Preparation of bispecific antibody-protein adducts by site-specific chemo-enzymatic conjugation

Bartels, Lina; Ploegh, Hidde L.; Spits, Hergen; Wagner, Kay Uwe

doi:10.1016/j.ymeth.2018.07.013

Cited by 19 publications

(28 citation statements)

References 76 publications

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“…Generation, purification, and stability of bTCE AT1413 bTCE and AT1002 bTCE molecules were generated and purified as described previously (25). bTCE stability was assessed by incubation of 0.1 mmol/L bTCE in PBS or IMDM þ 8% FBS at 37 C for up to 21 days followed by SDS-PAGE and flow cytometric analysis.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…To convert AT1413 into a bTCE, we chose to chemo-enzymatically link a CD3e-binding scFv, derived from the antibody UCHT1 (22), to the C-termini of the AT1413 IgG heavy chains in a C-to-C orientation. C-to-C fusion of the IgG and UCHT1 scFv is achieved by combining sortase transpeptidation and click chemistry (23)(24)(25). To abolish interaction with other non-T-cell immune effector cells, we removed Fc-gamma receptor (FcgR) interaction sites by introducing two point mutations G236R and L328R into the AT1413 heavy chains (AT1413-Fc0), as described previously (26).…”

Section: Introductionmentioning

confidence: 99%

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A Chemo-enzymatically Linked Bispecific Antibody Retargets T Cells to a Sialylated Epitope on CD43 in Acute Myeloid Leukemia

et al. 2019

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Acute myeloid leukemia (AML) is a high-risk disease with a poor prognosis, particularly in elderly patients. Because current AML treatment relies primarily on untargeted therapies with severe side effects that limit patient eligibility, identification of novel therapeutic AML targets is highly desired. We recently described AT1413, an antibody produced by donor B cells of a patient with AML cured after allogeneic hematopoietic stem cell transplantation. AT1413 binds CD43s, a unique sialylated epitope on CD43, which is weakly expressed on normal myeloid cells and overexpressed on AML cells. Because of its selectivity for AML cells, we considered CD43s as a target for a bispecific T-cell-engaging antibody (bTCE) and generated a bTCE by coupling AT1413 to two T-cell-targeting fragments using chemo-enzymatic linkage. In vitro, AT1413 bTCE efficiently induced T-cell-mediated cytotoxicity toward different AML cell lines and patient-derived AML blasts, whereas endothelial cells with low binding capacity for AT1413 remained unaffected. In the presence of AML cells, AT1413 bTCE induced upregulation of T-cell activation markers, cytokine release, and T-cell proliferation. AT1413 bTCE was also effective in vivo. Mice either coinjected with human peripheral blood mononuclear cells or engrafted with human hematopoietic stem cells [human immune system (HIS) mice] were inoculated with an AML cell line or patient-derived primary AML blasts. AT1413 bTCE treatment strongly inhibited tumor growth and, in HIS mice, had minimal effects on normal human hematopoietic cells. Taken together, our results indicate that CD43s is a promising target for T-cell-engaging antibodies and that AT1413 holds therapeutic potential in a bTCE-format. Significance: These findings offer preclinical evidence for the therapeutic potential of a bTCE antibody that targets a sialylated epitope on CD43 in AML.

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Section: Protein Expression and Purificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Chemo-enzymatically Linked Bispecific Antibody Retargets T Cells to a Sialylated Epitope on CD43 in Acute Myeloid Leukemia

et al. 2019

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“…IL2, the model bioactive payload of choice, was coupled to AAZ, a known nanomolar binder of CAIX. The product was gener-ated by SrtA mediated conjugation [26][27][28][29][30] , which allowed to obtain a site-specific functionalization of the IL2 cytokine with the AAZ targeting ligand ( Figure 1A) The sortagged AAZ-LPETGG substrate was obtained by solid phase peptide synthesis and "on resin" click chemistry of the AAZ moiety, as previously described 14 followed by standard solid phase peptide synthesis (SPPS) procedures to attach the LPETGG Sortag (FigureS1A and S1B). Prior to the enzymatic coupling, the identity and purity of the produced AAZ-LPETGG material was analyzed by LC-MS, confirming the presence of a single peak (MW 986 Da) ( Figure S1D).…”

Section: Resultsmentioning

confidence: 99%

“…Due to its high specificity for the Sortag and broad substrate tolerance, SrtA-mediated transpeptidation has emerged as a powerful method for the site-specific modification of proteins. Recently SrtA has been used for the site specific conjugation of antibodies to generate products such as imaging probes [26][27][28] , bispecific antibodies 29 and antibody drug conjugates 30 .…”

Section: Introductionmentioning

confidence: 99%

Sortase-mediated site-specific modification of interleukin-2 for the generation of a tumor targeting acetazolamide-cytokine conjugate

Gouyou

Millul

Villa

et al. 2020

Preprint

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1.AbstractSmall ligands specific to tumor-associated antigens can be used as alternatives to antibodies for the delivery of small payloads such as radionuclides, cytotoxic drugs and fluorophores. Their use as delivery moiety of bioactive proteins like cytokines remains largely unexplored. Here, we describe the preparation and in vivo characterization of the first small molecule-cytokine conjugate targeting carbonic anhydrase IX (CAIX), a marker of renal cell carcinoma and hypoxia. Site-specific conjugation between interleukin-2 and acetazolamide was obtained by Sortase A-mediated transpeptidation. Binding of the conjugate to the cognate CAIX antigen was confirmed by surface plasmon resonance. The in vivo targeting of structures expressing carbonic anhydrase IX was assessed by biodistribution experiments in tumor bearing mice. Optimization of manufacturability and tumor targeting performance of acetazolamide-cytokine products will be required in order to enable industrial applications.Graphical abstract

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“…SrtA has been employed for the generation of bi-and multi-specific antibodies of several formats, e.g., conjugations of Fab [55] or scFv fragments [58] as well as C-terminally linked antibody heterodimers in combination with click chemistry [59]. For a high throughput screening approach, the group of Plückthun recently established a one-pot SrtA-mediated coupling reaction for the alternative binding protein DARPin (designed ankyrin repeat proteins) with compatibility to functional assays, due to minimal levels of monovalent side products.…”

Section: Microbial Transglutaminase and Sortase Amentioning

confidence: 99%

Greatest Hits—Innovative Technologies for High Throughput Identification of Bispecific Antibodies

Hofmann

Krah

Sellmann

et al. 2020

IJMS

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Recent years have shown a tremendous increase and diversification in antibody-based therapeutics with advances in production techniques and formats. The plethora of currently investigated bi- to multi-specific antibody architectures can be harnessed to elicit a broad variety of specific modes of actions in oncology and immunology, spanning from enhanced selectivity to effector cell recruitment, all of which cannot be addressed by monospecific antibodies. Despite continuously growing efforts and methodologies, the identification of an optimal bispecific antibody as the best possible combination of two parental monospecific binders, however, remains challenging, due to tedious cloning and production, often resulting in undesired extended development times and increased expenses. Although automated high throughput screening approaches have matured for pharmaceutical small molecule development, it was only recently that protein bioconjugation technologies have been developed for the facile generation of bispecific antibodies in a ‘plug and play’ manner. In this review, we provide an overview of the most relevant methodologies for bispecific screening purposes—the DuoBody concept, paired light chain single cell production approaches, Sortase A and Transglutaminase, the SpyTag/SpyCatcher system, and inteins—and elaborate on the benefits as well as drawbacks of the different technologies.

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Preparation of bispecific antibody-protein adducts by site-specific chemo-enzymatic conjugation

Cited by 19 publications

References 76 publications

A Chemo-enzymatically Linked Bispecific Antibody Retargets T Cells to a Sialylated Epitope on CD43 in Acute Myeloid Leukemia

A Chemo-enzymatically Linked Bispecific Antibody Retargets T Cells to a Sialylated Epitope on CD43 in Acute Myeloid Leukemia

Sortase-mediated site-specific modification of interleukin-2 for the generation of a tumor targeting acetazolamide-cytokine conjugate

Greatest Hits—Innovative Technologies for High Throughput Identification of Bispecific Antibodies

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