Fab Chains As an Efficient Heterodimerization Scaffold for the Production of Recombinant Bispecific and Trispecific Antibody Derivatives

Schoonjans, Reinhilde; Willems, An; Schoonooghe, Steve; Fiers, Walter; Grooten, Johan; Mertens, Nico

doi:10.4049/jimmunol.165.12.7050

Cited by 65 publications

(41 citation statements)

References 26 publications

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“…11 To evaluate CH1-CK heterodimerization strength in the context of 4Dm2m, we subcloned the N-terminal region of 4Dm2m, resulting in a heterodimeric construct (designated MD) composed of mD1.22-CH1 and m36.4-CK with total calculated molecular weight (cMW) of 52 kDa (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

“…Another previous study demonstrated that the use of Fab, but not CH1-CK, as a dimerization scaffold led to efficient production of bispecific and trispecific antibodies. 11 This prompted us to engineer CH1-CK to compensate for the loss of heterodimerization strength contributed by VH-VL. By combining structureguided design and library selection technologies, we identified six CH1-CK mutants that resulted in formation of more stable heterodimers of the MD variants than the wild-type MD (Figs.…”

Section: Discussionmentioning

confidence: 99%

“…4 However, our preliminary study in mice showed that 4Dm2m had a markedly shorter in vivo half-life than CD4-Ig. In addition to the potential difference in stability and specificity of binding moieties between 4Dm2m and CD4-Ig, we hypothesized that the use of CH1-CK heterodimerization scaffold and multiple long (G4S)3 linkers in 4Dm2m might contribute to the reduced pharmacokinetics because CH1-CK heterodimerization had previously been shown to be inefficient 11 and unstable, 12,13 and the (G4S)3 linker may be sensitive to proteolysis. In this study, we demonstrated that the pharmacokinetics of 4Dm2m was improved by stabilizing CH1-CK and modifying the length and composition of the polypeptide linkers.…”

Section: Introductionmentioning

confidence: 99%

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Improving the CH1-CK heterodimerization and pharmacokinetics of 4Dm2m, a novel potent CD4-antibody fusion protein against HIV-1

Chen

Bardhi

Yang

et al. 2016

mAbs

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(2016) Improving the CH1-CK heterodimerization and pharmacokinetics of 4Dm2m, a novel potent CD4-antibody fusion protein against HIV-1, mAbs, 8:4, 761-774, DOI: 10.1080/19420862.2016 To link to this article: https://doi.org/10. 1080/19420862.2016 ABSTRACTWe previously described 4Dm2m, an exceptionally potent broadly neutralizing CD4-antibody fusion protein against HIV-1. It was generated by fusing the engineered single human CD4 domain mD1.22 to both the N and C termini of the human IgG1 heavy chain constant region and the engineered single human antibody domain m36.4, which targets the CD4-induced coreceptor binding site of the viral envelope glycoprotein, to the N terminus of the human antibody kappa light chain constant region via the (G4S)3 polypeptide linkers. However, therapeutic use of 4Dm2m was limited by its short in vivo half-life. Here, we show that a combination of three approaches have successfully increased the persistence of 4Dm2m in mice. First, to stabilize the scaffold, we enhanced heterodimerization between the heavy chain constant domain 1 (CH1) and kappa light chain constant domain (CK) by using structure-guided design and phage-display library technologies. Second, to address the possibility that long polypeptide linkers might render fusion proteins more susceptible to proteolysis, we shortened the (G4S)3 linkers or replaced them with the human IgG1 hinge sequence, which is naturally designed for both flexibility and stability. Third, we introduced two amino acid mutations into the crystallizable fragment (Fc) of the scaffold previously shown to increase antibody binding to the neonatal Fc receptor (FcRn) and prolong half-lives in vivo. Collectively, these approaches markedly increased the serum concentrations of 4Dm2m in mice while not affecting other properties of the fusion protein. The new 4Dm2m variants are promising candidates for clinical development to prevent or treat HIV-1 infection. To our knowledge, this is the first report on stabilized CH1-CK, which is potentially useful as a new heterodimerization scaffold for generation of bispecific and multispecific antibodies or proteins with a more favorable pharmacokinetic profile.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Improving the CH1-CK heterodimerization and pharmacokinetics of 4Dm2m, a novel potent CD4-antibody fusion protein against HIV-1

Chen

Bardhi

Yang

et al. 2016

mAbs

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“…Typically, even simple scFv constructs cannot be expressed at high levels in mammalian cells; however, we have had considerable success in producing IgG or Fab fragments with high productivity in myeloma cultures. Since the low yield of hBS14 was likely due to its lack of the C H 1 domain, which is critical for the folding and transporting of IgG, we decided to develop an alternative type of trivalent bsMAb, based on the ''tribody'' design, 110 where two scFvs derived from the variable domains of hMN-14 are fused to h679 Fab. Once again, several varieties were generated by having an hMN-14 scFv fused to the carboxyl terminal end of both the Fd and light chain of an h679 Fab fragment.…”

mentioning

confidence: 99%

Improved Cancer Therapy and Molecular Imaging with Multivalent, Multispecific Antibodies

Sharkey

Rossi

Chang

et al. 2010

Cancer Biotherapy and Radiopharmaceuticals

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SummationAntibodies are highly versatile proteins with the ability to be used to target diverse compounds, such as radionuclides for imaging and therapy, or drugs and toxins for therapy, but also can be used unconjugated to elicit therapeutically beneficial responses, usually with minimal toxicity. This update describes a new procedure for forming multivalent and=or multispecific proteins, known as the dock-and-lock (DNL) technique. Developed as a procedure for preparing bispecific antibodies capable of binding divalently to a tumor antigen and monovalently to a radiolabeled hapten-peptide for pretargeted imaging and therapy, this methodology has the flexibility to create a number of other biologic agents of therapeutic interest. A variety of constructs, based on anti-CD20 and CD22 antibodies, have been made, with results showing that multispecific antibodies have very different properties from the respective parental monospecific antibodies. The technique is not restricted to antibody combination, but other biologics, such as interferon-a2b, have been prepared. These types of constructs not only allow small biologics to be sustained in the blood longer, but also to be selectively targeted. Thus, DNL technology is a highly flexible platform that can be used to prepare many different types of agents that could further improve cancer detection and therapy.Key words: recombinant antibodies, bispecific antibodies, non-Hodgkin's lymphoma, pretargeting, molecular imaging, antibody therapy, radioimmunotherapy Introduction A ntibodies might not be the proverbial ''magic bullet'' as initially hoped, but they undoubtedly have played a very important role in the detection and treatment of cancer, helping pave the way for a new era of targeted therapy. As is often the case when new discoveries are made, our visions of what they can be are ahead of our ability to put them into practice. For example, studies first performed in the early 1950s established the feasibility of specifically targeting radionuclides to tumors in animal models, but at the time, there were not any suitable targets known to be associated with human tumors.1-3 Many other technical hurdles, such as better-defined antibodies and improved ways to radiolabel proteins, would not become available for 20-30 years. Even in the 1970s, when the localization of a radiolabeled antibody to a human tumor-associated antigen was finally demonstrated, still, affinity-purified polyclonal antibodies were the state of the art, and computer-assisted subtraction technology was required to visualize uptake. [4][5][6][7][8] By the 1980s, new chemistries for radiolabeling proteins were being developed that greatly expanded the possibilities for a variety of imaging and therapeutic radionuclides, and, importantly, murine monoclonal antibodies became available. [9][10][11][12][13][14][15][16][17][18][19] Hybridoma technology ultimately ushered in a new age of molecular engineering that has revolutionized the applications of antibodies and biologics alike. Murine monoclonal antibo...

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“…To overcome this problem, a number of improvements could be made. The size can be increased by modifications such as PEGylation or the addition of an extra scFv [20,21]. By addition of protein domains like the human serum albumin, the interaction with the FcRn can be enabled [22].…”

Section: Introductionmentioning

confidence: 99%

Dual-Targeting for the Elimination of Cancer Cells with Increased Selectivity

2012

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Here we review recombinant proteins with a capability for dual-targeting. These molecules address two different antigens on the same tumor cell and therefore are called “dual-targeting agents”. By virtue of binding a chosen pair of antigens on the malignant cell, preferential binding to antigen double-positive over single-positive cells can be achieved when both are present in the same environment. Therapeutic effects of such agents are based on different modes of action: (1) They can act as pro-apoptotic agents or by inhibiting pro-survival signals; (2) The dual recognition moiety can be fused to effector-domains, such as bacterial toxins or other drugs, leading to the generation of bispecific antibody-drug conjugates (ADCs); (3) Dual-targeting agents can further be used to redirect an effector-cell to the tumor. A new generation of scFv-derived fusion proteins are the tandem single chain triplebodies (sctbs), which carry two scFv binding sites for antigens on the tumor cell plus a third, specific for a trigger molecule on an effector cell. The ability of preferential or selective targeting of antigen double-positive over single-positive cells opens attractive new perspectives for the use of dual-targeting agents in cancer therapy, and possibly also for the treatment of certain inflammatory and autoimmune disorders.

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Fab Chains As an Efficient Heterodimerization Scaffold for the Production of Recombinant Bispecific and Trispecific Antibody Derivatives

Cited by 65 publications

References 26 publications

Improving the CH1-CK heterodimerization and pharmacokinetics of 4Dm2m, a novel potent CD4-antibody fusion protein against HIV-1

Improving the CH1-CK heterodimerization and pharmacokinetics of 4Dm2m, a novel potent CD4-antibody fusion protein against HIV-1

Improved Cancer Therapy and Molecular Imaging with Multivalent, Multispecific Antibodies

Dual-Targeting for the Elimination of Cancer Cells with Increased Selectivity

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