT-Ig, Fabs-in-tandem immunoglobulins, FIXa, factor IXa, FX, factor X, GPC3, glypican-3, GPCs, cell-surface glypicans, GS, glutamine synthetase, HC, heavy chain, HCCHN, squamous cell carcinoma of the head and neck, HER2, human epidermal growth factor receptor 2, HER3, human epidermal growth factor receptor 3, HGFR, hepatocyte growth factor receptor, HLE-BITE, half-life extended BITE, IAA, immune-associated antigen, IgG, immunoglobulin G, IND, investigational new drug, IO, immuno-oncology, ITab, immune-therapy antibody, KiH, knobs-into-holes, LC, light chain, LAG-3, lymphocyte activation gene-3, mAb, monoclonal antibody, MHC, major histocompatibility complex, MOA, mechanism of action, MSX, methionine sulfoximine, MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3 carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, MTT, 3-(4,5-dimethylazol-2-yl)-2,5-dipheny-tetrazoliumbromide, NFAT, nuclear factors of activated T-cells, NMPA, the China National Medical Products Administration, NSCLC, non-small-cell lung cancer, PD, pharmacodynamics, PD-1, programmed cell death protein 1, PD-L1, programmed death-ligand 1, PK, pharmacokinetics, RBC, red blood cell, scFv, single-chain variable fragment, SIRPα, signal regulatory protein α, TAA, tumor-associated antigen, TCR, T-cell receptor, TGFBRII, TGF beta receptor II, TGF-β, transforming growth factor β, TNF-α, T-cell-generated tumor necrosis factor-α, TKI, tyrosine kinase inhibitors, TME, tumor microenvironment, VCD, viable cell density, VHH, or single-domain antibody (SDA): antigen-binding immunoglobulin variable domain of “heavy-chain antibodies”, WST-1, 4-[3-(4-iodophenyl)-2(4-nitrophenyl)-2H-5-tetrazolio] -1,3benzene disulfonate, XTT, sodium,3-[1-[phenylamino-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate