Dual action Fab (DAF) is a dual-specific antibody capable of recognizing two different antigens simultaneously. DAF is a novel bispecific antibody format with higher antigen-binding capacity and functionality compared to conventional monospecific antibodies or antibody combinations. DAF is prepared by genetic engineering or protein engineering, and its structure consists of a single-chain variable fragment (scFv) and a Fab fragment, wherein the scFv and Fab fragments recognize different antigens respectively. The structural design of DAF enables it to simultaneously interact with two different cell surface molecules or two different ligands, thereby achieving dual specificity. DAF has multiple advantages, such as enhancing antigen binding affinity, expanding antigen coverage, and improving antibody function and safety. DAF can effectively block a variety of signaling pathways, induce cytotoxicity or apoptosis, and enhance immune effector functions. DAF can also avoid some common shortcomings of bispecific antibodies, such as interchain mismatches, instability, low expression, and high immunogenicity. DAF has broad application prospects in a variety of tumor treatment fields. At present, several DAFs have entered the clinical trial or marketing stage, mainly targeting EGFR, HER3, CD3, CD20 and other targets. These targets play an important role in various tumors, such as head and neck cancer, colorectal cancer, breast cancer, and lymphoma. DAF can effectively inhibit the growth and metastasis of tumor cells and improve the survival rate and quality of life of patients.
The structural composition of DAF consists of a single-chain variable fragment (scFv) and a Fab fragment, where the scFv and Fab fragments recognize different antigens, respectively. The scFv of DAF is connected to the Fab fragment through a flexible connecting peptide to form a dual-specific antibody molecule. The Fc part of DAF is the same as ordinary IgG, retaining the immune effector function. The antigen-binding site of DAF consists of variable domains (VH and VL) of scFv and Fab fragments, each of which contains three complementarity determinants (CDRs), namely CDR1, CDR2 and CDR3. CDR is the main part of antibody binding to antigen, which determines the specificity and affinity of antibody. DAF has four independent antigen-binding sites and is capable of binding to two different antigens simultaneously.
The dual specificity of DAF is based on the ability of scFv and Fab fragments to recognize different antigens. DAF can achieve dual specificity in two ways: one is to interact with two different cell surface molecules at the same time, thereby achieving the blocking or activation of intercellular or intracellular signaling pathways; the other is to simultaneously interact with two different Ligands interact to achieve competition or cooperation between ligands or between ligands and receptors. For example, a DAF antibody targeting EGFR and HER3 can exert dual-specific effects in the following ways: 1) simultaneously bind to two EGFRs, blocking EGFR homodimerization and signal transduction; 2) simultaneously Bind to two HER3s, block homologous or heterologous dimerization and signal transduction of HER3; 3) bind to one EGFR and one HER3 at the same time, block heterologous dimerization and signal transduction between EGFR and HER3. Since EGFR and HER3 often co-exist on the same cell, DAF antibodies can take advantage of the high affinity generated by their bivalence, which is not affected by the expression level of each target. Another example is a DAF antibody against CD3 and CD20, which can exert dual specific effects in the following ways: 1) Simultaneously bind to CD3 on T cells and CD20 on B cells, inducing T cells to B cells 2) Simultaneously bind to CD3 and hemolysin on T cells, activate T cells and enhance their proliferation ability; 3) Simultaneously bind to CD20 and hemolysin on B cells, induce apoptosis or lysis of B cells. Since CD3 and CD20 have different expression levels in different types of lymphomas, DAF antibodies can choose the most effective binding method according to different situations.
The preparation methods of DAF mainly include techniques such as genetic engineering, protein engineering and molecular evolution. Genetic engineering is to artificially design and synthesize the gene sequence of DAF, and then transfect or transform it into a suitable host cell to express the protein of DAF. Protein engineering is to modify the structure or function of DAF protein by chemical or enzymatic modification. Molecular evolution is to obtain DAF variants with higher affinity or specificity through random or directional mutations of DAF genes or proteins, and then by screening or sorting methods.
For example, a DAF antibody against EGFR and HER3 is prepared by the following steps: 1) Screen an anti-EGFR antibody from a human antibody phage library, which has an invariant light chain and a variable Heavy chain CDR; 2) Increase the affinity of the antibody to EGFR by introducing mutations in the light chain CDR-L3 to obtain Fab D1.5; 3) Combine the heavy chain VH of Fab D1.5 with an anti-HER3 single chain variable The fragments (scFv) are linked to form a dual-specific DAF antibody.
| Preparation method | Advantages | Disadvantages | Other information |
|---|---|---|---|
| Genetic engineering | It can flexibly design and synthesize the gene sequence of DAF, achieving different target combinations and selections; It can use different host cells to express DAF, improving its yield and stability; It can use gene editing technology to increase or decrease the glycosylation or other modifications of DAF, improving its pharmacokinetics and immunogenicity. | It needs to optimize the gene sequence of DAF, avoiding introducing unnecessary mutations or hybridizations; It needs to select suitable host cells and expression systems, ensuring the correct folding and function of DAF; It needs to perform strict purification and detection, ensuring the quality and safety of DAF. | Genetic engineering can prepare various forms of DAF, such as Fc fusion proteins, single-chain variable fragments (scFv), bispecific antibodies (bsAb), etc. |
| Protein engineering | It can use chemical or enzymatic modifications to increase or decrease the molecular weight, charge, hydrophilicity and other physicochemical properties of DAF, affecting its distribution and clearance in the body; It can connect different functional molecules, such as fluorophores, radioactive isotopes, toxins, etc., endowing DAF with new biological functions; It can change the connection mode or conformation of DAF, regulating its binding mode or affinity with targets. | It needs to select suitable modifiers or linkers, avoiding damaging the structure or function of DAF; It needs to control the position and degree of modification or connection, maintaining the dual specificity of DAF; It needs to evaluate the biological activity and toxic side effects of modified or connected DAF. | Protein engineering can prepare various types of DAF, such as antibody-drug conjugates (ADC), antibody-oligonucleotide conjugates (AOC), antibody-enzyme conjugates (AEC), etc. |
| Molecular evolution | It can use random or directed mutations to produce a large number of DAF variants, increasing its diversity and plasticity; It can use screening or sorting techniques to quickly select advantageous variants with higher affinity or specificity from a large number of DAF variants; It can use multiple iterative evolution to continuously optimize the performance and function of DAF. | It needs to design suitable mutation strategies, avoiding producing invalid or harmful variants; It needs to establish an effective screening or sorting system, ensuring the reliability and accuracy of the screening results; It needs to verify the difference and similarity between evolved DAF and original DAF. | Molecular evolution can prepare various shapes of DAF, such as bifunctional Fab (DFAb), dual-specificity T cell redirecting |
Currently, there are three DAF that have been approved worldwide. Blinatumomab is A bispecific T-cell engager that targets CD19 and CD3, approved in 2014 in the United States for the treatment of relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). The drug works by bringing T cells and B cells together, activating the killing function of T cells and eliminating leukemia cells. The common adverse reactions of the drug include fever, infection, neurotoxicity, etc. Emicizumab, a dual-function Fab (DFAb) that targets factor IXa and factor X, is approved in 2017 in the United States, European Union, Japan and other regions for the prevention of bleeding in patients with hemophilia A. The drug works by mimicking the function of the missing factor VIII, promoting blood coagulation and reducing the risk of bleeding. The common adverse reactions of the drug include injection site reactions, headache, joint pain, etc. And Dostarlimab, a bispecific antibody (bsAb) that targets PD-1 and LAG-3, is approved in 2021 in the United States for the treatment of dMMR/MSI-H endometrial cancer. The drug works by blocking two immune checkpoint molecules simultaneously, enhancing the attack ability of T cells against tumor cells. The common adverse reactions of the drug include fatigue, nausea, diarrhea, rash, etc.
At present, there are many DAF in different stages of clinical trials. For example, REGN1979, a bispecific antibody (bsAb) that targets CD20 and CD3, is currently in phase III clinical trials for the treatment of relapsed or refractory non-Hodgkin lymphoma (NHL). The drug works by bringing T cells and B cells together, activating the killing function of T cells and eliminating lymphoma cells. The drug showed high efficacy and tolerability in phase II clinical trials. Also, teclistamab is a bispecific T-cell engager that targets BCMA and CD3, currently in phase II clinical trials for the treatment of relapsed or refractory multiple myeloma (MM). The drug works by bringing T cells and myeloma cells together, activating the killing function of T cells and eliminating myeloma cells. The drug showed promising results in a phase I clinical trial, with nearly two-thirds of participants having at least a partial response and almost 40% having a complete remission of their cancer. The drug was recently approved for use in the European Union for adults with MM that did not respond to or came back after at least three other treatments for their disease. Moreover, REGN5458, a bispecific antibody (bsAb) that targets BCMA and CD3, is currently in phase I clinical trials for the treatment of relapsed or refractory MM. The drug works by bringing T cells and myeloma cells together, activating the killing function of T cells and eliminating myeloma cells. The drug showed encouraging results in a phase I clinical trial, with more than half of participants having at least a partial response and 28% having a very good partial response or better.
| DAF name | Target | Indication | Clinical stage | Approval time | Approval region |
|---|---|---|---|---|---|
| Blinatumomab | CD19/CD3 | B-ALL, B-NHL, B-CP ALL | Completed phase III | 2014 | USA, EU, Japan etc. |
| Emicizumab | FIXa/FX | Hemophilia A | Completed phase III | 2017 | USA, EU, Japan etc. |
| Dostarlimab | PD-1/LAG-3 | dMMR/MSI-H endometrial cancer | Completed phase II | 2021 | USA |
| REGN1979 | CD20/CD3 | Relapsed or refractory NHL | Ongoing phase III | - | - |
| Teclistamab | BCMA/CD3 | Relapsed or refractory MM | Ongoing phase II | - | - |
| REGN5458 | BCMA/CD3 | Relapsed or refractory MM | Ongoing phase I | - | - |
| Epcoritamab (DuoBody-CD3xCD20) | CD20/CD3 | Relapsed or refractory B-cell malignancies | Ongoing phase III | - | - |
| Cusatuzumab (ARGX-110) | CD70/CD3 | Acute myeloid leukemia (AML) | Ongoing phase II | - | - |
| XmAb20717 (DuoBody-PD-1xLAG-3) | PD-1/LAG-3 | Solid tumors | Ongoing phase I | - | - |
References
1. Eigenbrot C, et al. Two-in-One antibodies with dual action Fabs. Curr Opin Chem Biol. 2013 Jun;17(3):400-5.
2. Koenig P, et al. Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration. J Biol Chem. 2015 Sep 4;290(36):21773-86.
3. Klein C, et al. Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies. MAbs. 2012 Nov-Dec;4(6):653-63.
4. Ljungars A, et al. A bispecific IgG format containing four independent antigen binding sites. Sci Rep. 2020 Jan 31;10(1):1546.
5. Dugal-Tessier J, et al. Antibody-Oligonucleotide Conjugates: A Twist to Antibody-Drug Conjugates. J Clin Med. 2021 Feb 18;10(4):838.
6. Li H, et al. Challenges and strategies for next-generation bispecific antibody-based antitumor therapeutics. Cell Mol Immunol. 2020 May;17(5):451-461.
7. Yuan M, et al. Preparation of an Antidanofloxacin Monoclonal Antibody and Development of Immunoassays for Detecting Danofloxacin in Meat. ACS Omega. 2020 Jan 2;5(1):667-673.
8. Mullard A. FDA approves 100th monoclonal antibody product. Nat Rev Drug Discov. 2021 Jul;20(7):491-495.
9. Dahlén AD, et al. Trends in Antidiabetic Drug Discovery: FDA Approved Drugs, New Drugs in Clinical Trials and Global Sales. Front Pharmacol. 2022 Jan 19;12:807548.
10. Baeuerle PA, et al. Bispecific T-cell engaging antibodies for cancer therapy. Cancer Res. 2009;69(12):4941-4944.
11. Brinkmann U, et al. The making of bispecific antibodies. MAbs. 2017;9(2):182-212.
12. Chari A, et al. Results of an early access treatment protocol of daratumumab in United States patients with relapsed or refractory multiple myeloma. Blood. 2018;131(19):2120-2130.
13. Davila ML, et al. CD19-targeted CAR T cells as novel cancer immunotherapy for relapsed or refractory B-cell acute lymphoblastic leukemia. Clin Adv Hematol Oncol. 2016;14(10):802-808.
14. Dreier T, et al. Extremely potent, rapid and costimulation-independent cytotoxic T-cell response against lymphoma cells catalyzed by a single-chain bispecific antibody. Int J Cancer. 2002;100(6):690-697.
15. Gökbuget N, et al. Blinatumomab for minimal residual disease in adults with B-cell precursor acute lymphoblastic leukemia. Blood. 2018;131(14):1522-1531.
Welcome! For price inquiries, we will get back to you as soon as possible.
INQUIRY
SERVICES
PRODUCTS
PLATFORMS
